Production of motif-specific and context-independent antibodies using peptide libraries as antigens

• US 9,738,711 B2
• Filed: 12/17/2014
• Issued: 08/22/2017
• Est. Priority Date: 09/04/1998
• Status: Expired due to Fees

• Current Assignee: Cell Signaling Technology Incorporated
• Original Assignee: Cell Signaling Technology Incorporated
• Inventors: Comb, Michael J., Tan, Yi, Hornbeck, Peter
• Primary Examiner(s): ROARK, JESSICA HOPE
• Application Number:
  US14/573,635
• Publication Number:
  US 20150259405A1
• Time in Patent Office: 979 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 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 2317/20 characterized by taxonomic origin

Abstract

A method is provided for producing motif-specific, context-independent antibodies that recognize a plurality of peptides or proteins within a genome that contain the same post-translationally modified motif. The method includes the step of immunizing a host with a degenerate peptide library antigen featuring (i) a fixed target motif containing one or more invariant amino acids including at least one modified amino acid, and (ii) a plurality of degenerate amino acids flanking the motif. Motif-specific, context-independent antibodies produced by the disclosed method are also provided. The method encompasses motifs consisting of a single modified amino acid, as well as short motifs comprising multiple invariant amino acids including one or more modified amino acids, such as all or part of kinase consensus substrate motifs, protein-protein binding motifs, or other cell signaling motifs. Methods of using the antibodies, e.g. for genome-wide profiling, are also provided.

Claims

1. A motif-specific, context-independent antibody that specifically binds a modified motif, which is recurring in a plurality of non-homologous peptides or proteins within an organism, consisting of (i) two to six invariant amino acids including at least one phosphorylated, acetylated, or methylated amino acid, and, optionally, (ii) one or more degenerate amino acid position(s), said antibody specifically binding said motif in a plurality of non-homologous peptides or proteins within an organism in which it recurs.

2. The antibody of claim 1, wherein said motif is selected from the group consisting of methylarginine-G-G, methylarginine-G-G-methylarginine-G-G (SEQ ID NO:

317), methylarginine-X-methylarginine, methylarginine-G, and G-methylarginine-G.

3. The antibody of claim 1, wherein said motif is selected from the group consisting of XXkXXXK, XXGkXX, XXkXE, XXKkXX, XXEkXX, XXkLXX, and XXkXEXKXX, wherein k=acetyl-lysine.

4. The antibody of claim 1, wherein said motif is a phosphorylated kinase consensus substrate motif selected from the group consisting of XX[s/t]PX, X[S/T]X[s/t]XX, XX[s/t]XXX[S/T]X, XX[s/t]X[D/E]X, X[S/T]XX[s/t]XX, XX[s/t]XXX[E/D]X, XX[s/t]XXXXPX, XX[s/t][L/I]X, XD[s/t]XX, XX[s/t]DX, XX[s/t]QX, X[V/L/I]Yxx, X[D/E]XXyXX, XyXX[V/L]X, X[D/E]XyXX, XXyXX[I/M]X, XXyXNX, XNXyXX, XYyXX, XHyXX, XXyXXCX, XyXXXXCX, and XyXXXWX, wherein s=phosphoserine, t=phosphothreonine, and y=phosphotyrosine.

5. The antibody of claim 1, wherein said motif is a phosphorylated kinase consensus substrate motif selected from the group consisting of an AGC kinase consensus substrate motif, an Akt kinase consensus substrate motif, an AMPK kinase consensus substrate motif, an ATM/ATR kinase consensus substrate motif, a CaMK kinase consensus substrate motif, a Cdc2 kinase consensus substrate motif, a CDK kinase consensus substrate motif, a Chk1 kinase consensus substrate motif, a CK kinase consensus substrate motif, a CLK kinase consensus substrate motif, a DAPK3 kinase consensus substrate motif, a DCAMKL1 kinase consensus substrate motif, a DMPK kinase consensus substrate motif, a Dual specificity JNK kinase consensus substrate motif, a Dual specificity MAPK kinase consensus substrate motif, a Dual specificity p38 kinase consensus substrate motif, and a DYRK1A kinase consensus substrate motif.

6. The antibody of claim 1, wherein said motif is a phosphorylated kinase consensus substrate motif selected from the group consisting of an ERK1 kinase consensus substrate motif, a GSK3 kinase consensus substrate motif, a Histone H kinase consensus substrate motif, a KSR kinase consensus substrate motif, a LKB1 kinase consensus substrate motif, a MAPKAPK-2 kinase consensus substrate motif, a MAPKKK kinase consensus substrate motif, a Nek1 kinase consensus substrate motif, a PAK kinase consensus substrate motif, a PDK1 kinase consensus substrate motif, a Phosphorylase kinase consensus substrate motif, a Pim1 kinase consensus substrate motif, a PKA kinase consensus substrate motif, a PKC kinase consensus substrate motif, a PKG kinase consensus substrate motif, a Proline-directed kinase consensus substrate motif, a SLK1 kinase consensus substrate motif and a TGF-beta kinase consensus substrate motif.

7. The antibody of claim 1, wherein said motif is a phosphorylated protein binding motif selected from the group consisting of a MDC1 binding motif, a CDC4/WD40 binding motif, a BRCA1 binding motif, a Chk2/FHA binding motif, a14-3-3 binding motif, a BRCT binding motif, a BARD1 binding motif, a WW binding motif, a RAD9 binding motif, a SCF-Beta/TROP binding motif, a Plk1/Polobox binding motif, a PP1 binding motif, and a FHA binding motif.

8. The antibody of claim 1, wherein said motif is a phosphorylated phosphatase consensus substrate motif selected from the group consisting of a PP2C delta consensus substrate motif, a MKP1 consensus substrate motif, a MKP3 consensus substrate motif, and a Calcinuerin consensus substrate motif.

9. The antibody of any one of claims 1-8 produced by a method comprising the steps of:

(a) constructing a degenerate peptide library comprising (i) a fixed target motif comprising two to six invariant amino adds including at least one modified amino add, and, optionally, one or more degenerate amino add position(s), and (ii) a plurality of degenerate amino adds flanking said motif;

(b) immunizing a host with said peptide library to raise a context-independent antibody specific for all or part of said target motif; and

(c) isolating antisera from said host, and purifying the motif-specific, context-independent antibody of step (b) from said antisera, said antibody specifically binding said motif in a plurality of peptides or proteins within an organism in which it recurs.

10. The method of claim 9, further comprising the step of utilizing spleen cells from the host of step (b) to generate at least one monoclonal;

motif-specific, context-independent antibody.

11. The method of claim 9, wherein said peptide library is from 6 to 20 amino adds long.

12. The method of claim 9, wherein said peptide library is from 6 to 14 amino adds long.