Humanized antibodies and methods for making them

• US 6,054,297 A
• Filed: 05/09/1995
• Issued: 04/25/2000
• Est. Priority Date: 06/14/1991
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Presta, Leonard G., Carter, Paul J.
• Primary Examiner(s): Hutzell, Paula K.
• Assistant Examiner(s): BURKE, JULIE ELAINE
• Application Number:
  US08/437,642
• Time in Patent Office: 1,813 Days
• Field of Search: 530/387.3, 530/387.7, 530/388.8, 530/389.7, 424/130.1, 424/133.1, 424/138.1, 424/155.1, 424/174.1, 435/328, 435/344, 435/69.6, 435/69.7
• US Class Current: 435/69.6
• 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 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• 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 2317/31 multispecific
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• 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
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: G06F 15/00 Digital computers in general details G06F1/00 – G06F13/00; Data processing equipment in general

Abstract

Variant immunoglobulins, particularly humanized antibody polypeptides are provided, along with methods for their preparation and use. Consensus immunoglobulin sequences and structural models are also provided.

Claims

1. A method for making a humanized antibody comprising non-human, import Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues, comprising the steps of:

(a) obtaining the amino acid sequences of an import variable domain and of a V_H subgroup III consensus human variable domain;

(b) identifying CDR amino acid sequences in the import and the human variable domain sequences;

(c) substituting import CDRs for the corresponding human CDRs;

(d) aligning the amino acid sequences of a FR of the import antibody and the corresponding FR of the consensus variable domain;

(e) identifying import antibody FR residues in the aligned FR sequences that are non-homologous to the corresponding consensus variable domain residues;

(f) determining if the non-homologous import amino acid residue is expected to have at least one of the following effects;

(1) non-covalently binds antigen directly;

(2) interacts with a CDR;

or

(3) participates in the V_L -V_H interface;

(g) for any non-homologous import antibody amino acid residue which is expected to have at least one of these effects, substituting that residue for the corresponding amino acid residue in the consensus variable domain FR sequence; and

(h) preparing a humanized antibody which binds antigen, wherein the humanized antibody comprises an amino acid sequence determined according to the above steps.

2. The method of claim 1, having an additional step of determining if any such non-homologous residues are exposed on the surface of the domain or buried within it, and if the residue is exposed, retaining the consensus residue.

3. The method of claim 1, having the additional steps of searching the import variable domain sequence for glycosylation sites, determining if any such glycosylation site is expected to affect the antigen binding or affinity of the antibody, and if so, substituting the glycosylation site into the consensus sequence.

4. The method of claim 1, having the additional steps of searching the consensus variable domain sequence for glycosylation sites which are not present at the corresponding amino acid in the import sequence, and if the glycosylation site is not present in the import sequence, substituting the import amino acid residues for the amino acid residues comprising the consensus glycosylation site.

5. The method of claim 1, having an additional step which comprises aligning import antibody and consensus variable domain FR sequences, identifying import antibody FR residues which are non-homologous with the aligned consensus FR sequence, and for each such non-homologous import antibody FR residue, determining if the corresponding consensus variable domain residue represents a residue which is highly conserved across all species at that site, and if it is so conserved, preparing a humanized antibody which comprises the consensus amino acid residue at that site.

6. The method of claim 1, wherein the corresponding consensus residues are selected from the group consisting of 4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L 87L, 98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H, and 103H.

7. A method for making a humanized antibody comprising non-human Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues, comprising providing an import, non-human antibody variable domain amino acid sequence having CDR amino acid residues and FR amino acid residues;

obtaining the amino acid sequence of a V_H subgroup III consensus human antibody variable domain having CDR amino acid residues and FR amino acid residues;

substituting non-human CDR amino acid residues for human CDR amino acid residues in the consensus human antibody variable domain;

substituting an amino acid residue for the consensus amino acid residue at at least one of the following sites;

4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H, 91H, 92H, 93H. and 103H; and

preparing a humanized antibody which binds an antigen, wherein the humanized antibody comprises an amino acid sequence determined according to the above steps.

8. The method of claim 7, wherein the substituted residue is the residue found at the corresponding location of the non-human antibody.

