Methods and reagents for creating monoclonal antibodies

Patent metadata

Field	Value
Publication number	US-9920110-B2
Application number	US-201213416582-A
Country	US
Kind code	B2
Filing date	Mar 9, 2012
Priority date	Mar 9, 2011
Publication date	Mar 20, 2018
Grant date	Mar 20, 2018

Abstract

In some embodiments, the invention relates to methods for creating a monoclonal antibody that specifically binds to antigen. The method may start from a polyclonal population of antibodies such as a non-specific polyclonal population or a polyclonal population of antibodies that specifically bind to the antigen. The method includes obtaining nucleic acid molecules encoding heavy and light immunoglobulin chains (or variable regions thereof) of multiple immunoglobulins from an animal; obtaining mass spectra information of peptide fragments of a population of polyclonal immunoglobulins that specifically bind to an antigen of choice; comparing and/or correlating the mass spectra information of the peptide fragments of the polyclonal immunoglobulins with predicted mass spectra information of predicted amino acid sequences encoded by the nucleic acid molecules, and then assembling the heavy and light chains to create an antibody (or variable region thereof) that specifically binds to the antigen.

First claim

What is claimed is:

1. A method for obtaining nucleic acid sequences or amino acid sequences of heavy or light chains of immunoglobulins that specifically bind to an antigen of interest, comprising:
(a) providing nucleic acid sequences encoding immunoglobulin chains of white blood cells of at least one animal, and deriving predicted mass spectra information from predicted amino acid sequences encoded by said nucleic acid sequences;
(b) isolating a population of polyclonal immunoglobulins, wherein said population of polyclonal immunoglobulins is a purified population of polyclonal immunoglobulins obtained by antigen affinity purification with said antigen of interest, digesting said population of polyclonal immunoglobulins with one or more proteases to obtain peptide fragments, and performing mass spectrometry analysis to obtain mass spectra information of said peptide fragments;
(c) correlating the mass spectra information of said peptide fragments obtained in step (b) with the predicted mass spectra information in step (a), identifying nucleic acid sequences encoding immunoglobulin chains that comprise amino acid sequences corresponding to one or more of said peptide fragments, determining heavy or light variable regions and CDR3 regions of the immunoglobulin chains encoded by the identified nucleic acid sequences; and for the heavy or light variable region and the CDR3 region encoded by each identified nucleic acid sequence, calculating the amino acid sequence coverage of the heavy or light variable region by said peptide fragments, and calculating the amino acid sequence coverage of the CDR3 region by said peptide fragments;
(d) selecting nucleic acid sequences having an amino acid sequence coverage of the heavy or light variable region of at least 60% and an amino acid sequence coverage of the CDR3 region of at least 60% from the identified nucleic acid sequences to obtain nucleic acid sequences or amino acid sequences of heavy or light chains of immunoglobulins that specifically bind to said antigen of interest; and
(e) producing a heavy or light chain encoded by a nucleic acid sequence obtained in step (d) by recombinant molecular biology techniques or gene synthesis techniques.

2. The method of claim 1 , wherein said at least one animal of step (a) is an animal exposed to said antigen of interest.

3. The method of claim 1 , wherein the nucleic acid sequences provided in step (a) are expressed nucleic acid sequences.

4. The method of claim 1 , wherein the nucleic acid sequences encoding immunoglobulin chains are obtained from said white blood cells of said at least one animal by:
(1) isolating nucleic acid molecules from said white blood cells from said at least one animal; and
(2) amplifying nucleic acid molecules encoding immunoglobulin chains using primers specific for polynucleotide sequences adjacent to said nucleic acid molecules encoding immunoglobulin chains, and
(3) obtaining nucleic acid sequences of said amplified nucleic acid molecules encoding immunoglobulin chains.

5. The method of claim 4 , wherein the nucleic acid molecules are RNA molecules and said amplification step includes an initial reverse transcription step.

6. The method of claim 4 , wherein said polynucleotide sequences in step (2) are selected from the group consisting of genomic DNA flanking immunoglobulin genes, immunoglobulin chain constant region-encoding polynucleotide sequences, and immunoglobulin chain framework region-encoding polynucleotide sequences.

