CONJUGATED COMPOUNDS COMPRISING CYSTEINE-ENGINEERED ANTIBODIES

• US 20180169255A1
• Filed: 04/10/2015
• Published: 06/21/2018
• Est. Priority Date: 04/11/2014
• Status: Active Grant

• Current Assignee: Medimmune LLC (Astrazeneca PLC)
• Original Assignee: Medimmune LLC (Astrazeneca PLC)
• Inventors: Gao, Changshou, Rainey, Godfrey, Gao, Cuihua
• Granted Patent: US 10,744,204 B2
• Time in Patent Office: Days
• 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 47/68031 the drug being an auristatin
• CPC Class Codes: A61K 47/6811 the drug being a protein or peptide, e.g. transferrin or bleomycin
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• 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 35/00 Antineoplastic agents
• 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 16/30 from tumour cells
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/71 Decreased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• 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

This disclosure provides conjugate compounds comprising antibodies and fragments thereof engineered with one or more reactive cysteine residues and more specifically to conjugate compounds with therapeutic or diagnostic applications. The conjugate compounds comprise cysteine-engineered antibodies or fragments thereof conjugated, for example, with chemotherapeutic drugs, toxins, and detection labels such as radionuclides or fluorophores. The disclosure also provides methods of using the disclosed conjugate compounds for in vitro, in situ, ex vivo, and in vivo diagnosis or treatment of mammalian cells, or associated pathological conditions.

Claims

1. A conjugate compound comprising a cysteine-engineered antibody or Fc fusion protein and at least one heterologous moiety, wherein:

(i) the Fc domain of the antibody or Fc fusion protein comprises a cysteine amino acid insertion between positions 239 and 240, wherein the amino acid position numbering is according to the EU index as set forth in Kabat; and

(ii) wherein at least one heterologous moiety is conjugated to the cysteine amino acid insertion between positions 239 and 240.

2. The conjugate compound of claim 94, wherein the engineered cysteine amino acid is selected from cysteine amino acid substitutions at amino acid positions 241, 243, 251, 253, 258, 264, 271, 285, 288, 291, 296, 301, 307, 309, 311, 329, 385, 387, 433, or 435, and combinations thereof.

3. The conjugate compound of claim 2, wherein the engineered cysteine amino acid is selected from cysteine amino acid substitutions at amino acid positions 258 or 435, and combinations thereof.

4. The conjugate compound according to claim 1, comprising:

a Cysteine (C) inserted between the Serine (S) located at position 239 and the Valine (V) located at position 240; and

(a) a Cysteine (C) substituting the Glutamic acid (E) located at position 258;

(b) a Cysteine (C) substituting the Histidine (H) located at position 435;

(c) a Cysteine (C) substituting the Arginine (R) located at position 435;

or

(d) a combination thereof,

wherein the amino acid position numbering is according to the EU index as set forth in Kabat.

5. The conjugate compound according to claim 14, wherein the Fc domain further comprises at least one engineered cysteine residue selected from cysteine amino acid substitutions at amino acid positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443 and 446.

10. The conjugate compound according to claim 1, wherein each one of the heterologous moieties is conjugated to an engineered cysteine.

16. The conjugate compound according to claim 1, wherein the Fc domain of the antibody or Fc fusion protein is a human IgG Fc domain.

17. The conjugate compound according claim 16, wherein the human IgG is an IgG1, IgG2, IgG3, or IgG4.

19. The conjugate compound according to claim 1, wherein the Fc fusion protein comprises an antigen binding domain selected from the group consisting of:

(a) an scFv;

(b) a diabody;

(c) an Fd fragment;

(d) an Fv fragment;

(e) a TANDAB®

;

(f) a F(ab′

)₂ fragment;

(g) a FCAB™

, and (h) a F(ab) fragment.

23. The conjugate compound according to claim 1, wherein the antibody is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a human antibody, or a humanized antibody.

24. The conjugate compound according to claim 1, wherein at least one heterologous moiety is a toxin, drug, radionuclide, immunomodulator, cytokine, lymphokine, chemokine, growth factor, tumor necrosis factor, hormone, hormone antagonist, enzyme, oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, peptide nucleic acid, photoactive therapeutic agent, anti-angiogenic agent, pro-apoptotic agent, non-natural amino acid, peptide, lipid, carbohydrate, scaffolding molecule, fluorescent tag, visualization peptide, biotin, serum half-life extender, capture tag, chelating agent, solid support, or a combination thereof, and wherein conjugation is at one of the engineered cysteines.

26. The conjugate compound according to claim 24, wherein the drug is an auristatin, a tubulysin, a pyrrolobenzodiazepine (PBD), or a maytansinoid.

55. The conjugate compound according to claim 1, wherein the cysteine-engineered antibody or Fc fusion protein specifically binds to at least one target.

62. The conjugate compound according to claim 55, wherein the target is a tumor antigen and the heterologous moiety is an antitumor agent.

94. The conjugate compound of claim 1, further comprising one or more engineered cysteines at 241, 243, 251, 253, 258, 264, 269, 271, 272, 274, 280, 281, 285, 288, 291, 293, 294, 296, 301, 307, 309, 311, 318, 329, 340, 341, 345, 357, 385, 386, 387, 401, 402, 411, 417, 433, 435, or 439 wherein the amino acid position numbering is according to the EU index as set forth in Kabat.

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