9. A humanized antibody variable domain having a functional antigen binding region, said humanized antibody variable domain comprising non-human Complementarity Determining Region (CDR) amino acid residues incorporated into a V_H subgroup III consensus human antibody variable domain, and further comprising an amino acid substitution at a site selected from the group consisting of:

4L, 35L, 36L, 38L, 43L, 44L, 46L, 58L, 62L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 68H, 69H, 70H, 73H, 74H, 75H, 76H, 78H, 92H, and 93H.

10. The humanized antibody variable domain of claim 9, wherein the substituted residue is the residue found at the corresponding location of the non-human antibody from which the non-human CDR amino acid residues are obtained.

11. The humanized antibody variable domain of claim 9, wherein no human (FR) Framework Region (FR) residue other than those set forth in the group has been substituted.

12. A method for making a humanized antibody comprising introducing Complementarity Determining Region (CDR) amino acid residues from an import antibody variable domain into a V_H subgroup III consensus human antibody variable domain.

13. A humanized antibody variable domain having a functional antigen binding region, said humanized antibody variable domain comprising non-human Complementarity Determining Region (CDR) amino acid residues incorporated into a V_H subgroup III consensus human antibody variable domain and further comprising a non-human import Framework Region (FR) residue, wherein the non-human import FR residue introduces a glycosylation site which affects the antigen binding or affinity of the humanized antibody variable domain.

14. A humanized antibody which binds the HER2 receptor with an affinity of about 4.7 nM K_d or better affinity and comprises a heavy chain variable domain which comprises non-human import antibody Complementarity Determining Region (CDR) amino acid residues incorporated into a V_H subgroup III consensus human variable domain.

15. A humanized variant of a non-human parent antibody, wherein the humanized variant comprises non-human Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues, and:

(a) binds the HER2 receptor with an affinity of about 4.7 nM K_d or better affinity;

(b) mediates specific cell lysis of SK-BR-3 calls in the presence of IL-2 activated human peripheral blood lymphocytes at least about four fold more effectively than the non-human parent antibody; and

(c) mediates Antibody Dependent Cellular Cytotoxicity (ADCC) selective for cell types which overexpress p185^HER2 at least about two fold more effectively than for cell types which express low levels of p185^HER2.

16. The humanized variant of claim 15 which binds the HER2 receptor with an affinity of about 0.82 nM K_d or better affinity.

17. The humanized variant of claim 16 which binds the HER2 receptor with an affinity of about 0.10 nM K_d.

18. The humanized variant of claim 15 which inhibits proliferation of SK-BR-3 cells incubated for 96 hr with the antibody.

19. The humanized variant of claim 18 wherein the antibody inhibits proliferation of SK-BR-3 cells to about 66% of untreated control or greater inhibition.

20. A humanized variant of a non-human parent antibody, wherein the humanized variant comprises non-human Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues, and;

(a) binds the HER2 receptor with an affinity of about 4.7 nM K_d or better affinity; and

(b) comprises a Framework Region (FR) amino acid substitution at a site selected from the group consisting of 73H, 78H, 93H and 66L, utilizing the numbering system set forth in Kabat.

21. The humanized variant of claim 20 which consists of about 1 to about 5 FR substitutions.

22. The humanized variant of claim 20 which comprises a FR substitution at site 73H.

23. The humanized variant of claim 20 which comprises a FR substitution at site 78H.

24. The humanized variant of claim 20 which comprises a FR substitution at site 93H.

25. The humanized variant of claim 20 which comprises a FR substitution at site 66L.

26. The humanized variant of claim 20 which further comprises a FR substitution at site 71H.

27. The humanized variant of claim 26 which comprises FR substitutions at sites 71H, 73H, 78H, 93H and 66L.

28. A humanized variant of a non-human parent antibody, wherein the humanized variant comprises non-human Complementarity Determining Region (CDR) amino acid residues and human Framework Region (FR) amino acid residues;

binds the HER2 receptor with better affinity than the non-human parent antibody; and

comprises Framework Region (FR) amino acid substitutions at sites 71H, 73H, 78H, 93H and 66L, utilizing the numbering system set forth in Kabat.

29. An antibody which binds an antigen and comprises non-human heavy chain variable domain Complementarity Determining Region (CDR) amino acid residues which bind said antigen and V_H subgroup III consensus human variable domain Framework Region (FR) amino acid residues;

and further comprises non-human light chain variable domain CDR amino acid residues which bind said antigen.

30. The antibody of claim 29, further comprising V_L κ

subgroup I consensus human variable domain FR amino acid residues.