CYSTEINE ENGINEERED ANTIBODIES AND CONJUGATES

• US 20110301334A1
• Filed: 06/07/2011
• Published: 12/08/2011
• Est. Priority Date: 06/08/2010
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Junutula, Jagath R., Bhakta, Sunil
• Granted Patent: US 9,000,130 B2
• Time in Patent Office: Days
• US Class Current: 530/387.3
• CPC Class Codes: A61K 47/6801 Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• 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 47/6819 Plant toxins
• CPC Class Codes: A61K 47/6829 Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 47/6855 the tumour determinant being from breast cancer cell
• 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: A61K 51/1051 the tumor cell being from breast, e.g. the antibody being herceptin
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/04 Antipruritics
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• 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 25/16 : Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 : for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 : Ophthalmic agents
• CPC Class Codes: A61P 27/16 : Otologicals
• 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 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/02 : Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 5/38 : of the suprarenal hormones
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 16/3092 : against tumour-associated mucins
• CPC Class Codes: C07K 16/32 : against translation products of oncogenes
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2319/31 : fusions, other than Fc, for prolonged plasma life, e.g. albumin

Abstract

Cysteine engineered antibodies comprising a free cysteine amino acid in the heavy chain or light chain are prepared by mutagenizing a nucleic acid sequence of a parent antibody and replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody; expressing the cysteine engineered antibody; and isolating the cysteine engineered antibody. Certain highly reactive cysteine engineered antibodies were identified by the PHESELECTOR assay. Isolated cysteine engineered antibodies may be covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

Claims

1. An isolated cysteine engineered antibody comprising a free cysteine amino acid and a sequence in the heavy chain selected from SEQ ID NOS 50-98 or a sequence in the light chain selected from SEQ ID NOS 99-147 wherein a cysteine in the sequence is the free cysteine amino acid.

2. The isolated cysteine engineered antibody of claim 1 prepared by a process comprising:

(i) mutagenizing a nucleic acid sequence of a parent antibody by replacing one or more amino acid residues by cysteine to encode the cysteine engineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating the cysteine engineered antibody.

3. The cysteine engineered antibody of claim 2 wherein mutagenizing comprises site-directed mutagenesis.

4. The cysteine engineered antibody of claim 2 wherein the cysteine engineered antibody is expressed on a viral particle selected from a phage or a phagemid particle.

5. The cysteine engineered antibody of claim 2 further comprising:

(i) reacting the cysteine engineered antibody with a thiol-reactive affinity reagent to generate an affinity labelled, cysteine engineered antibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineered antibody to a capture media.

6. The cysteine engineered antibody of claim 5 wherein the thiol-reactive affinity reagent comprises a biotin moiety.

7. The cysteine engineered antibody of claim 5 wherein the thiol-reactive reagent comprises a maleimide moiety.

8. The cysteine engineered antibody of claim 5 wherein the capture media comprises streptavidin.

9. The cysteine engineered antibody of claim 1 wherein the cysteine engineered antibody is a fusion protein comprising the albumin-binding peptide (ABP).

10. The cysteine engineered antibody of claim 9 wherein the ABP comprises a sequence selected from SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, and SEQ ID NO;

5.

11. The cysteine engineered antibody of claim 1 wherein the cysteine engineered antibody is selected from a monoclonal antibody, an antibody fragment, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

12. The cysteine engineered antibody of claim 11 wherein the antibody fragment is a Fab fragment.

13. The cysteine engineered antibody of claim 11 wherein the cysteine engineered antibody is an anti-HER2 antibody.

14. The cysteine engineered antibody of claim 1 wherein the cysteine engineered antibody binds to one or more of receptors (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB);

(2) E16 (LAT1, SLC7A5);

(3) STEAP1 (six transmembrane epithelial antigen of prostate);

(4) 0772P (CA125, MUC16);

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B);

(8) PSCA hlg (2700050C12R1k, C530008O16R1k, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene);

(9) ETBR (Endothelin type B receptor);

(10) MSG783 (RNF124, hypothetical protein FLJ20315);

(11) STEAP2 (HGNC_—

8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein);

(12) TrpM4 (BR22450, F1120041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member

     4);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor);

(14) CD21 (CR2 (Complement receptor

     2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792);

(15) CD79b (CD79B, CD79β

, IGb (immunoglobulin-associated beta), B29);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C);

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20Rα

;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3;

(27) CD22 (B-cell receptor CD22-B isoform);

(28) CD79a (CD79A, CD79α

, immunoglobulin-associated alpha, a B cell-specific protein);

(29) CXCR5 (Burkitt'"'"'s lymphoma receptor 1, a G protein-coupled receptor);

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen);

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel

     5);

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2);

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family);

(34) FcRH1 (Fc receptor-like protein

     1);

(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated

     2); and

(36) TENB2 (putative transmembrane proteoglycan).

15. The cysteine engineered antibody of claim 1 wherein the antibody is covalently attached to a capture label, a detection label, a drug moiety, or a solid support.

16. The cysteine engineered antibody of claim 15 wherein the antibody is covalently attached to a biotin capture label.

17. The cysteine engineered antibody of claim 15 wherein the antibody is covalently attached to a fluorescent dye detection label.

18. The cysteine engineered antibody of claim 17 wherein the fluorescent dye is selected from a fluorescein type, a rhodamine type, dansyl, Lissamine, a cyanine, a phycoerythrin, Texas Red, and an analog thereof.

19. The cysteine engineered antibody of claim 15 wherein the antibody is covalently attached to a radionuclide detection label selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, and ²¹³Bi.

20. The cysteine engineered antibody of claim 15 wherein the antibody is covalently attached to a detection label by a chelating ligand.

21. The cysteine engineered antibody of claim 20 wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA.

22. The cysteine engineered antibody of claim 15 wherein the antibody is covalently attached to a drug moiety selected from a maytansinoid, an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin, enediyne antibiotics, a taxane, and an anthracycline to form an antibody-drug conjugate having Formula I:

Ab-(L-D)_p 

 

I

where Ab is the antibody, L is a linker, D is the drug moiety, and p is 1, 2, 3, or 4.

23. The cysteine engineered antibody of claim 22 wherein the antibody-drug conjugate has the structure:

24. The cysteine engineered antibody of claim 22 wherein D is a maytansinoid having the structure:

25. The cysteine engineered antibody of claim 24 wherein D is selected from the structures:

26. The cysteine engineered antibody of claim 25 having the structure:

27. The cysteine engineered antibody of claim 22 wherein D is a monomethylauristatin drug moiety MMAE or MMAF having the structures:

28. The cysteine engineered antibody of claim 27 wherein the antibody-drug conjugate is selected from the structures: