Engineered Antibody Constant Regions for Site-Specific Conjugation and Methods and Uses Therefor

• US 20160008485A1
• Filed: 12/19/2012
• Published: 01/14/2016
• Est. Priority Date: 12/23/2011
• Status: Active Grant

• Current Assignee: Pfizer Inc.
• Original Assignee: Pfizer Inc.
• Inventors: Marquette, Kimberly, Tchistiakova, Lioudmila, Tumey, L. Nathan, Graziani, Edmund, Bennett, Eric, Bikker, Jack, Calabro, Valerie
• Granted Patent: US 11,147,852 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/08 Peptides having 5 to 11 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• 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 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/32 specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/77 Internalization into the cell
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

The present invention is directed to antibodies, and antigen-binding portions thereof, engineered to introduce amino acids for site-specific conjugation. The invention relates to engineered antibody constant region (Fc, Cγ, Cκ, and Cλ) polypeptides, and portions thereof, and antibodies comprising the polypeptides. Further, the invention relates to Fc fusion proteins comprising an engineered Fc region. The invention also relates to methods and uses of the engineered antibodies and portions for, among other things, production of antibody-drug conjugate therapeutics.

Claims

1. An engineered antibody constant domain polypeptide, or a portion thereof, wherein the engineered constant domain comprises at least one amino acid substitution to introduce a cysteine residue useful for conjugation, and wherein the constant domain polypeptide is selected from the group consisting of:

(a) an engineered human IgG heavy chain constant domain (Cγ

) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K246, D249, D265, 5267, D270, N276, Y278, E283, R292, E293, E294, Y300, V302, V303, L314, N315, E318, K320, I332, E333, K334, 1336, E345, Q347, 5354, R355, M358, K360, Q362, K370, Y373, D376, A378, E380, E382, Q386, E388, N390, K392, T393, D401, F404, T411, D413, K414, R416, Q418, Q419, N421, M428, A431, L432, T437, Q438, K439, L443, and S444, According to the EU Index of Kabat;

(b) an engineered human lambda light chain constant domain (Cλ

) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of K110, A111, L125, K149C, V155, G158, T161, Q185, 5188, H189, 5191, T197, V205, E206, K207, T208 and A210, according to the numbering of Kabat;

(c) an engineered human kappa light chain constant domain (Cκ

) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of A111, K183, and N210, according to the numbering of Kabat;

(d) an engineered Cγ

polypeptide, or portion thereof, comprising at least one amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NOs;

97-100, 102, 104, 107-127, and 129-163;

(e) an engineered Cκ

polypeptide, or portion thereof, comprising at least one amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NOs;

90, 92, 95, 164, 166, and 169; and

(f) an engineered Cλ

polypeptide, or portion thereof, comprising at least one amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NOs;

172-186.

2. The engineered Cγ

polypeptide of claim 1(a), wherein the polypeptide comprises at least one mutation selected from the group consisting of a mutation at amino acid position, K334, Q347, Y373, E388, K392 and L443, according to the EU index of Kabat.

3. The engineered Cγ

polypeptide of claim 1(a), wherein the polypeptide comprises one or more of the following pairs of amino acid substitutions;

a) E380 and L443;

b) L398 and L443;

c) V422 and L443;

d) E380 and L398;

e) L398 and V422;

f) E380 and V422;

g) K392 and L443;

h) F404 and L443;

i) Q347 and L443; and

j) K392 and F404.

4. The engineered constant domain polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of (a) the amino acid sequence of SEQ ID NO:

107;

(b) the amino acid sequence of SEQ ID NO;

102;

(c) the amino acid sequence of SEQ ID NO;

131;

(d) the amino acid sequence of SEQ ID NO;

134;

(e) the amino acid sequence of SEQ ID NO;

141;

(f) the amino acid sequence of SEQ ID NO;

146; and

(g) the amino acid sequence of SEQ ID NO;

166.

5. The engineered Cγ

polypeptide of claim 1, wherein the polypeptide is selected from an IgG1, IgG2, IgG3, or an IgG4 subclass.

6. The engineered antibody constant domain polypeptide, or a portion thereof, of claim 1, wherein the engineered polypeptide is conjugated to one or more of a cytotoxic agent, cytostatic agent, chemotherapeutic agent, toxin, radionuclide, DNA, RNA, siRNA, microRNA, peptide nucleic acid, non-natural amino acid, peptide, enzyme, fluorescent tag, and biotin, wherein the conjugation is at the substituted cysteine.

