ANTIBODY-DRUG CONJUGATE INCLUDING ANTIBODY AGAINST HUMAN CLDN18.2, AND USE THEREOF

Abstract

The present disclosure relates to a novel antibody-drug conjugate (ADC) targeting claudin 18 isoform 2 (CLDN18.2), an active metabolite of the ADC, a method of producing the ADC, use of the ADC for the treatment and/or prevention of diseases, and use of the ADC for producing a drug for the treatment and/or prevention of diseases, more specifically hyperproliferative and/or angiogenic diseases, for example, cancer, and more particularly, to an antibody-drug conjugate including a novel antibody or antigen-binding fragment thereof that binds to CLDN18.2, and a pharmaceutical composition including the same.

Claims

1. A conjugate represented by General Formula I or a pharmaceutically acceptable salt or solvate thereof:
Ab-[LINKER-(B).sub.I].sub.m[General Formula I] wherein, Ab is an anti-Claudin 18 isoform 2 (CLDN18.2) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein, the heavy chain variable region comprises a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 2, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 4, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6, and the light chain variable region comprises a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 9, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 13, LINKER is a linker, B is an active agent, and I and m are each independently an integer selected from 1 to 20, wherein, when I is an integer of 2 or more, at least two of B are identical to or different from each other.

2. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the heavy chain variable region comprises a heavy chain FR of a human antibody, and a gene encoding the heavy chain FR is derived from germline V gene IGHV3-23.

3. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 2, wherein the heavy chain FR comprises a heavy chain FR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a heavy chain FR2 consisting of the amino acid sequence represented by SEQ ID NO: 3, a heavy chain FR3 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain FR4 consisting of the amino acid sequence represented by SEQ ID NO: 7.

4. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the light chain variable region comprises a light chain FR of a human antibody, and a gene encoding the light chain FR is derived from germline V gene IGKV3-11 or IGKV3-15.

5. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 4, wherein the light chain FR comprises: a light chain FR1 consisting of the amino acid sequence represented by SEQ ID NO: 8; a light chain FR2 consisting of the amino acid sequence represented by SEQ ID NO: 10; a light chain FR3 consisting of the amino acid sequence represented by SEQ ID NO: 12; and a light chain FR4 consisting of the amino acid sequence represented by SEQ ID NO: 14, or a light chain FR4 consisting of the amino acid sequence represented by SEQ ID NO: 20.

6. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to CLDN18.2 comprises: a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 15 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15; and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 16 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 16.

7. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is any one selected from a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab fragment, a F(ab)2 fragment, an scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a bispecific antibody, and a multispecific antibody.

8. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the linker between the antibody and an active agent is cleavable.

9. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein the conjugate has the structure of General Formula IIa: ##STR00059## wherein, Ab is an anti-Claudin 18 isoform 2 (CLDN18.2) antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; each B is independently an active agent, wherein the active agents are identical to or different from each other; ##STR00060## G and G are each independently a glucuronic acid moiety or R.sup.3 is hydrogen or a carboxyl-protecting group, and each R.sup.4 is independently hydrogen or a hydroxyl-protecting group; R.sup.1 and R.sup.2 are each independently hydrogen, C.sub.1-8 alkyl, or C.sub.3-8 cycloalkyl; each W is independently C(O), C(O)NR, C(O)O, SO.sub.2NR, P(O)RNR, SONR, or PO.sub.2NR, wherein C, S, or P is directly bonded to a phenyl ring, NR is bonded to L, and R and R are each independently hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkylthio, mono- or di-C.sub.1-8 alkylamino, C.sub.3-20 heteroaryl, or C.sub.6-20 aryl; each Z is independently hydrogen, C.sub.1-8 alkyl, halogen, cyano, or nitro; n is an integer from 0 to 3 wherein, when n is an integer of 2 or more, at least two of Z are identical to or different from each other; each L is independently any one selected from A) or B) below: A) C.sub.1-50 alkylene or 1-50 atom heteroalkylene, satisfying at least one of the following: (i) L comprises one or more unsaturated bonds; (ii) two atoms in L are substituted with a bivalent substituent, which completes a heteroarylene; (iii) L is a 1-50 atom heteroalkylene; or (iv) the alkylene is further substituted with at least one C.sub.1-20 alkyl; or B) at least one isoprenyl derivative unit of General Formula III which is recognizable by an isoprenoid transferase: ##STR00061## and m is an integer selected from 1 to 20.

