ANTIBODY-DRUG CONJUGATES THAT TARGET DLK1 AND USES THEREOF

Abstract

The present invention relates to new antibody-drug conjugates (ADCs) targeting DLK1, active metabolites of such ADCs, methods for preparation of such ADCs, uses for such ADCs in treatment and/or prevention of illnesses, and uses for such ADCs in production of drugs for treatment and/or prevention of diseases, more specifically, uses for such ADCs in producing drugs for treatment and/or prevention of proliferative and/or angiogenetic diseases, for example, cancer. More particularly, the present invention relates to an antibody-drug conjugate comprising an antibody binding with DLK1 or an antigen-binding fragment thereof, and a pharmaceutical composition comprising the same.

Claims

1. An antibody conjugate represented by General Formula I, or a pharmaceutically acceptable salt thereof:
Ab−(X).sub.y   [General Formula I] wherein: Ab is an antibody or antigen-binding fragment comprising: a heavy chain variable region comprising at least one heavy chain CDR1 comprising a sequence selected from SEQ ID NO. 2, 16, 30, 44, 58, 72 and 86; at least one heavy chain CDR2 comprising a sequence selected from SEQ ID NO. 4, 18, 32, 46, 60, 74 and 88; at least one heavy chain CDR3 comprising a sequence selected from SEQ ID NO. 6, 20, 34, 48, 62, 76 and 90; a light chain variable region comprising at least one light chain CDR1 comprising a sequence selected from SEQ ID No. 9, 23, 37, 51, 65, 79, 93, 115 and 121; at least one light chain CDR2 comprising a sequence selected from SEQ ID No. 11, 25, 39, 53, 67, 81 and 95; and at least one light chain CDR3 comprising a sequence selected from SEQ ID No. 13, 27, 41, 55, 69, 83, 97, 116 and 125; each X is independently a chemical moiety comprising at least one active agent and a linker, wherein the linker links the antibody and to the at least one active agent; and y is 1 to 20.

2. The antibody conjugate of claim 1, wherein the antibody comprises: at least one heavy chain FR1 comprising a sequence selected from SEQ ID No. 1, 15, 29, 43, 57, 71, 85 and 119; at least one heavy chain FR2 comprising a sequence selected from SEQ ID No. 3, 17, 31 , 45, 59, 73 and 87; at least one heavy chain FR3 comprising a sequence selected from SEQ ID No. 5, 19, 33, 47, 61, 75 and 89; at least one heavy chain FR4 comprising a sequence selected from SEQ ID No. 7, 21, 35, 49, 63, 77 and 91; at least one light chain FR1 comprising a sequence selected from SEQ ID No. 8, 22, 36, 50, 64, 78, 92, 117 and 120; at least one light chain FR2 comprising a sequence selected from SEQ ID No. 10, 24, 38, 52, 66, 80, 94 and 122; at least one light chain FR3 comprising a sequence selected from SEQ ID No. 12, 26, 40, 54, 68, 82, 96, 118 and 123; and at least one light chain FR4 comprising a sequence selected from SEQ ID No. 14, 28, 42, 56, 70, 84, 98 and 125.

3. The antibody conjugate of claim 1, wherein the antibody comprises a heavy chain variable region comprising a sequence that has at least 90% sequence identity to a sequence selected from SEQ ID No. 99, 101, 103, 105, 107, 109, 111 and 127.

4. The antibody conjugate of claim 1, wherein the antibody comprises a light chain variable region comprising a sequence that has at least 90% sequence identity to a sequence selected from SEQ ID No. 100, 102, 104, 106, 108, 110, 112, 126 and 128.

5. The antibody conjugate of claim 1, wherein at least one X has a structure represented by General Formula II or a pharmaceutically acceptable salt thereof: ##STR00037## wherein: G is a glucuronic acid moiety or ##STR00038## R.sup.3 is hydrogen or a carboxyl protecting group; each R.sup.4 is independently hydrogen or a hydroxyl protecting group; B is an active agent; R.sub.1 and R.sub.2 are each independently hydrogen, (C1-C8) alkyl or (C3-C8) cycloalkyl; W is —C(O)—, —C(O)NR′—, —C(O)O—, —SO2NR′—, —P(O)R″NR′—, —SONR′— or —PO.sub.2NR′—, wherein the C, S or P is directly bound to the phenyl ring; R′ and R″ are each independently hydrogen, (C1-C8) alkyl, (C3-C8) cycloalkyl, (C1-C8) alkoxy, (C1-C8) alkylthio, mono- or di-(C1-C8) alkylamino, (C3-C20) heteroaryl, or (C6-C20) aryl; each Z is independently (C1-C8) alkyl, halogen, cyano or nitro; n is 1 to 3; L is comprises: A) a C.sub.1-C.sub.50 alkylene or a 1 to 50 atom heteroalkylene, and satisfies at least one of the following: (i) L comprises at least one unsaturated bond (ii) 2 atoms within L are substituted with a divalent substituent the same as in a substituent, which completes heteroarylene; (iii) L is a 1 to 50 atom heteroalkylene, and; (iv) The alkylene is substituted by at least one C.sub.1-20 alkyl; or B) at least one isoprenyl group represented by General Formula III: ##STR00039## and the bond overlaid with the wavy line represents a connection to the antibody.

6-9. (canceled)

10. The antibody conjugate of claim 5, wherein L comprises a peptide comprising 2 to 20 amino acids selected from alanine, aspartate, asparagine, glutamate, glutamine, glycine, lysine, ornithine, proline, serine and threonine.

11. The antibody conjugate of claim 5, wherein W is —C(O)NR′—.

12. (canceled)

13. The antibody conjugate of claim 5, wherein L is coupled to the antibody by a thioether bond that comprises a sulfur atom of a cysteine of the antibody.

14. (canceled)

15. The antibody conjugate of claim 13, wherein the antibody comprises the amino acid sequence CYYX, wherein: C is cysteine; Y is an aliphatic amino acid selected from of alanine, isoleucine, leucine, methionine and valine; X is selected from the glutamine, glutamate, serine, cysteine, methionine, alanine and leucine; and the thioether bond comprises a sulfur atom of cysteine of the amino acid sequence.

16. The antibody conjugate of claim 15, wherein the amino acid sequence comprises CVIM (SEQ ID NO: 130) or CVLL (SEQ ID NO: 131).

17. (canceled)

18. (canceled)

19. The antibody conjugate of claim 5, wherein L comprises the amino sequence GGGGGGGCVIM (SEQ ID NO: 132) at the C-terminal.

20-22. (canceled)

23. The antibody conjugate of claim 5, wherein comprises ##STR00040##

24. The antibody conjugate of claim 5, wherein L further comprises a connecting unit represented by General Formula VIII or General Formula IX:
—(CH.sub.2).sub.r(V(CH.sub.2).sub.p).sub.q—  [General Formula VIII]
—(CH.sub.2CH.sub.2X).sub.w—  [General Formula IX] wherein: V is a single bond, —O—, —S—, —NR.sup.21—, —C(O)NR.sup.22—, NR.sup.23C(O)—, NR.sup.24SO.sub.2—, or —SO.sub.2NR.sup.25—; X is —O—, C.sub.1-C.sub.8 alkylene or —NR.sup.21—; R.sup.21 to R.sup.25 are each independently hydrogen, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkyl (C.sub.6-C.sub.20) aryl, or (C.sub.1-C.sub.6) alkyl (C.sub.3-C.sub.20) heteroaryl; r is 0 to 10; p is 0 to 10; q is 1 to 20; and w is 1 to 20.

25. The antibody conjugate of claim 5, wherein L comprises 1 to 20 polyethylene glycol units.

26-29. (canceled)

30. The antibody conjugate of claim 5, wherein L further comprises a binding unit represented by General Formulae IV, V, VI or VII: ##STR00041## wherein: L.sup.1 is a single bond or a C.sub.1-C.sub.30 alkylene; and R11 is hydrogen or a C.sub.1-C.sub.10 alkyl.

