ANTIBODY-SN-38 IMMUNOCONJUGATES WITH A CL2A LINKER

• US 20140227180A1
• Filed: 04/17/2014
• Published: 08/14/2014
• Est. Priority Date: 12/13/2012
• Status: Active Grant

• Current Assignee: Immunomedics, Inc.
• Original Assignee: Immunomedics, Inc.
• Inventors: Govindan, Serengulam V., Goldenberg, David M., Gale, Jonathan B., Holman, Nicholas J.
• Granted Patent: US 9,107,960 B2
• Time in Patent Office: Days
• US Class Current: 424/1.49
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/507 Comprising a combination of two or more separate antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/337 having four-membered rings, e.g. taxol
• CPC Class Codes: A61K 31/4184 condensed with carbocyclic rings, e.g. benzimidazoles
• CPC Class Codes: A61K 31/4375 the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
• CPC Class Codes: A61K 31/454 containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
• CPC Class Codes: A61K 31/4745 condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48
• CPC Class Codes: A61K 31/513 having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• CPC Class Codes: A61K 31/675 having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 38/395 Alveolar surfactant peptides; Pulmonary surfactant peptides
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• 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 47/6849 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: A61K 47/6853 Carcino-embryonic antigens
• CPC Class Codes: A61K 47/6855 : the tumour determinant being from breast cancer cell
• CPC Class Codes: A61K 47/6857 : the tumour determinant being from lung cancer cell
• CPC Class Codes: A61K 47/6859 : the tumour determinant being from liver or pancreas cancer cell
• CPC Class Codes: A61K 47/6863 : the tumour determinant being from stomach or intestines cancer cell
• CPC Class Codes: A61K 47/6867 : the tumour determinant being from a cell of a blood cancer
• CPC Class Codes: A61K 47/6869 : the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
• 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: A61N 2005/1098 : Enhancing the effect of the particle by an injected agent or implanted device
• CPC Class Codes: A61P 31/00 : Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 35/04 : specific for metastasis
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: B01D 15/08 : Selective adsorption, e.g. chromatography
• CPC Class Codes: C07K 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2833 : against MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/2851 : against the lectin superfamily, e.g. CD23, CD72
• CPC Class Codes: C07K 16/2887 : against CD20
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 16/3007 : Carcino-embryonic Antigens
• CPC Class Codes: C07K 16/3015 : Breast
• CPC Class Codes: C07K 16/3023 : Lung
• CPC Class Codes: C07K 16/303 : Liver or Pancreas
• CPC Class Codes: C07K 16/3046 : Stomach, Intestines
• CPC Class Codes: C07K 16/3061 : Blood cells
• CPC Class Codes: C07K 16/3092 : against tumour-associated mucins
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/77 : Internalization into the cell
• 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: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention concerns improved methods and compositions for preparing SN-38 conjugates of proteins or peptides, preferably immunoconjugates of antibodies or antigen-binding antibody fragments. More preferably, the SN-38 is attached to the antibody or antibody fragment using a CL2A linker, with 1-12, more preferably 6 or less, most preferably 1-5 SN-38 moieties per antibody or antibody fragment. Most preferably, the immunoconjugate is prepared in large scale batches, with various modifications to the reaction scheme to optimize yield and recovery in large scale. Other embodiments concern optimized dosages and/or schedules of administration of immunoconjugate to maximize efficacy for disease treatment and minimize side effects of administration.

Claims

1. A method to produce a compound, CL2A-SN-38, of the structure,

2. The method of claim 1, further comprising reacting SN-38 with tert-butyldimethylsilyl chloride (TBDMS-Cl) to produce 10-O-TBDMS-SN-38 (intermediate 4), wherein the reaction is carried out in dichloromethane solvent.

3. The method of claim 1, further comprising reacting 10-O-TBDMS-SN-38 (intermediate 4) with triphosgene and DMAP to make 10-O-TBDMS-SN-38-20-O-chloroformate (reactive intermediate 5), wherein the reaction is performed by adding triphosgene to a dichloromethane reaction mixture that contains intermediate 4, and the triphosgene is added in portions, to reduce exothermic reaction during large scale manufacture and maintain high reaction yield.

4. The method of claim 1, further comprising precipitating Lys(MMT)-PABOH (intermediate 2) with heptane.

5. The method of claim 4, further comprising assaying residual diethylamine in the precipitated intermediate 2 and purifying the precipitate by chromatography if the presence of diethylamine was detected.

6. The method of claim 1, further comprising reacting azido-PEG-Lys(MMT)-PABO-CO-20-O-SN-38 (intermediate 7) with MCC-Yne (intermediate 8) by a copper catalyzed cycloaddition reaction to make MCC-PEG-Lys(MMT)-PABO-CO-20-O-SN-38 (intermediate 9), wherein the reaction is carried out for 14 h to improve product yield.

7. The method of claim 6, wherein intermediate 9 is first purified by silica gel chromatography, followed by extraction with EDTA to remove copper.

8. The method of claim 1, further comprising reacting a maleimide moiety of CL2A-SN38 with a protein or peptide to make an SN38-conjugated protein or peptide.

9. The method of claim 8, wherein the maleimide moiety reacts with a reduced sulfhydryl on the protein or peptide.

10. The method of claim 8, wherein the protein or peptide is an antibody or an antigen-binding antibody fragment and the SN-38 conjugated antibody or antibody fragment is an immunoconjugate.

11. The method of claim 10, further comprising purifying the immunoconjugate by tangential flow filtration (TFF).

12. The method of claim 10, wherein the TFF is performed with a 50,000 dalton molecular weight cut-off membrane using 25- to 30-diafiltration volumes of buffer.

13. The method of claim 12, further comprising formulating the immunoconjugate in Good'"'"'s biological buffer at a pH of 6.0 to 7.0, and lyophilizing the immunoconjugate for storage.

14. The method of claim 13, wherein the Good'"'"'s biological buffer is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and 1,4-piperazinediethanesulfonic acid (PIPES), in the pH range of 6-7, preferably in the pH range of 6.5 to 7, and at a buffer concentration of 10-100 mM, preferably 25 mM.

15. The method of claim 14, wherein the buffer is 25 mM MES buffer, pH 6.5.

16. The method of claim 10, wherein the antibody is a bispecific antibody or a monoclonal antibody.

17. The method of claim 10, wherein the antibody fragment is selected from the group consisting of F(ab′

)₂, F(ab)₂, Fab′

, Fab, Fv, scFv, single domain antibody and half-molecule of IgG4 antibody.

18. The method of claim 10, wherein the antibody or antibody fragment is attached to between 1 and 12 copies of CL2A-SN38.

19. The method of claim 10, wherein the antibody or antibody fragment is attached to 6 copies of CL2A-SN38.

20. The method of claim 10, wherein the antibody or antibody fragment is attached to between 1 and 5 copies of CL2A-SN38.

21. The method of claim 10, wherein the antibody is an anti-cancer antibody, an anti-infectious disease antibody, or an anti-autoimmune disease antibody.

22. The method of claim 10, wherein the antibody is selected from the group consisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), RS7 (anti-EGP-1), PAM4 (anti-MUC5AC), KC4 (anti-mucin), A19 (anti-CD19), A20 (anti-CD20), MN-14 (anti-CEACAM5), MN-15 (anti-CEACAM6), MN-3 (anti-CEACAM6), R1 (anti-IGF-1R), Mu-9 (anti-CSAp), Immu 31 (anti-AFP), CC49 (anti-TAG-72), J591 (anti-PSMA), HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX) and hL243 (anti-HLA-DR).

23. The method of claim 10, wherein the antibody or antibody fragment binds to an antigen selected from the group consisting of carbonic anhydrase IX, alpha-fetoprotein (AFP), α

-actinin-4, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α

, colon-specific antigen-p (CSAp), CEACAM5, CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO-β

, H2B, H3, H4, HLA-DR, HM1.24, human chorionic gonadotropin (HCG), HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ

, IFN-α

, IFN-β

, IFN-λ

, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PD-1, PD-L1, PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α

, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, bc1-2, bc1-6, Kras, and an oncogene product.

24. A method of treating a disease comprising:

a) producing CL2A-SN-38 according to claim 1;

b) conjugating the CL2A-SN-38 to an antibody or antigen-binding antibody fragment to make an immunoconjugate; and

c) administering the immunoconjugate to a subject with a disease.