7. The engineered polypeptide of claim 6, wherein the cytotoxic agent is conjugated to the polypeptide via a linker.

8. The engineered polypeptide of claim 7, where the linker is selected from the group consisting of mc (maleimidocaproyl), val-cit (valine-citrulline), mc-val-cit (maleimidocaproyl-valine-citrulline), mc-val-cit-PABC (maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate), Mal-PEG2C2 (maleimido-[CH₂CH₂O]₂CH₂CH₂C(═

O)), Mal-PEG3C2 (maleimido-[CH₂CH₂O]₃CH₂CH₂C(═

O)), and Mal-PEG6C2 (maleimido-[CH₂CH₂O]₆CH—

₂CH2C(═

O)).

10. The engineered polypeptide of claim 8, wherein the linker and the cytotoxic agent are selected from the group consisting of maleimidocaproyl-monomethyl auristatin D (mcMMAD), maleimidocaproyl-0101 (mc0101), maleimidocaproyl-3377 (mc3377), maleimidocaproyl-8261 (mc8261), valine-citrulline-monomethyl auristatin D (vcMMAD), valine-citrulline-0101 (vc0101), valine-citrulline-3377 (vc3377), valine-citrulline-8261 (vc8261), mcValCitPABCMMAD (maleimidocaproyl-valine-citrulline-monomethyl auristatin D), mcValCit0101 (maleimidocaproyl-valine-citrulline-0101), mcValCit3377 (maleimidocaproyl-valine-citrulline-3377), mcValCit8261 (maleimidocaproyl-valine-citrulline-8261), Mal-PEG2C2-MMAD, Mal-PEG3C2-MMAD, Mal-PEG6C2-MMAD, Mal-PEG2C2-0101, Mal-PEG3C2-0101, Mal-PEG6C2-0101, Mal-PEG2C2-3377, Mal-PEG3C2-3377, and Mal-PEG6C2-3377, Mal-PEG2C2-8261, Mal-PEG3C2-8261, and Mal-PEG6C2-8261.

11. An antibody, or antigen-binding portion thereof, comprising the engineered Cγ

polypeptide of claim 1(a) wherein the amino acid substitution is selected from the group consisting of K334, Q347, Y373, E388, K392, and L443.

16. An antibody, or antigen-binding portion thereof, comprising the engineered Cκ

polypeptide of claim 1(c) wherein the amino acid substitution is K183.

17. An antibody, or antigen-binding portion thereof, comprising an engineered constant domain, or portion thereof, of claim 1.

18. The antibody, or antigen-binding portion thereof, of claim 17, comprising at least one engineered constant domain selected from the group consisting of:

(a) an engineered heavy chain constant domain (Cγ

) polypeptide, or portion thereof, comprising at least one amino acid substitution selected from the group consisting of, K334, Q347, Y373, E388, K392, and L443, according to the EU index of Kabat;

and

(b) an engineered human kappa light chain constant domain (Cκ

) polypeptide, or portion thereof, comprising the amino acid substitution K183, according to the numbering of Kabat.

20. An Fc fusion protein comprising the engineered Cγ

polypeptide of claim 1(a).

21. A pharmaceutical composition comprising the antibody, or antigen-binding portion thereof, of claim 17 and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition comprising the antibody, or antigen-binding portion thereof, of claim 18 and a pharmaceutically acceptable carrier.

23. A method of treating cancer, autoimmune, inflammatory, or infectious diseases or disorders in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, or an Fc fusion protein, wherein the antibody, or antigen-binding portion thereof, or the Fc fusion protein, comprises the an engineered constant domain polypeptide, or a portion thereof, of claim 1.

26. The method of claim 23, wherein the engineered constant domain polypeptide, or portion thereof, is conjugated to one or more of a cytotoxic agent, cytostatic agent, chemotherapeutic agent, toxin, radionuclide, DNA, RNA, siRNA, microRNA, peptide nucleic acid, non-natural amino acid, peptide, enzyme, fluorescent tag, and biotin, and wherein the conjugation is at the substituted amino acid.

27. The method of claim 23, wherein the antibody comprises an engineered constant domain polypeptide, or portion thereof, of claim 10.

28. A nucleic acid encoding the engineered constant domain polypeptide of claim 1.

29. A nucleic acid encoding the engineered Fc polypeptide of claim 20.

30. A host cell comprising the nucleic acid of claim 28.

32. A host cell comprising the nucleic acid of claim 30.

36. A method of producing an engineered antibody, or antigen-binding portion thereof, comprising incubating the host cell of claim 28 under suitable conditions for expressing the antibody, or antigen-binding portion thereof, and isolating the antibody or antigen-binding portion.

9. (canceled)

12-15. -15. (canceled)

19. (canceled)

24. (canceled)

25. (canceled)

31. (canceled)

33-35. -35. (canceled)