31. (canceled)

32. The antibody conjugate of claim 5, wherein L comprises ##STR00042## wherein V is a single bond, —O—, —S—, —NR.sup.21—, —C(O)NR.sup.22—, NR.sup.23C(O)—, NR.sup.24SO.sub.2—, or —SO.sub.2NR.sup.25—; R.sup.21 to R.sup.25 are each independently hydrogen, (C.sub.1-6) alkyl, (C.sub.1-6) alkyl (C.sub.6-20) aryl, or (C.sub.1-6) alkyl (C.sub.3-20) heteroaryl; r is 0 to 10; p is 0 to 10; q is 1 to 20; and L.sup.1 is a single bond.

33. The antibody conjugate of claim 1, wherein at least one X is represented by ##STR00043## B is an active agent; n is an integer of 0 to 20; and the bond overlaid with the wavy line represents a connection to the antibody.

34-44. (canceled)

45. The antibody conjugate of claim 1, wherein the active agent is is a chemotherapeutic agent or a toxin.

46. The antibody conjugate of claim 1, wherein the active agent is an immunoregulatory compound, anti-cancer agent, antiviral agent, antibacterial agent, antifungal agent, or antiparasitic agent.

47. The antibody conjugate of claim 1, wherein the active agent is selected from: (a) erlotinib, bortezomib, fulvestrant, sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, meturedopa, uredopa, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, camptothecin, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, dolastatin, duocarmycin, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotoxin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, antrymycin, azaserine, bleomycins, catcinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tudercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguianine, ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone, propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaqizuone, 2,2′,2″-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, thiotepa, paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, docetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates or acids thereof; (b) monokine, lympokine, traditional polypeptide hormone, parathyroid hormone, thyroxine, relaxin, prorelaxin, glycoprotein hormone, follicle stimulating hormone, thyroid stimulating hormone, luteinizing hormone, hepatic growth factor fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor, tumor necrosis factor-a, tumor necrosis factor-β, Mullerian inhibiting substance, mouse gonadotropin associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-α, interferon-β, interferon-γ, colony stimulating factor (CSF), macrophage-CSF, granulocyte-macrophage-CSF), granulocyte-macrophage-CSF, granulocyte-CSF, interleukin (IL), IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factor, TNF-α, TNF-β, polypeptide factor, LIF, kit or ligand; (c) diphtheria toxin, botulinum toxin, tetanus toxin, dysentery toxin, cholera toxin, amanitin, α-amatinin, pyrrolobenzodiazepine, pyrrolobenzodiazepine derivative, indolinobenzodiazepine, pyridobenzodiazepine, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamicin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analog, auristatin, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065 analogs or derivatives, duocarmycin, enediyne antibiotic, esperamicin, epothilone, or toxoid; (d) affinity ligand, where the affinity ligand is a substrate, inhibitor, neurotransmitter, or radioactive isotope; (e) radioactive label, 32P, 35S, fluorescent dye, enzyme, biotin, streptavidin, dioxigenin, hapten, immunogenic protein, or nucleic acid molecule with a sequence complementary to a target; (f) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone or toremifene; (g) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole or anastrozole; (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (j) aromatase inhibitor; (k) protein kinase inhibitor; (l) lipid kinase inhibitor; (m) antisense oligonucleotide; (n) ribozyme; (o) vaccine; and (p) anti-angiogenic agent.

48. The antibody conjugate of claim 1, wherein the active agent is a pyrrolobenzodiazepine dimer.

49. (canceled)

