Humanized immunoglobulins and methods of making the same

• US 6,180,370 B1
• Filed: 06/07/1995
• Issued: 01/30/2001
• Est. Priority Date: 12/28/1988
• Status: Expired due to Term

• Current Assignee: PDL BioPharma, Inc.
• Original Assignee: Protein Design Labs, Inc. (PDL BioPharma, Inc.)
• Inventors: Selick, Harold E., Queen, Cary L.
• Primary Examiner(s): BURKE, JULIE ELAINE
• Application Number:
  US08/484,537
• Time in Patent Office: 2,064 Days
• Field of Search: 424/133.1, 424/143.1, 435/328, 435/69.6, 435/172.3, 530/387.3, 530/388.2
• US Class Current: 435/69.6
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/12 Antivirals
• 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 37/06 Immunosuppressants, e.g. drugs for graft rejection
• 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 16/087 Herpes simplex virus
• CPC Class Codes: C07K 16/088 Varicella-zoster virus
• CPC Class Codes: C07K 16/089 Cytomegalovirus
• CPC Class Codes: C07K 16/249 Interferons
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/465 with additional modified FR-residues
• 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 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/732 : Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/02 : containing a signal sequence
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

Novel methods for producing, and compositions of, humanized immunoglobulins having one or more complementarity determining regions (CDR'"'"'s) and possible additional amino acids from a donor immunoglobulin and a framework region from an accepting human immunoglobulin are provided. Each humanized immunoglobulin chain will usually comprise, in addition to the CDR'"'"'s, amino acids from the donor immunoglobulin framework that are, e.g., capable of interacting with the CDR'"'"'s to effect binding affinity, such as one or more amino acids which are immediately adjacent to a CDR in the donor immunoglobulin or those within about about 3 Å as predicted by molecular modeling. The heavy and light chains may each be designed by using any one or all of various position criteria. When combined into an intact antibody, the humanized immunoglobulins of the present invention will be substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound containing an epitope.

Claims

1. A method of producing a humanized immunoglobulin which specifically binds to an antigen, the method comprising:

providing a cell containing DNA segments encoding humanized light and heavy chain variable regions; and

expressing the DNA segments in the cell to produce the humanized immunoglobulin;

wherein the cell containing the DNA segments was produced by;

(1) comparing the sequence of a donor immunoglobulin heavy chain variable region against a collection of sequences of human heavy chain variable regions;

(2) selecting a human heavy chain variable region from the collection of human heavy chain variable regions to provide an acceptor heavy chain variable region, wherein the selected variable region framework is at least 65% identical to the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering;

(3) synthesizing the DNA segment encoding the humanized heavy chain variable region, comprising complementarity determining regions (CDRs) from the donor immunoglobulin heavy chain variable region and a variable region framework from the selected acceptor heavy chain variable region;

(4) introducing the DNA segment encoding the humanized immunoglobulin heavy chain variable region and the DNA segment encoding the humanized immunoglobulin light chain variable region into the cell,

wherein the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acid residues identical to those in the acceptor immunoglobulin heavy chain variable region framework.

2. A method of producing a humanized immunoglobulin, the method comprising:

providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 10⁸ M<sup>−

1</sup> and no greater than about four-fold that of the donor immunoglobulin, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, and the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acid residues identical to those in the acceptor human immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering; and

expressing the DNA segments in the cell to produce the humanized immunoglobulin.

25. A method of claim 2, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 70% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering.

3. A method of producing a humanized immunoglobulin, the method comprising:

providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having hypervariable regions from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 10⁸ M<sup>−

1</sup> and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs, or

(III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences;

expressing the DNA segments in the cell to produce the humanized immunoglobulin.

4. A method of producing a humanized immunoglobulin having hypervariable regions and framework regions, the method comprising:

providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy or light chain frameworks, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs; and

expressing the DNA segments in the cell to produce the immunoglobulin.

5. A method of producing a pharmaceutical composition, comprising;

formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition, wherein the humanized immunoglobulin was produced by;

(1) comparing the sequence of a donor immunoglobulin heavy chain variable region against a collection of sequences of human heavy chain variable regions;

(2) selecting a human heavy chain variable region from the collection of human heavy chain variable regions to provide an acceptor heavy chain variable region, wherein the selected variable region framework is at least 65% identical to the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering;

(3) synthesizing a DNA segment encoding a humanized heavy chain variable region, comprising complementarity determining regions (CDRs) from the donor immunoglobulin heavy chain variable region and a variable region framework from the selected acceptor heavy chain variable region;

(4) introducing the DNA segment encoding the humanized immunoglobulin heavy chain variable region and a DNA segment encoding a humanized immunoglobulin light chain variable region into a cell; and

(5) expressing the DNA segments in the cell to produce the humanized immunoglobulin,

wherein the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acid residues identical to those in the acceptor immunoglobulin heavy chain variable region framework.

