Soluble “heavy-chain only” antibodies

• US 8,883,150 B2
• Filed: 03/19/2010
• Issued: 11/11/2014
• 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:
  US13/259,472
• Publication Number:
  US 20120151610A1
• Time in Patent Office: 1,698 Days
• Field of Search: None
• US Class Current: 424/133.1
• 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 method for obtaining nucleic acid encoding a heavy chain-only antibody chain comprising:

a) obtaining long-lived plasma cells or memory B-cells from a transgenic mammal including a heavy chain-only 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 consist of VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-33, VH3-30, VH3-23, VH1-18, VH3-15, VH4-39, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human D gene segments comprise at least 20 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, which mammal has been challenged with an antigen; and

b) isolating nucleic acid encoding a heavy chain-only antibody chain specific for the antigen from the long-lived plasma cells or memory B-cells.

2. The method of claim 1, wherein the long-lived plasma cells or memory B-cells are purified from secondary lymph glands, bone marrow or spleen of the antigen-challenged, non-human transgenic mammal.

3. The method of claim 1, wherein the long-lived plasma cells or memory B-cells are purified using cell surface markers on the cells.

4. The method of claim 3, wherein the cell surface marker is the CD138 marker.

5. The method of claim 1, further comprising isolating a population of mRNAs from the cells and producing a population of cDNAs encoding heavy chain-only antibody from said population of mRNAs.

6. The method of claim 5, further comprising:

a) cloning the population of cDNAs into an expression vector;

b) optionally purifying sequences cloned into the cDNA expression vector by transfecting said expression vectors into bacteria and selecting colonies comprising said vectors for amplification;

c) transforming a cell-line capable of expressing, accumulating, secreting or presenting a protein encoded by the expression vector with the expression vector; and

d) selecting a cell-line which expresses, accumulates, secretes or presents a heavy chain-only antibody chain which recognises the antigen.

7. The method of claim 1, further comprising isolating a population of mRNAs from the cells and producing a population of cDNAs which encode the VH domains of any antibody produced by the cells from said population of mRNAs.

8. The method of claim 7, further comprising:

a) cloning the population of cDNAs which encode the VH domains of any antibody produced by the cells into an expression vector;

b) transforming a cell-line capable of expressing, accumulating, secreting or presenting a protein encoded by the expression vector with the expression vector; and

c) selecting a cell-line which expresses, accumulates, secretes or presents a VH domain which recognises the antigen.

9. The method of claim 6 wherein the cell-line is a microbial cell-line or a mammalian cell-line.

10. A method of producing a heavy chain-only antibody comprising a soluble VH and which binds specifically to an antigen comprising:

(a) immunising a non-human transgenic mammal comprising a heavy chain-only antibody 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 consist of VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-33, VH3-30, VH3-23, VH1-18, VH3-15, VH4-39, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human D gene segments comprise at least 20 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, with the antigen, wherein the mammal expresses heavy chain-only antibodies comprising soluble VHs and 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 of step (c) into an expression vector and expressing the cDNA in a cell-line; and

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

11. The method of claim 10, wherein the isolated long-lived plasma cells or memory B-cells are immortalised prior to mRNA isolation.

12. The method of claim 10 wherein any heavy chain-only antibody locus includes at least one dominant selective marker gene.

13. The method of claim 10, which includes the step of immortalising antibody producing cells from the mammal.

14. The method of claim 13, wherein the cells are immortalised by fusion with a myeloma cell.

15. The method of claim 13, wherein the cells are immortalised by transformation with a virus.

31. The method of claim 15 or 21, wherein the virus is EBV.

16. A method of producing a heavy chain-only antibody comprising a soluble VH and which binds specifically to an antigen comprising:

(a) immunising a non-human transgenic mammal comprising a heavy chain-only antibody 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 consist of VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-33, VH3-30, VH3-23, VH1-18, VH3-15, VH4-39, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human D gene segments comprise at least 20 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, with the antigen, wherein the mammal expresses heavy chain-only antibodies comprising soluble VHs and 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 cDNAs which encode a VH domains derived from the mRNA of step (c) into expression vector such that a VH fusion protein, which may comprise a heavy chain effector region, is expressed in the cell-line; and

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

17. The method of claim 16, wherein the isolated long-lived plasma cells or memory B-cells are immortalised prior to mRNA isolation.

18. The method of claim 16, wherein any heavy chain-only antibody locus includes at least one dominant selective marker gene.

19. The method of claim 16, which includes the step of immortalising antibody producing cells from the mammal.

20. The method of claim 19, wherein the cells are immortalised by fusion with a myeloma cell.

21. The method of claim 19, wherein the cells are immortalised by transformation with a virus.

22. A method for obtaining nucleic acid encoding a soluble VH domain comprising:

a) obtaining long-lived plasma cells or memory B-cells from a transgenic mammal including a heavy chain-only 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 consist of VH1-69, VH4-59, VH3-53, VH3-49, VH4-34, VH3-48, VH3-33, VH3-30, VH3-23, VH1-18, VH3-15, VH4-39, VH1-8, VH3-07, VH2-5, VH4-4, VH1-2, and VH6-1, the human D gene segments comprise at least 20 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, which mammal has been challenged with an antigen; and

b) isolating nucleic acid encoding a soluble VH domain specific for the antigen from the long-lived plasma cells or memory B-cells.

23. The method of claim 22, wherein the long-lived plasma cells or memory B-cells are purified from the secondary lymph glands, bone marrow or spleen of the antigen-challenged, non-human transgenic mammal.

24. The method of claim 22, wherein the long-lived plasma cells or memory B-cells are purified using cell surface markers on the cells.

25. The method of claim 24, wherein the cell surface marker is the CD138 marker.

26. The method of claim 22, further comprising isolating a population of mRNAs from the cells and producing a population of cDNAs encoding heavy chain-only antibody from said population of mRNAs.

27. The method of claim 26, further comprising:

a) cloning the population of cDNAs into an expression vector;

b) optionally purifying sequences cloned into the cDNA expression vector by transfecting said expression vectors into bacteria and selecting colonies comprising said vectors for amplification;

c) transforming a cell-line capable of expressing, accumulating, secreting or presenting a protein encoded by the expression vector with the expression vector; and

d) selecting a cell-line which expresses, accumulates, secretes or presents a heavy chain-only antibody chain which recognises the antigen.

28. The method of claim 22, further comprising isolating a population of mRNAs from the cells and producing a population of cDNAs which encode the VH domains of any antibody produced by the cells from said population of mRNAs.

29. The method of claim 28, further comprising:

a) cloning the population of cDNAs which encode the VH domains of any antibody produced by the cells into an expression vector;

b) transforming a cell-line capable of expressing, accumulating, secreting or presenting a protein encoded by the expression vector with the expression vector; and

c) selecting a cell-line which expresses, accumulates, secretes or presents a VH domain which recognises the antigen.

30. The method of claim 29 wherein the cell-line is a microbial cell-line or a mammalian cell-line.