50. The antibody conjugate of claim 48, wherein the pyrrolobenzodiazepine dimer has a structure represented by represented by General Formula X or General Formula XI: ##STR00044## wherein: the dotted line represents an optional double bond, as permitted by valency; the bond overlaid with the wavy line represents a connection to L; R.sup.X1 and R.sup.X1′ are independently selected from H, OH, ═O, ═CH.sub.2CN, R.sup.mOR.sup.m, ═CH—R.sup.m′, ═C(R.sup.m′).sub.2, O—SO.sub.2—R.sup.m, CO.sub.2R.sup.m, COR.sup.m, halo and dihalo; R.sup.m′ is selected from R.sup.m, CO.sub.2R.sup.m, COR.sup.m, CHO, CO.sub.2H and halo; each R.sup.m is independently selected from of C.sub.1-12 alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.5-20 aryl, C.sub.5-20 heteroaryl, C.sub.3-6 cycloalkyl, 3 to 7 membered heterocyclyl, 3 to 7 membered heterocycloalkyl and 5 to 7 membered heteroaryl; R.sup.X2, R.sup.X2′, R.sup.X3, R.sup.X3′, R.sup.X5 and R.sup.X5′ are each independently selected H, R.sup.m, OH, OR.sup.m, SH, SR.sup.m, NH.sub.2, NHR.sup.m, NR.sup.m.sub.2, NO.sub.2, Me.sub.3Sn and halo; R.sup.X4 and R.sup.X4′ are each independently selected from H, R.sup.m, OH, OR.sup.m, SH, SR.sup.m, NH.sub.2, NHR.sup.m, NR.sup.m.sub.2, NO.sub.2, Me.sub.3Sn, halo, C.sub.1-6 alkyl, C.sub.1-6 alkenyl, C.sub.2-6 alkynyl, C.sub.2-6 aryl, C.sub.3-6 heteroaryl, C.sub.3-6 cycloalkyl, 3 to 7 membered heterocycloalkyl, C.sub.5-12 aryl, 5 to 7 membered heteroaryl, —CN, —NCO, —OR.sup.n, —OC(O)R.sup.n, —OC(O)NR.sup.nR.sup.n′, —OS(O)R.sup.n, —OS(O).sub.2R.sup.n, —SR.sup.n, —S(O)R.sup.n, —S(O).sub.2R.sup.n, —S(O)NR.sup.nR.sup.n′, —S(O).sub.2NR.sup.nR.sup.n′, —OS(O)NR.sup.nR.sup.n′, —OS(O).sub.2NR.sup.nR.sup.n′, —NR.sup.nR.sup.n′, —NR.sup.nC(O)R.sup.o, —NR.sup.nC(O)OR.sup.o, —NR.sup.nC(O)NR.sup.oR.sup.o′, —NR.sup.nS(O)R.sup.o, —NR.sup.nS(O).sub.2R.sup.o, —NR.sup.nS(O)NR.sup.oR.sup.o′, —NR.sup.nS(O).sub.2NR.sup.oR.sup.o′, —C(O)R.sup.n, —C(O)OR.sup.n and —C(O)NR.sup.nR.sup.n′; R.sup.X and R.sup.X′ are each independently selected from H, OH, N.sub.3, CN, NO.sub.2, SH, NH.sub.2, ONH.sub.2, NHNH.sub.2, halo, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkylthio, C.sub.3-20 heteroaryl, C.sub.5-20 aryl and mono- or di-Ci.sub.1-8 alkylamino; Y and Y′ are each independently selected from O, S and N(H); R.sup.x6 is C.sub.3-12 alkylene, C.sub.3-12 alkenylene, or C.sub.3-12 heteroalkylene; R.sup.X7 and R.sup.X7′ are each independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, 3 to 7 membered heterocycloalkyl, C.sub.6-10 aryl, 5 to 7-membered