26. A method of claim 5, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 70% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering.

6. A method of producing a pharmaceutical composition, comprising:

formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition, wherein the humanized immunoglobulin was produced by a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 10⁸ M<sup>−

1</sup> and no greater than about four-fold that of the donor immunoglobulin, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, and the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acid residues identical to those in the acceptor human immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering.

27. A method of claim 6, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 70% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering.

7. A method of producing a pharmaceutical composition, comprising:

formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition, wherein the humanized antibody has complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 10⁸ M<sup>−

1</sup> and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs, or

(III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences,

with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

8. A method of producing a pharmaceutical composition, comprising:

formulating a humanized immunoglobulin with a carrier to form a pharmaceutical composition,

wherein the humanized antibody has complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs,

with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

9. A humanized immunoglobulin having hypervariable regions from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 10⁸ M⁻

1 and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs,

wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab′

)₂.

10. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin framework outside the hypervariable regions that replace the corresponding amino acids in the acceptor immunoglobulin heavy or light chain frameworks, and each of these said donor amino acids:

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs, or

(III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences,

wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab′

)₂.

14. A humanized immunoglobulin according to claim 10, wherein said donor amino acids are from the donor immunoglobulin heavy chain framework.

18. A humanized immunoglobulin according to any one of claims 9 to 16, which comprises at least 3 said amino acids from the donor immunoglobulin framework.

19. A humanized immunoglobulin according to any one of claims 9 to 16, which comprises two light chain/heavy chain dimers.

20. A humanized immunoglobulin according to any one of claims 9 to 16, which is substantially pure.

21. A pharmaceutical composition comprising a humanized immunoglobulin according to claim 20 in a pharmaceutically acceptable carrier.

22. A humanized immunoglobulin according to any one of claims 9 to 16, wherein said humanized immunoglobulin has an affinity for antigen, wherein said affinity is greater than the affinity of another immunoglobulin which has the same sequence as the humanized immunoglobulin except without the donor immunoglobulin framework amino acids that replace the corresponding amino acids in the acceptor immunoglobulin.

11. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin framework outside the Kabat and Chothia CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy or light chain frameworks, and each of these said donor amino acids is capable of interacting with the CDRs,

wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab′

)₂.

15. A humanized immunoglobulin according to claim 11, wherein said donor amino acids are from the donor immunoglobulin heavy chain framework.

17. A humanized immunoglobulin according to any one of claims 9, 11, or 12, further comprising an amino acid from the donor immunoglobulin framework that replaces the corresponding amino acid in the acceptor immunoglobulin heavy or light chain frameworks, wherein said amino acid is typical at its position in human immunoglobulin sequences and said corresponding amino acid in the acceptor immunoglobulin is rare at its position in human immunoglobulin sequences.

12. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least 10⁸ M⁻

1 and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs,

with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

16. A humanized immunoglobulin according to claim 12, wherein said humanized immunoglobulin is an antibody tetramer, Fab, or (Fab′

)₂.

13. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor heavy chain immunoglobulin framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs, or

(III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences,

with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

23. A method of producing a humanized immunoglobulin, the method comprising:

providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs, or

(III) is typical at its position for human immunoglobulin sequences, and the replaced amino acid is rare at its position for human immunoglobulin sequences; and

expressing the DNA segments in the cell to produce the humanized immunoglobulin, with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

24. A method of producing a humanized immunoglobulin, the method comprising:

providing a cell containing DNA segments encoding heavy and light chain variable regions of a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from acceptor immunoglobulin heavy and light chains, which humanized immunoglobulin specifically binds to an antigen with an affinity constant of at least about 10⁸ M<sup>−

1</sup> and no greater than about four-fold that of the donor immunoglobulin, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids;

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs;

and expressing the DNA segments in the cell to produce the immunoglobulin, with the proviso that each of these said donor amino acids is outside Chothia CDR H1 (amino acids 26-32).

28. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks, which humanized immunoglobulin specifically binds to an antigen, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 70% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, and the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acids identical to those in the acceptor human immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering.

29. A humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks, which humanized immunoglobulin specifically binds to an antigen, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, and the humanized immunoglobulin heavy chain variable region framework comprises at least 70 amino acids identical to those in the acceptor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering and wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin heavy chain framework outside the Kabat CDRs that replace the corresponding amino acids in the acceptor immunoglobulin heavy chain framework, and each of these said donor amino acids:

(I) is adjacent to a CDR in the donor immunoglobulin sequence, or

(II) is capable of interacting with the CDRs.

30. A humanized immunoglobulin according to claim 29, wherein the sequence of the acceptor immunoglobulin heavy chain variable region framework is at least 70% identical to the sequence of the donor immunoglobulin heavy chain variable region framework, wherein percentage sequence identity is determined by aligning amino acids in said frameworks by Kabat numbering.