Soluble heavy-chain only antibodies

• US 9,365,655 B2
• Filed: 03/15/2013
• Issued: 06/14/2016
• Est. Priority Date: 03/24/2009
• Status: Active Grant

• Current Assignee: Harbour Antibodies BV (Harbour BioMed Shanghai Co., Ltd.)
• Original Assignee: Erasmus University Medical Center Rotterdam
• Inventors: Craig, Roger Kingdon, Grosveld, Franklin Gerardus, Janssens, Richard Wilhelm, Drabek, Dubravka, Chen, Tao, De Boer, Ernie
• Primary Examiner(s): SZPERKA, MICHAEL EDWARD
• Application Number:
  US13/815,812
• Publication Number:
  US 20130323235A1
• Time in Patent Office: 1,187 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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 16/1018 Orthomyxoviridae, e.g. influenza virus
• CPC Class Codes: C07K 16/1271 from Micrococcaceae (F), e.g. Staphylococcus
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

Abstract

The present invention provides a high affinity, antigen-specific, soluble heavy chain-only antibody which: lacks hallmark camelid-related amino acid substitutions and has FR2 substitutions which are not found in antibodies which comprise heavy and light chain; shows increased net hydrophobicity within CDR1 and an increased number of charged amino acids present in CDR3; and comprises one or more amino acid substitutions within the framework β-pleated sheet leading to increased net hydrophobicity within FR1 and increased number of charged amino acids present in FR3. Also provided are VH domains having the same properties, gene segments for their production, methods for their production, transgenic animals and uses of the antibody of the VH domains in therapy.

Claims

1. A transgenic non-human mammal comprising a transgene, the transgene comprising at least one heterologous immunoglobulin heavy chain locus comprising human VH gene segments, one or more human D gene segments, human J gene segments, and at least one mouse constant region gene segment wherein the human VH gene segments comprise up to 20 VH gene segments and all of said human VH gene segments are naturally occurring, the human D gene segments comprise at least 5 human D gene segments, the human J gene segments comprise all six human J gene segments, and the mouse constant region gene segment comprises a Cγ

lacking CH1.

2. The transgenic non-human mammal according to claim 1, wherein said locus further comprises a selectable marker.

3. The transgenic non-human mammal according to claim 1, wherein the mammal is a rodent.

4. The transgenic non-human mammal according to claim 3, wherein the rodent is a mouse.

5. A method of producing a heavy chain-only antibody which binds specifically to an antigen comprising:

(a) immunising the non-human transgenic mammal of claim 1 with the antigen, wherein the mammal expresses heavy chain-only antibodies which lack CH1 functionality in the transcribed and processed heavy chain mRNA;

(b) isolating long-lived plasma cells or memory B-cells from the immunised mammal;

(c) isolating an mRNA population from cells derived from step (b);

(d) cloning a cDNA population derived from the mRNA isolated in step (c) into an expression vector and expressing the cDNA in a cell-line of choice; and

(e) selecting at least one cell-line which produces a heavy chain-only antibody which binds specifically to the antigen.

6. A method of producing a heavy chain-only antibody which binds specifically to an antigen comprising:

(a) immunising the non-human transgenic mammal of claim 1 with the antigen, wherein the mammal expresses heavy chain-only antibodies which lack CH1 functionality in the transcribed and processed heavy chain mRNA;

(b) isolating long-lived plasma cells or memory B-cells from the immunised mammal;

(c) isolating an mRNA population from cells derived from step (b);

(d) cloning a cDNA population comprising VH domains derived from the mRNA isolated in step (c) into expression vector of choice such that a VH fusion protein, which may comprise a heavy chain effector region, is expressed in a cell-line of choice; and

(e) selecting at least one cell-line which produces a VH fusion protein which binds specifically to the antigen.

7. A method of deriving a human, antibody from a hybrid antibody comprising:

(a) carrying out the steps of claim 5 or 6;

(b) cloning and sequencing the V_H domain from the cell line;

(c) recloning selected sequences comprising the V_H binding domain coding sequences with human constant effector domains of choice; and

(e) expressing the recloned sequences using an expression vector in a cell type of choice.

8. A vector comprising an immunoglobulin heavy chain locus comprising human VH gene segments, one or more human D gene segments, human J gene segments, and at least one mouse constant region gene segment wherein the human VH gene segments comprise up to 20 VH gene segments and all of said human VH gene segments are naturally occurring, the human D gene segments comprise at least 5 human D gene segments, the human J gene segments comprise all six human J gene segments, and the mouse constant region gene segment comprises a Cγ

lacking CH1.