heteroaryl, —OR.sup.r, —OC(O)R.sup.r, —OC(O)NR.sup.rR.sup.r′, —OS(O)R.sup.r, —OS(O).sub.2R.sup.r, —SR.sup.r, —S(O)R.sup.r, —S(O).sub.2R.sup.r, —S(O)NR.sup.rR.sup.r′, —S(O).sub.2NR.sup.rR.sup.r′, —OS(O)NR.sup.rR.sup.r′, —OS(O).sub.2NR.sup.rR.sup.r′, —NR.sup.4R.sup.r′, —NR.sup.rC(O)R.sup.s, —NR.sup.rC(O)OR.sup.s, —NR.sup.rC(O)NR.sup.sR.sup.s′, —NR.sup.rS(O)R.sup.s, —NR.sup.rS(O).sub.2R.sup.s, —NR.sup.rS(O)NR.sup.sR.sup.s′, —NRr.sup.s(O).sub.2NR.sup.sR.sup.s, —C(O)R.sup.r, —C(O)OR.sup.s and —C(O)NR.sup.rR.sup.r′; each R.sup.r, R.sup.r′, R.sup.s and R.sup.s′ is independently selected from H, C.sub.1-7 alkyl, C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.3-13 cycloalkyl, 3 to 7 membered heterocycloalkyl, C.sub.5-10 aryl and 5 to 7 membered heteroaryl; each R.sup.X8 and R.sup.X8′ is independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 heteroalkyl, 3 to 7 membered heterocycloalkyl, C.sub.5-10 aryl, 5 to 7 membered heteroaryl, —S(O)R.sup.m, —S(O).sub.2R.sup.m, —S(O)NR.sup.mR.sup.m′, —S(O).sub.2NR.sup.mR.sup.m′, —NR.sup.mR.sup.m′, —NR.sup.mC(O)R.sup.m, —NR.sup.mC(O)OR.sup.n, —NR.sup.mC(O)NR.sup.nR.sup.n′, —NR.sup.mS(O)R.sup.n, —NR.sup.mS(O).sub.2R.sup.n, —NR.sup.mS(O)NR.sup.nR.sup.n′, —NR.sup.mS(O).sub.2NR.sup.nR.sup.n′, —C(O)R.sup.m, —C(O)OR.sup.m and —C(O)NR.sup.mR.sup.m′; Z.sup.a is OR.sup.X12a, NR.sup.X12aR.sup.X12a, or SR.sup.X12a; Z.sup.b is OR.sup.X13a, NR.sup.X13aR.sup.X13a, or SR.sup.X13a; Z.sup.a′ is OR.sup.X12a, NR.sup.X12aR.sup.X12a, or SR.sup.X12a; Z.sup.b′ is OR.sup.X13a′, NR.sup.X13a′R.sup.X13a′, or SR.sup.X13a′; each R.sup.X12a, R.sup.X12a′, R.sup.X13a′ and R.sup.x13a′ independently selected from H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, 3 to 7 membered heterocycloalkyl, C.sub.5-10 aryl, 5 to 7 membered heteroaryl, —C(O)R.sup.X14a+, —C(O)OR.sup.X15a and —C(O)NR.sup.X15aR.sup.X15a′; and each R.sup.X15a and R.sup.x15a′ is independently selected from C.sub.1-12 alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.5-20 aryl, C.sub.5-20 heteroaryl, C.sub.3-6 cycloalkyl, 3 to 7 membered heterocyclyl, 3 to 7 membered heterocycloalkyl, and 5 to 7 membered heteroaryl; and ##STR00045## X.sup.a and X.sup.a′ are independently selected from a bond or C.sub.1-6 alkylene; Z.sup.X′ and Z.sup.X are independently selected from among hydrogen, C.sub.1-8 alkyl, halogen, cyano, nitro, ##STR00046## or —(CH.sub.2).sub.m—OCH.sub.3; the respective R.sup.80, R.sup.90 and R.sup.100 are selected from hydrogen, C.sub.1-8 alkyl, C.sub.2-6 alkenyl and C.sub.1-6 alkoxy; and m is an integer of 0 to 12.