7. The method of claim 1 , wherein the predicted mass spectra information is obtained using a method comprising the steps of:
(i) performing a theoretical digest of predicted amino acid sequences encoded by the nucleic acid sequences with one or more proteases and/or one or more chemical protein cleavage reagents to generate virtual peptide fragments; and
(ii) creating predicted mass spectra of said virtual peptide fragments.

8. The method of claim 1 , wherein the nucleic acid sequences of step (a), predicted amino acid sequences, and predicted mass spectra derived from said nucleic acid sequences are located within a genetic material database.

9. The method of claim 1 , wherein said population of polyclonal immunoglobulins of step (b) is obtained from a body fluid sample or fraction thereof of an animal.

10. The method of claim 9 , wherein said body fluid is selected from the group consisting of blood, cerebrospinal fluid, synovial fluid, peritoneal fluid, mucosal secretions, tears, nasal secretions, saliva, milk, and genitourinary secretions.

11. The method of claim 9 , wherein the animal is an animal previously exposed to said antigen of interest.

12. The method of claim 11 , wherein the animal previously exposed to said antigen of interest is an animal previously immunized with said antigen of interest.

13. The method of claim 9 , wherein the animal from which the body fluid sample or fraction thereof is obtained is the same as said at least one animal in step (a).

14. The method of claim 1 , wherein said population of polyclonal immunoglobulins of step (b) is obtained from the medium of cultured white blood cells in vitro.

15. The method of claim 1 , wherein the peptide fragments of step (b) are obtained from the population of polyclonal immunoglobulins by digesting the population with multiple proteases.

16. The method of claim 1 , wherein the selection in step (d) is additionally based on at least one parameter selected from the group consisting of the number of unique peptides mapped, spectrum share, total peptide count, unique peptide count, frequency of the encoding nucleic acid sequences, and clonal relatedness.

17. The method of claim 1 , wherein step (e) comprises:
making a heavy chain and a light chain based on the obtained nucleic acid sequences or amino acid sequences of step (d) by recombinant molecular biology techniques or gene synthesis techniques, and
assembling said heavy chain with said light chain to create an immunoglobulin that specifically binds to said antigen of interest.

18. The method of claim 17 , further comprising evaluating the immunoglobulin in an immunoassay to confirm that said immunoglobulin specifically binds to said antigen of interest.

19. The method of claim 18 , wherein the immunoassay is selected from the group consisting of a flow cytometry assay, an enzyme-linked immunosorbent assay (ELISA), a Western blotting assay, an immunohistochemistry assay, an immunofluorescence assay, a radioimmunoassay, a neutralization assay, a binding assay, an affinity assay, a protein immunoprecipitation assay, and a peptide immunoprecipitation assay.

20. A method for obtaining nucleic acid sequences or amino acid sequences of immunoglobulin chain variable regions of immunoglobulins that specifically bind to an antigen of interest comprising:
(a) providing nucleic acid sequences encoding immunoglobulin chain variable regions of multiple immunoglobulins of white blood cells of at least one animal, and deriving predicted mass spectra information from predicted amino acid sequences encoded by said nucleic acid sequences;
(b) isolating a population of polyclonal immunoglobulins, wherein said population of polyclonal immunoglobulins is a purified population of polyclonal immunoglobulins obtained by antigen affinity purification with said antigen of interest, digesting said population of polyclonal immunoglobulins with one or more proteases to obtain peptide fragments of immunoglobulin chain variable regions of said population, and performing mass spectrom

Assignees

• Polakiewicz Roberto
• Cheung Wan Cheung
• Rush Ii John Edward
• Beausoleil Sean Andre
• Cell Signaling Technology Inc

Inventors

• Polakiewicz Roberto
• Cheung Wan Cheung
• Rush Ii John Edward
• Beausoleil Sean Andre

Classifications

• C07K16/18

  against material from animals or humans · CPC title

• C07K16/082

  Hepadnaviridae (F), e.g. hepatitis B virus · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2317/565

  Complementarity determining region [CDR] · CPC title

• C07K16/44

  against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids} · CPC title