Combinatorial antibody libraries and uses thereof

• US 9,221,902 B2
• Filed: 11/04/2009
• Issued: 12/29/2015
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: Taurus Biosciences LLC (Ligand Pharmaceuticals, Inc.)
• Original Assignee: Fabrus, LLC. (Eloxx Pharmaceuticals, Inc.)
• Inventors: Smider, Vaughn, Graziano, James, Mao, Helen Hongyuan, Song, Byeong Doo, Chase, Tyson, Bazirgan, Omar
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Weiler, Karen S
• Application Number:
  US13/128,219
• Publication Number:
  US 20120058906A1
• Time in Patent Office: 2,246 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2869 against hormone receptors for antibodies against neuromediator receptors C07K16/286
• CPC Class Codes: C07K 16/2884 against CD44
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• 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 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/74 : Inducing cell proliferation
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/1037 : Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/08 : Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/08 : Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

Methods for making a combinatorial antibody library from human germline segments are provided. Also provided are libraries of nucleic acid molecules compiled from germline segments encoding VL chains and libraries of nucleic acid molecules encoding VH chains, and resulting antibody libraries. The libraries are provided as addressable libraries. Methods for screening antibody libraries against a target protein antigen, and the identified or selected antibodies are provided.

Claims

1. A method of generating a combinatorial human antibody library comprising a plurality of antibody or antigen-binding antibody fragment species, comprising:

a) for each antibody or antigen-binding antibody fragment species in the library, combining a human V_H and a J_H or a human V_H, a D_H, and a J_H germline segment or portion thereof in frame to generate a nucleic acid molecule encoding a VH chain or a portion thereof, wherein, optionally, the VH chain is modified by replacement or insertion of one amino acid, optionally the replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR) selected from among a CDRH1, CDRH2, CDRH3, wherein the CDR optionally is CDRH3;

b) for each antibody or antigen-binding antibody fragment species in the library, combining a human V_κ

and a J_κ

germline segment or portion thereof, or a human V_λ

and a J_λ

germline segment or portion thereof in frame to generate a nucleic acid molecule encoding a VL chain or a portion thereof, wherein, optionally, the VL chain is modified by replacement or insertion of one amino acid, optionally the replacement or insertion of at least one amino acid into at least one complementarity determining region (CDR) selected from among a CDRL1, CDRL2, and CDRL3,

wherein each nucleic acid molecule of part (a) encoding the VH chain or portion thereof comprises a different combination of V_H and J_H or V_H, D_H and J_H germline segments yielding a different nucleic acid sequence encoding the VH chain or portion thereof and/or each nucleic acid molecule of part (b) encoding the VL chain or portion thereof comprises a different combination of V_κ

and J_κ

germline segments or V_λ

and J_λ

germline segments yielding a different nucleic acid sequence encoding the VL chain; and

c) expressing nucleic acid molecules of parts (a) and (b) and generating the antibody or antigen-binding antibody fragment species that contain a VL chain and a VH chain or a sufficient portion thereof to form an antigen binding site, thereby generating the combinatorial human antibody library,

wherein the library comprises at least about 50 or 100 or more different antibody or antigen-binding antibody fragment species in an addressable format, wherein the antibody or antigen-binding antibody fragment species at each address is the same antibody or antigen-binding antibody fragment species and is different from the antibody or antigen-binding antibody fragment species at all other addresses, and wherein the amino acid sequence identity of each antibody or antigen-antibody fragment species is known based on the address of the antibody or antigen-binding antibody fragment species; and

optionally further comprising,

d) purifying the antibody or antigen-binding antibody fragment species, optionally to about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity.

2. A method according to claim 1, wherein the antibody species are full-length antibody species and the antigen-binding antibody fragment species are Fab fragments.