10. A conjugate represented by General Formula IIa or a pharmaceutically acceptable salt or solvate thereof: ##STR00062## wherein, Ab is an antibody or antigen-binding fragment thereof that specifically binds to claudin 18 isoform 2 (CLDN18.2); each G is independently a glucuronic acid moiety or ##STR00063## R.sup.3 is hydrogen or a carboxyl-protecting group, and each R.sup.4 is independently hydrogen or a hydroxyl-protecting group; R.sup.1 and R.sup.2 are each independently hydrogen, C.sub.1-8 alkyl, or C.sub.3-8 cycloalkyl; each W is independently C(O), C(O)NR, C(O)O, SO.sub.2NR, P(O)RNR, SONR, or PO.sub.2NR, wherein C, S, or P is directly bonded to a phenyl ring, NR is bonded to L, and R and R are each independently hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkylthio, mono- or di-C.sub.1-8 alkylamino, C.sub.3-20 heteroaryl, or C.sub.6-20 aryl; each Z is independently hydrogen, C.sub.1-8 alkyl, halogen, cyano, or nitro; n is an integer from 0 to 3; and each L is independently any one selected from A) or B) below: A) C.sub.1-50 alkylene or 1-50 atom heteroalkylene, satisfying at least one of the following: (i) L comprises one or more unsaturated bonds; (ii) two atoms in L are substituted with a bivalent substituent, which completes a heteroarylene; (iii) L is a 1-50 atom heteroalkylene; or (iv) the alkylene is substituted with at least one C.sub.1-20 alkyl; or B) at least one isoprenyl derivative unit of General Formula III which is recognizable by an isoprenoid transferase: ##STR00064## B is an active agent; Ab is bonded to a site indicated as a wave symbol; and I and m are each independently an integer selected from 1 to 20, wherein, when I is 2 or more, the active agents are identical to or different from each other.

11. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein each G is independently ##STR00065## R.sup.3 is hydrogen or a carboxyl-protecting group, and each R.sup.4 is independently hydrogen or a hydroxyl-protecting group.

12. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein: R.sup.1 and R.sup.2 are each hydrogen; n is 0; and each W is independently C(O)NR, C is directly bonded to a phenyl ring, and R is hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkylthio, mono- or di-C.sub.1-8 alkylamino, C.sub.3-20 heteroaryl, or C.sub.6-20 aryl, wherein NR is bonded to L.

13. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L is a 1-5 atom heteroalkylene comprising nitrogen, L comprises two or more atoms of a hydrophilic amino acid, and the nitrogen forms a peptide bond with a carbonyl of the hydrophilic amino acid.

14. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L is covalently bonded to the antibody by a thioether bond, wherein the thioether bond comprises a sulfur atom of a cysteine of the antibody.

15. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 14, wherein the antibody comprises an amino acid motif recognizable by an isoprenoid transferase at the C-terminus of the antibody, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

16. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 15, wherein: the amino acid motif has a CYYX sequence, wherein C is cysteine, Y is an aliphatic amino acid, and X is any one selected from glutamine, glutamate, serine, cysteine, methionine, alanine, and leucine; and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

17. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 15, wherein the amino acid motif has a CYYX sequence, wherein Y is any one selected from alanine, isoleucine, leucine, methionine, and valine; or a CVIM or CVLL sequence.

18. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 15, wherein at least one of 1 to 20 amino acids preceding the amino acid motif is glycine.

19. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L comprises the amino acid sequence of GGGGGGGCVIM at the C-terminus.

20. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L is 3 to 50 heteroalkylene containing an oxime, the oxygen atom of the oxime is on the side of L linked to W, the carbon atom of the oxime is on the side of L linked to Ab, or the carbon atom of the oxime is on the side of L linked to W, and the oxygen atom of the oxime is on the side of L linked to Ab.

21. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim, wherein L comprises an oxime, and at least one isoprenyl unit covalently bonds the oxime to Ab.

22. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L further comprises a second 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] 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, alkylene, or NR.sup.21; 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 an integer from 0 to 10; p is an integer from 0 to 10; q is an integer from Ito 20; and w is an integer from 1 to 20.

23. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 22, wherein: q is an integer from Ito 10; r and p are each 1 or 2; and V is O.

24. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 22, wherein: X is O; and w is an integer from 1 to 10.

25. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 21, wherein L comprises at least one polyethylene glycol unit represented by ##STR00066##

26. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 21, wherein L comprises an oxime, and at least one polyethylene glycol unit covalently bonds the oxime to an active agent.

27. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L further comprises a third unit formed by a reaction between an alkyne and an azide or between an aldehyde or ketone group and hydrazine or hydroxylamine.

28. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L further comprises L or a third unit represented by General Formula IVa, IVb, IVc, IVd or IVe below: ##STR00067## wherein, L.sub.1 and L.sub.2 are each independently a single bond or C.sub.1-30 alkylene; and R.sub.11 is hydrogen or C.sub.1-10 alkyl.

29. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 28, wherein L.sub.1 and L.sub.2 are each independently a single bond, C.sub.11 alkylene, or C.sub.12 alkylene.

30. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

31. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein L comprises one or more branched linkers covalently bonded to Ab, wherein: i) each branched linker comprises a fifth unit covalently bonded to Ab by a primary linker (PL); ii) each branched linker comprises a first branch (B1) in which a first active agent is covalently bonded to the fifth unit by a secondary linker (SL) and a cleavage group (CG); and iii) each branched linker comprises: a) a second branch (B2) in which a second active agent is covalently bonded to the fifth unit by a secondary linker (SL) and the cleavage group (CG); or b) a second branch (B2) in which a polyethylene glycol moiety is covalently bonded to the fifth unit, and each cleavage group is hydrolyzed to release an active agent from the conjugate.

32. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 31, wherein the fifth unit is ##STR00068## wherein L.sup.2, L.sup.3, and L.sup.4 are each independently a bond or wherein n is an integer from 1 to 30; G.sup.1, G.sup.2, and G.sup.3 are each independently a bond, ##STR00069## R.sup.30 is hydrogen or C.sub.1-30alkyl; R.sup.40 is hydrogen or L.sup.5-COOR.sub.6; and L.sup.5 is a bond or C.sub.n.Math.H.sub.2n.Math., wherein n is an integer from 1 to 10; and R.sub.6 is hydrogen or C.sub.1-30alkyl.

33. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 31, wherein the cleavage group is cleavable in a target cell, or is capable of releasing one or more active agents.

34. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 33, wherein: the conjugate comprises Ab, and 1, 2, 3, 4 or more branched linkers covalently bonded to Ab; and each branched linker is bonded to one or more active agents, wherein the active agents are identical to or different from each other.

35. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 32, wherein each branched linker comprises a fifth unit, each active agent is bonded to the fifth unit via a secondary linker, and the fifth unit is bonded to the antibody via a primary linker.

36. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 35, wherein: the fifth unit is an amide, and the primary linker comprises: a carbonyl of the amide; or a lysine unit.

37. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 33, wherein the conjugate or the pharmaceutically acceptable salt or solvate thereof comprises Structural Formula below: ##STR00070## wherein B and B are each active agents which are identical to or different from each other; n1 to n3 each independently are an integer from 0 to 30; and AA is an amino acid group.