52. The antibody conjugate of claim 51, wherein Z.sup.Z′ and Z.sup.X are —(CH.sub.2).sub.m—OCH.sub.3. OCH3.

53. The antibody conjugate of claim 1, wherein the active agent is: R.sup.X12a and R.sup.X13a join the atoms to which they are attached to form a 3 to 7 membered heterocyclyl, 3 to 7 membered heterocycloalkyl, or 3 to 7 membered heteroaryl, and the R.sup.X12a′ and R.sup.X13a′ join the atoms to which they are attached to form 3 to 7 membered heterocyclyl, 3 to 7 membered heterocycloalkyl, or 3 to 7 membered heteroaryl; and each of the R.sup.n, R.sup.n′, R.sup.o, R.sup.o′, R.sup.p and R.sup.p′ is independently selected from H, C.sub.1-7 alkyl, C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.3-13 cycloalkyl, 3 to 7 membered heterocycloalkyl, C.sub.5-10 aryl, and 5 to 7 membered heteroaryl.

51. The antibody conjugate of claim 50, wherein the pyrrolobenzodiazepine dimer has a structure represented by ##STR00047## ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052## and the bond overlaid with a dashed line represents a connection point to L.

54-56. (canceled)

57. A pharmaceutical composition comprising the antibody conjugate of claim 1 and a pharmaceutically acceptable excipient.

58-60. (canceled)

61. A method of treating a proliferative disease, angiogenic disease, or cancer in a subject comprising administering to the subject an antibody conjugate of claim 1 or a pharmaceutically acceptable salt thereof.

62. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0282] FIG. 1 is a diagram confirming the efficacy of the ADC according to the present invention in a patient-derived tumor animal model.

BEST MODE(S) FOR CARRYING OUT THE INVENTION

[0283] In the following, the present invention is described in further detail through embodiments and experimental examples.

[0284] The following embodiments and experimental examples are intended to aid in understanding the present invention, and are not intended to limit the scope of the present invention thereto.

<Example 1> Preparation of Compounds 1, 2, 3 and 4

[0285] ##STR00031## ##STR00032##

[0286] Compounds 1, 2, 3 and 4 were prepared using the method stated in International Patent Publication WO2017-089895.

[0287] In compounds 1, 2, 3 and 4, the structure of MMAE is as follows.

##STR00033##

<Example 2> Preparation of Compounds 5, 6, 7 and 8

[0288] ##STR00034##

[0289] Compounds 5, 6 and 7 were prepared using the method stated in Korean Laid-open Patent No. 10-2018-0110645.

##STR00035##

[0290] Compound 8 was prepared using the method stated in Korean Patent Application No. 10-2019-0000514.

<Example 3> Preparation of ADC

[0291] ADC was prepared through the following two steps, and the LCB14-0511 and LCB14-0606 used commonly were prepared using the method stated in Korean Laid-open Patent No. 10-2014-0035393. The structural formulae of LCB14-0511 and LCB14-0606 are as follows:

##STR00036##

Step 1: Preparation of Prenylated Antibody

[0292] A prenylation reaction mixture of an antibody (18A5) was prepared using the method stated in Korean Patent Application No. 10-2018-0107639 and reacted for 16 hours at 30° C. The reaction mixture was comprised of 24 μM antibody (18A5), 200 nM FTase (Calbiochem #344145) and a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl.sub.2, 10 μM ZnCl.sub.2, 0.144 mM DTT) containing 0.144 mM LCB14-0511 or LCB14-0606. After the reaction was completed, the prenylated antibody was desalted using a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer solution.

Step 2: Method of Drug-Conjugation

Conjugation by Oxime Bond Formation

[0293] The mixture for oxime bond-forming reaction between prenylated antibody and linker-drug was prepared by mixing 100mM Na-acetate buffer solution pH 5.2, 10% DMSO, 20 μM antibody and 200 μM linker-drug (in house, compounds 1, 2, 3, 4, 5, 7 and 8 from Embodiments 1 and 2), and stirred lightly at 30° C. After reacting for 6 or 24 hours, an FLPC (AKTA purifier, GE healthcare) process was carried out to remove the surplus small compounds used. The protein fraction was collected and concentrated.

Conjugation by Click Reaction

[0294] The mixture for oxime bond-forming reaction between prenylated antibody and linker-drug was 10% DMSO, 20 μM antibody and 200 μM linker-drug (in house, compound 6 from Embodiment 2), 1 mM copper (II) sulfate pentahydrate, 2 mM (BimC.sub.4A).sub.3 (Sigma-Aldrich 696854), 10 mM sodium ascorbate and 10 mM aminoguanidine hydrochloride, reacted for 3 hours at 25° C., then treated with 2.0 mM EDTA and reacted for 30 minutes. After reacting, an FLPC (AKTA purifier, GE healthcare) process was carried out to remove the surplus small compounds used. The protein fraction was collected and concentrated.

