BINDING MOLECULES

• US 20130344057A1
• Filed: 03/15/2013
• Published: 12/26/2013
• Est. Priority Date: 07/22/2004
• Status: Active Grant

• Current Assignee: Harbour Antibodies BV (Harbour BioMed Shanghai Co., Ltd.)
• Original Assignee: Erasmus University Medical Center Rotterdam
• Inventors: Grosveld, Franklin Gerardus, Janssens, Richard Wilhelm, Drabek, Dubravka, Craig, Roger Kingdon
• Granted Patent: US 9,346,877 B2
• Time in Patent Office: Days
• US Class Current: 424/132.1
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• 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: A61P 9/12 : Antihypertensives
• 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 16/1054 : gag-pol, e.g. p17, p24
• CPC Class Codes: C07K 16/1063 : env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
• CPC Class Codes: C07K 16/1232 : from Escherichia (G)
• CPC Class Codes: C07K 16/241 : Tumor Necrosis Factors
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 2317/22 : from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/31 : multispecific
• CPC Class Codes: C07K 2317/53 : Hinge
• CPC Class Codes: C07K 2317/569 : Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/64 : comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/73 : containing coiled-coiled motif (leucine zippers)

Abstract

The present invention relates to the manufacture of a diverse repertoire of functional heavy chain-only antibodies that undergo affinity maturation, and uses thereof. The invention also relates to the manufacture and use of a diverse repertoire of class-specific heavy chain-only antibodies and to the manufacture and use of multivalent polypeptide complexes with antibody heavy chain functionality, preferably antibody heavy chain binding functionality, constant region effector activity and, optionally, additional effector functions.

The present invention also relates to a method of generation of fully functional heavy chain-only antibodies in transgenic mice in response to antigen challenge. In particular, the present invention relates to a method for the generation of human antigen-specific, high affinity, heavy chain-only antibodies of any class, or mixture of classes and the isolation and expression of fully functional VH antigen-binding domains.

Claims

1. A method for the production of a soluble, antigen-specific V_H binding domain comprising:

(a) immunising a transgenic rodent expressing a heterologous V_H heavy chain locus with an antigen wherein;

(i) the V_H heavy chain locus comprises a variable region comprising at least one V_H gene segment, at least one D gene segment, at least one J gene segment and at least one heavy chain constant region;

(ii) each constant region does not encode a functional C_H1 domain;

(iii) a V gene segment, a D gene segment and a J gene segment are capable of recombining to form a VDJ coding sequence;

(iv) the recombined V_H heavy chain locus, when expressed upon antigen challenge, is capable of forming a soluble, heavy chain-only antibody comprising a soluble, antigen-specific V_H binding domain and a constant effector region devoid of a functional C_H1 domain with an antigen;

(b) cloning a V_H locus resulting from recombination between single V, D and J gene segments encoding a soluble, antigen-specific V_H binding domain from an antibody-producing cell of said immunised transgenic rodent after affinity maturation via somatic mutation; and

(c) producing said soluble, V_H binding domain from the clone of step (b).

2. The method of claim 1, wherein steps (b) to (d) of claim 1 are carried out by:

(a) injecting an antigen into said transgenic rodent;

(b) isolating a cell or tissue expressing an antigen-specific, heavy chain-only antibody of interest;

(c) producing a hybridoma from the cell or tissue of step (b);

(d) cloning the heavy chain-only antibody mRNA from said hybridoma; and

(e) producing said soluble, antigen-specific V_H binding domain by isolating and expressing mRNA encoding said soluble, antigen-specific heavy chain only antibody from the mRNA of step (d).

3. The method of claim 1, wherein steps (b) to (d) of claim 1 are carried out by:

(a) injecting an antigen into said transgenic rodent;

(b) isolating a cell or tissue expressing an antigen-specific, soluble heavy chain-only antibody of interest;

(c) cloning the V_H locus from mRNA derived from the isolated cell or tissue;

displaying the encoded VH domain using phage or similar library;

(d) identifying soluble, antigen-specific V_H binding domain(s); and

(e) expressing the soluble, antigen-specific V_H domain(s) alone or as a fusion protein.

4. The method of any one of claims 1-3, wherein the heterologous V_H heavy chain locus comprises naturally-occurring human V, D and J gene segments.

5. The method of claim 4, wherein the heterologous V_H heavy chain locus comprises more than one V gene segment, more than one D gene segment and more than one J gene segment.

6. The method of claim 5, wherein the heterologous V_H heavy chain locus comprises 20 or more human V gene segments, all human D gene segments, and all human J gene segments.

7. The method of claim 6, wherein the constant region is from the rat.

8. The method of any one of claims 1-3, wherein the endogenous heavy chain locus of the transgenic rodent has been silenced.