38. A conjugate represented by Structural Formula below and comprising a [LINKER-B] structure, or a pharmaceutically acceptable salt or solvate thereof: ##STR00071## wherein B and B are active agents which are identical to or different from each other; and m and n each independently are an integer from 0 to 30.

39. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein the active agent is a chemotherapeutic agent or a toxin.

40. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 9, wherein the active agent is an immunomodulatory compound, an anticancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof.

41. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim, wherein the active agent is any one selected from the following: (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, triethiylenethiophosphoramide, trimethylolomelamine, 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, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, calicheamicin, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, esperamicin, neocarzinostatin chromophore, aclacinomysins, actinomycin, antrmycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thiguanine, ancitabine, azacitidine, 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, edatraxate, 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, triaziquone, 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 a pharmaceutically acceptable salt, solvate or acid thereof; (b) monokines, lymphokines, traditional polypeptide hormones, parathyroid hormones, 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-?, tumor necrosis factor-?, mullerian inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, thrombopoietin, erythropoietin, osteoinductive factor, interferon, interferon-?, interferon-?, interferon-?, colony stimulating factor (CSF), macrophageCSF, granulocyte-macrophageCSF, granulocyteCSF, interleukin (IL), IL-1, IL-1?, 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, polypeptide factor, LIF, kit ligand, or a combination thereof; (c) diphtheria toxin, botulinum toxin, tetanus toxin, decentretoxin, cholera toxin, amanitin, ?-amanitin, pyrrolobenzodiazepines, pyrrolobenzodiazepine derivatives, indolinobenzodiazepines, pyridinobenzodiazepines, tetrodotoxin, brevetoxin, ciguatoxin, ricin, AM toxin, auristatin, tubulysin, geldanamycin, maytansinoid, calicheamycin, daunomycin, doxorubicin, methotrexate, vindesine, SG2285, dolastatin, dolastatin analog, cryptophycin, camptothecin, rhizoxin, rhizoxin derivatives, CC-1065, CC-1065, analogs or derivatives, duocarmycin, enediyne antibiotics, esperamicin, epothilone, toxoid, or a combination thereof; (d) an affinity ligand, wherein the affinity ligand is a substrate, an inhibitor, an active agent, a neurotransmitter, a radioisotope, or a combination thereof; (e) a radioactive label, 32P, 35S, a fluorescent dye, an electron dense reagent, an enzyme, biotin, streptavidin, dioxigenin, hapten, an immunogenic protein, a nucleic acid molecule with a sequence complementary to a target, or a combination thereof; (f) an immunomodulatory compound, an anticancer agent, an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, an anti-parasitic agent, or a combination thereof; (g) tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, or toremifene; (h) 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, letrozole, or anastrozole; (i) flutamide, nilutamide, bicalutamide, leuprolide, goserelin, or troxacitabine; (j) an aromatase inhibitor; (k) a protein kinase inhibitor; (l) a lipid kinase inhibitor; (m) an antisense oligonucleotide; (n) a ribozyme; (o) a vaccine; and (p) an anti-angiogenic agent.

42. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein: the active agent is a pyrrolobenzodiazepine dimer; position N10 of the pyrrolobenzodiazepine dimer is substituted with X or position N10 is substituted with X wherein X or X links the pyrrolobenzodiazepine dimer to the linker; X and X are each independently C(O)O* or C(O)*; and * refers to a binding site between the pyrrolobenzodiazepine dimer and the linker.

43. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 42, wherein the pyrrolobenzodiazepine dimer is represented by General Formula X or General Formula XI below: ##STR00072## wherein, a dotted line denotes an optional double bond between C1 and C2, between C2 and C3, between C1 and C2, or between C2 and C3; R.sup.X1 and R.sup.X1 are each independently selected from H, OH, ?O, ?CH.sub.2, CN, R.sup.m, OR.sup.m, ?CHR.sup.m, ?C(R.sup.m).sub.2, OSO.sub.2R.sup.m, CO.sub.2R.sup.m, COR.sup.m, halo, or dihalo; R.sup.X2, R.sup.X2, R.sup.X3, and R.sup.X3 are each independently selected from H, R.sup.m, OH, OR.sup.m, NR.sup.m.sub.2, NO.sub.2, 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, halo, and C.sub.1-6alkyl; R.sup.X5 and R.sup.X5 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, NR.sup.mR.sup.m, NO.sub.2, NR.sup.mC(O)R.sup.m, NR.sup.mC(O)OR.sup.m, NR.sup.mC(O)NR.sup.mR.sup.m, 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.mS(O)R.sup.m, NR.sup.mS(O).sub.2R.sup.m, P(O)R.sup.m, P(O).sub.2R.sup.m, P(O)NR.sup.mR.sup.m, P(O).sub.2NR.sup.mR.sup.m, NR.sup.mP(O)R.sup.m, NR.sup.mP(O).sub.2R.sup.m, and halo; Y and Y are each independently O, S, or N(H); R.sup.X6 is independently C.sub.3-12 alkylene, C.sub.3-12 alkenylene, or C.sub.3-12 heteroalkylene; R.sup.X6 is unsubstituted or substituted with NH.sub.2, NHR.sup.m, NHC(O)R.sup.m, NHC(O)CH.sub.2[OCH.sub.2CH.sub.2].sub.nR.sup.XX, or [CH.sub.2CH.sub.2O].sub.nR.sup.XX; R.sup.XX is 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.3-8cycloalkyl, C.sub.1-8 alkoxy, C.sub.1-8 alkylthio, C.sub.3-20 heteroaryl, C.sub.5-20 aryl, or mono- or di-C.sub.1-8 alkylamino, wherein n is an integer from 1 to 6; R.sup.X7 and R.sup.X7 are each independently H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C(O)R.sup.r, C(O)OR.sup.s, or C(O)NR.sup.rR.sup.r; R.sup.r, R.sup.r, and R.sup.s are each independently 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, or 5- to 7-membered heteroaryl; each R.sup.m is independently selected from R.sup.m, CO.sub.2R.sup.m, COR.sup.m, CHO, CO.sub.2H, and halo; and each R.sup.m is independently selected from the group consisting of H, OH, C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.2-12alkenyl, C.sub.2-12alkynyl, 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, wherein at least one hydrogen atom of C.sub.5-20aryl, 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 is substituted with OH, ?O, C.sub.1-12 alkyl, or C.sub.1-12 alkoxy.

44. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 43, wherein R.sup.X1 and R.sup.X1 are each independently selected from: ?CH.sub.2; C.sub.1-6 alkyl; C.sub.1-6 alkoxy; C.sub.2-6 alkenyl; C.sub.5-7 aryl; C.sub.3-6 heteroaryl; or C.sub.5-7 aryl substituted with C.sub.1-6 alkyl or C.sub.1-6 alkoxy.

45. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 43, wherein R.sup.X2, R.sup.X2, R.sup.X3, and R.sup.X3 are each independently selected from H or OH.

46. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 43, wherein R.sup.X5 and R.sup.X5 are each independently selected from the group consisting of H, OH, S(O)R.sup.m, S(O).sub.2R.sup.m, P(O)R.sup.m, and P(O).sub.2R.sup.m, wherein R.sup.m is H, OH, C.sub.1-12 alkyl, C.sub.1-12 alkoxy, C.sub.2-12 alkenyl, or C.sub.2-12 alkynyl.

47. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 43, wherein R.sup.X4 and R.sup.X4 are each independently C.sub.1-6alkoxy.

48. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 43, wherein Y and Y are O.

49. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 43, wherein: R.sup.X6 is C.sub.3-12 alkylene, C.sub.3-12 alkenylene, or C.sub.3-12 heteroalkylene, and R.sup.X6 is substituted with NH.sub.2, NHR.sup.m, NHC(O)R.sup.m, NHC(O)CH.sub.2[OCH.sub.2CH.sub.2].sub.nR.sup.XX, or [CH.sub.2CH.sub.2O].sub.nR.sup.XX; R.sup.XX is 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, or mono- or di-C.sub.1-8 alkylamino; and n is an integer from 1 to 6.

50. The conjugate or the pharmaceutically acceptable salt or solvate thereof of claim 1, wherein [Linker-(B).sub.I] is selected from the group consisting of: ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080## wherein MMAE is monomethyl auristatin E, and MMAF is monomethyl auristatin F.

51. A pharmaceutical composition for the prevention or treatment of hyperproliferation, cancer, or an angiogenic disease, the pharmaceutical composition comprising the conjugate of claim 1.

52. The pharmaceutical composition of claim 51, further comprising a pharmaceutically effective amount of a chemotherapeutic agent.

53. The pharmaceutical composition of claim 51, wherein the cancer is any one selected from lung cancer, small cell lung cancer, gastrointestinal cancer, colon cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, and melanoma.

54. A pharmaceutical preparation comprising the conjugate of claim 1, and a pharmaceutically acceptable carrier, the pharmaceutical preparation being selected from injections, tablets, pills, powders, granules, capsules, troches, suspending agents, liquids for internal use, emulsions, syrups, freeze-dried preparations, and suppositories.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0426] FIG. 1 illustrates the results of a cell binding experiment using a flow cytometer, performed to confirm the ability of an antibody to bind to cancer cells.

[0427] FIGS. 2 and 3 are graphs showing the in-vivo experimental results of ADC samples.

[0428] FIGS. 4 to 7 are schematic views illustrating a process of producing ADCs according to the present disclosure.

MODE OF DISCLOSURE

[0429] Hereinafter, the present disclosure will be described in more detail with reference to the following examples and experimental examples.

[0430] These examples and experimental examples are intended to aid in understanding of the present disclosure and are not intended to limit the scope of the present disclosure.

<Example 1> Production of Antibody Specific to Claudin 18 Isoform 2 (CLDN18.2)

[0431] An antibody that specifically binds to CLDN18.2 was produced by the method described in International Patent Application No. PCT/CN2020/118650. In addition, the amino acid sequences of the antibody that specifically binds to CLDN18.2 are shown in Table 1 below.

TABLE-US-00001 TABLE1 SEQ ID NO: PR002726-heavychain FR1 EVQLLESGGGLVQPGGSLRLSCAAS 1 CDR1 GFTFSSFVMS 2 FR2 WVRQAPGKGLEWVS 3 CDR2 TISGSGRSTYYADSVKG 4 FR3 RFTISRDNSKNTLHLQMNSLRAEDTAVYYCAK 5 CDR3 DAAAAGTKFDY 6 FR4 WGQGTLVTVSS 7 V.sub.H EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFVMSWVRQAPGKGLEWVSTISG 15 SGRSTYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKDAAAAGTKF DYWGQGTLVTVSS HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFVMSWVRQAPGKGLEWVSTISG 17 SGRSTYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYYCAKDAAAAGTKF DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK PR002726-lightchain FR1 EIVLTQSPATLSLSPGERATLSC 8 CDR1 RASQSVSSYLA 9 FR2 WYQQKPGQAPRLLIY 10 CDR2 DASNRAT 11 FR3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 12 CDR3 QQRSNWPLT 13 FR4 FGGGTKVEIK 14 V.sub.L EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRA 16 TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK LC EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRA 18 TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0432] For ADC synthesis, an antibody clone PR301839 was constructed by fusing a CaaX peptide moiety (GGGGGGGCVIM; SEQ ID NO: 19) to the C-terminus of the light chain (SEQ ID NO: 18) of the antibody (PR002726) shown in Table 1, and an antibody was produced through transient expression based on CHO cells. The produced PR301839 antibody was used for ADC synthesis.