TABLE-US-00002 TABLE 2 List of ADCs prepared ADCs Antibody Linker-toxin ADC1 18A5 Compound 1 ADC2 Compound 2 ADC3 Compound 3 ADC4 Compound 4 ADC5 Compound 5 ADC6 Compound 6 ADC7 Compound 7 ADC8 Compound 8

Experimental Example 1: In Vitro Cytotoxicity Assessment

[0295] The cancer cell line cell proliferation inhibition activity of the drugs stated in Table 3 below and the ADCs prepared in <Embodiment 3> above was measured. For this purpose, commercially available human pancreatic cancer cell line MIA-PaCa2 (DLK1 negative or normal) and MIA-PaCa-2-DLK1 over-expressing cell line were used. As the drug, MMAF-OMe was used as the ADC, and the ADCs of Table 2 were used. In a 96-well plate, each well was seeded with 4,000 to 5,000 of the respective cancer cell lines for the 72-hour treatment group, 2,000 to 3,000 cells for the 96-hour treatment group, and 800 to 1,000 cells for the 168-hour treatment group. After culturing for 24 hours, they were treated with the drug and ADC at a concentration of 0.0015 to 10.0 nM (serially diluted threefold). 72, 96 and 168 hours later, respectively, the number of live cells was measured using SRB (Sulforhodamine B) dye.

TABLE-US-00003 TABLE 3 IC50 (nM) MIA-PaCa2-DLK1 MIA-PaCa2-DLK1 negative positive Test samples 72 hr 96 h 168 hr 72 hr 96 h 168 hr MMAF-OMe 0.41 0.12 0.12 0.30 0.13 0.06 ADC1 >10 N.T >10 0.54 N.T 0.09 ADC2 >10 >10 >10 0.57 0.26 0.09 ADC3 >10 >10 >10 0.19 0.11 0.03 ADC4 N.T >10 N.T N.T 0.05 N.T ADC5 >10 N.T >10 0.22 0.01 0.003 ADC6 N.T N.T >10 N.T N.T 0.21 ADC7 >10 N.T >10 0.008 N.T 0.004 ADC8 N.T >10 N.T N.T 0.008 N.T *NT: not tested.

[0296] Most of the antibody-drug complexes were confirmed to have substantially improved cytotoxicity in pancreatic cancer cell lines with DLK1 over-expression than in pancreatic cancer cell lines without DLK1 expression. In pancreatic cancer cell lines over-expressing DLK1, pyrrolobenzodiazepine based antibody-drug complexes (ADCs 5, 7 and 8) were confirmed to exhibit stronger cytotoxicity than auristatin-based antibody-drug complexes (ADCs 1, 2, 3 and 4). Further, pyrrolobenzodiazepine based antibody-drug complexes was confirmed to exhibit stronger cytotoxicity compared to auristatin-based antibody-drug complexes with longer reaction time.

Experimental Example 2: In Vivo Efficacy Assessment

[0297] In a patient-derived tumor animal model, the efficacy of the ADCs prepared in <Embodiment 3> was assessed. Specifically, the patient-derived small cell lung cancer mouse model (PDX model) was provided by Champions Oncology. The control group of the PDX model was intravenously administered PBS in the tail, while the experimental groups were administered ADC4 at 6mpk/Q4D*4, 6 mpk/QW*4 and 9 mpk/QW*4, followed by measurement of tumor size.

[0298] In the results, as shown in FIG. 1, tumors disappeared from all 8 individual experimental animals in each of the 6 mpk/Q4D*4 group and the 9 mpk/QW*4 group (CR, complete remission), while tumors disappeared in 6 of the 8 experimental animals in the 6 mpk/QW*4 group.

INDUSTRIAL APPLICABILITY

[0299] The new antibody-drug conjugates (ADCs) targeting DLK1 and pharmaceutical compositions comprising the same may be used in production of drugs for treatment and/or prevention of diseases proliferative and/or angiogenetic diseases, for example, cancer.