25. The method of claim 24, wherein the subject is a human subject.

26. The method of claim 24, wherein the disease is selected from the group consisting of cancer, autoimmune disease, immune system dysfunction and infectious disease.

27. The method of claim 24, wherein the disease is cancer and the antibody or antigen-binding fragment thereof;

wherein the antibody or fragment thereof binds to an antigen selected from the group consisting of EGP-1 (TROP-2), CEACAM5, CEACAM6, CD74, CD19, CD20, CD22, CSAp, HLA-DR, MUCSac, and AFP (alpha-fetoprotein); and

wherein the immunoconjugate is administered at a dosage of between 3 mg/kg and 18 mg/kg.

28. The method of claim 27, wherein the dosage is selected from the group consisting of 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12 mg/kg, 16 mg/kg and 18 mg/kg.

29. The method of claim 23, wherein the antibody is selected from the group consisting of hLL1 (anti-CD74), hLL2 (anti-CD22), hRFB4 (anti-CD22), hRS7 (anti-TROP-2), hPAM4 (anti-MUCSac), hMN-3 (anti-CEACAM6), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hA19 (anti-CD19), hA20 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR) and hIMMU31 (anti-AFP).

30. The method of claim 25, wherein the cancer is a solid tumor and the treatment results in a reduction in tumor size of at least 15%, at least 20%, at least 30%, or at least 40%.

31. The method of claim 25, wherein the cancer is selected from the group consisting of B-cell lymphoma, B-cell leukemia, colon cancer, stomach cancer, esophageal cancer, medullary thyroid cancer, kidney cancer, breast cancer, lung cancer, pancreatic cancer, urinary bladder cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, prostate cancer, liver cancer, skin cancer, bone cancer, brain cancer, rectal cancer, and melanoma.

32. The method of claim 31, wherein the B-cell leukemia or B-cell lymphoma is selected from the group consisting of indolent forms of B-cell lymphoma, aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, non-Hodgkin'"'"'s lymphoma, Hodgkin'"'"'s lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse B-cell lymphoma, mantle cell lymphoma and multiple myeloma.

33. The method of claim 25, wherein the cancer is metastatic.

34. The method of claim 33, further comprising reducing in size or eliminating the metastases.

35. The method of claim 25, wherein the cancer is refractory to other therapies but responds to the immunoconjugate.

36. The method of claim 23, wherein the patient has failed to respond to at least one other therapy, prior to treatment with the immunoconjugate.

37. The method of claim 36, wherein the patient has failed to respond to therapy with irinotecan, prior to treatment with the immunoconjugate.

38. The method of claim 36, wherein the cancer is a solid tumor and the treatment results in a reduction in tumor size of at least 15%, at least 20%, at least 30%, or at least 40%.

39. The method of claim 23, wherein the antibody has an allotype selected from the group consisting of G1m3, G1m3,1, G1m3,2, G1m3,1,2, nG1m1, nG1m1,2 and Km3 allotypes.

40. The method of claim 23, wherein the immunoconjugate dosage is administered to the human subject once or twice a week on a schedule with a cycle selected from the group consisting of:

(i) weekly;

(ii) every other week;

(iii) one week of therapy followed by two, three or four weeks off;

(iv) two weeks of therapy followed by one, two, three or four weeks off;

(v) three weeks of therapy followed by one, two, three, four or five weeks off;

(vi) four weeks of therapy followed by one, two, three, four or five weeks off;

(vii) five weeks of therapy followed by one, two, three, four or five weeks off; and

(viii) monthly.

41. The method of claim 23, wherein the immunoconjugate is administered in combination with one or more therapeutic modalities selected from the group consisting of unconjugated antibodies, radiolabeled antibodies, drug-conjugated antibodies, toxin-conjugated antibodies, gene therapy, chemotherapy, therapeutic peptides, cytokine therapy, oligonucleotides, localized radiation therapy, surgery and interference RNA therapy.

42. The method of claim 41, wherein the drug, toxin or chemotherapeutic agent is selected from the group consisting of 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, crizotinib, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, flavopiridol, floxuridine (FUdR), 3′

,5′

-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839.