TABLE-US-00002 TABLE2 Clone FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 V.sub.H/V.sub.L PR301 EVQLL GFTFS WVR TISG RFTI DAAA WGQ EVQLLESGGGLVQPGGSLRLSCAA 839-V.sub.H ESGG SFVMS QAP SGR SRD AGTK GTLV SGFTFSSFVMSWVRQAPGKGLEWV GLVQP GKGL STYY NSKN FDY TVSS STISGSGRSTYYADSVKGRFTISRD GGSLR EWV ADSV TLHL NSKNTLHLQMNSLRAEDTAVYYCAK LSCAA S KG QMN DAAAAGTKFDYWGQGTLVTVSS S SLRA EDTA VYYC AK SEQID 1 2 3 4 5 6 7 15 NO: PR301 EIVLTQ RASQS WYQ DASN GIPA QQR FGG EIVLTQSPATLSLSPGERATLSCRAS 839-V.sub.L SPATL VSSYL QKP RAT RFSG SNW GTKV QSVSSYLAWYQQKPGQAPRLLIYDA SLSPG A GQA SGS PLT EIK SNRATGIPARFSGSGSGTDFTLTISS ERATL PRLLI GTDF LEPEDFAVYYCQQRSNWPLTFGGG SC Y TLTIS TKVEIK SLEP EDFA VYYC SEQID 8 9 10 11 12 13 20 21 NO:

<Example 2> Preparation of Compounds 1 and 2

[0433] ##STR00053##

[0434] Compounds 1 and 2 were prepared using the method described in Korean Patent Publication No. 10-2018-0110645.

<Example 3> Preparation of Compound 3

[0435] ##STR00054##

[0436] Compound 3 was prepared using the method described in International Patent Publication No. WO2017-089895.

[0437] The structure of MMAE in compound 3 is as follows:

##STR00055##

<Example 4> Preparation of Compounds 4 and 5

[0438] ##STR00056##

[0439] Compounds 4 and 5 were prepared using the method described in W02017-089890.

[0440] The structure of MMAE in compound 5 is as follows:

##STR00057##

<Example 5> Production of ADCs

[0441] ADCs were produced through the following two steps, and LCB14-0606 and LCB14-0512 used commonly were prepared using the method described in Korean Patent Publication No. 10-2014-0035393. The structural formulae of LCB14-0606 and LCB14-0512 are as follows:

##STR00058##

Step 1: Production of Prenylated Antibodies

[0442] A prenylation reaction mixture of the antibodies produced according to Example 1 was prepared and a reaction was allowed to occur at 30? C. for 16 hours. The reaction mixture consisted of a 24 ?M antibody, 400 nM FTase (Calbiochem #344145), and a buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl.sub.2, 10 ?M ZnCl.sub.2, and 0.5 mM DTT) containing 0.25 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: Drug Conjugation Method

<Conjugation by Oxime Bond Formation>

[0443] The mixture for oxime bond-formation reaction between the prenylated antibody and linker-drug was prepared by mixing 100 mM Na-acetate buffer solution pH 5.2, 10% DMSO, 24 ?M of the prenylated antibody, and 200 ?M linker-drug (in house, compounds 1, 2, 3, 4, 5, 7 and 8 of Examples 1 and 2), and stirred lightly at 30? C. After a reaction was allowed to occur for 6 hours or 24 hours, an FPLC (AKTA purifier, GE healthcare) process was carried out to remove the excess of small-molecule compounds used, and the protein fraction was collected and concentrated.

<Conjugation by Copper-Free Click Reaction>

[0444] For the production of ADCs using the copper-free click coupling reaction between the prenylated antibody and linker-drug, PBS buffer solution (pH 7.4), 1% DMSO, 10 ?M of the prenylated antibody, and 100 ?M linker-drug (in house, compound 2 of Example 1) were mixed to prepare a reaction mixture, and after reaction at 25? C. for 6 hours, the excess of small-molecule compounds used were removed through FPLC (AKTA purifier, GE healthcare) or a G25 Sepharose column process, and the protein fraction was collected and concentrated.

TABLE-US-00003 TABLE 3 List for ADC production ADCs Antibody linker-toxin ADC1 PR301839 Compound 1 ADC2 Compound 2 ADC3 Compound 3 ADC4 Compound 4 ADC5 Compound 5

<Experimental Example 1> Evaluation of Ability of Antibody to Bind to Cancer Cells

[0445] The ability of the anti-CLDN18.2 antibody (PR002726) and the antibody (PR301839), which was used in the production of the ADC in which a CaaX peptide is fused to the C-terminus of the light chain, to bind to cancer cells was examined through a cell binding experiment using a flow cytometer (FACS).

[0446] As cancer cells, gastric cancer cell lines SNU-601 and SNU-620, and a pancreatic cancer cell line PATU-8988s, which are known to express CLDN18.2, were used, and a group treated with human IgG and a group treated with only secondary antibodies were used as controls.

[0447] Through the experimental results, it was confirmed that PR002726 and PR301839 were strongly bound 22.95 times and 17.5 times, respectively, in SNU-601 compared to the IgG control. It was confirmed that PR002726 and PR301839 were strongly bound 15.09 times and 11.21 times in SNU-620 compared to the controls. It was confirmed that PR301839 was strongly bound 16.21 times in PATU-8988s compared to the controls. Taken together, it was confirmed that the two antibodies PR002726 and PR301839 can bind to cancer cells expressing CLDN18.2. (see FIG. 1)

<Experimental Example 2> In Vitro Cytotoxicity Evaluation

[0448] The cell proliferation inhibitory activity of drugs shown in Table 4 and the ADCs was measured. Gastric cancer cell lines NUGC-4, SNU-601, and SNU-602, and a pancreatic cancer cell line PATU-8988s, which are commercially available as cancer cell lines, were used, and normal cell lines HaCaT, Fa2N-4, HK-2, and HS738.st/Int were used. MMAE and SG2057 were used as drugs, and the ADCs shown in Table 3 were used as ADCs. As for test groups treated with each cancer cell line for 144 hours, cells were seeded into a 96-well plate at the rate of 2,500 cells/well to 10,000 cells/well and cultured for 24 hours, and then each cancer cell line was treated with each drug and each ADC at a concentration of 0.256 pM to 100 nM (5-fold serial diltuion), and each normal cell line was treated with each drug and each ADC at a concentration of 2.56 pM to 1,000 nM (5-fold serial dilution). The number of cells survived after 144 hours was quantified using a sulforhodamine B (SRB) dye, and the cell line Hs738.st/Int was quantified using Cell titer glo.

TABLE-US-00004 TABLE 4 IC50 (nM) Test SNU- SNU- PATU- samples NUGC-4 601 620 8988s HaCaT Fa2N-4 HK-2 Hs738.st/Int MMAE 0.29 0.51 0.71 0.145 0.14 3.0 21.9 N/D SG2057 0.03 0.003 0.097 ADC1 17.11 0.028 20.2 ADC2 9.73 0.022 20.55 ADC3 6.72 0.81 18.14 2.073 32.7 365.7 >1,000 >1,000 ADC4 4.122 9.297 420.7 ADC5 2.278 27.26 562.5

[0449] In a cytotoxicity test in which the reaction was allowed to occur for the same time period, it was confirmed that, in the NUGC-4 cell line, an auristatin-based antibody-drug conjugates (ADC3) exhibited equivalent or higher cytotoxicity than pyrrolobenzodiazepine-based antibody-drug conjugates (ADC1 and ADC2). Also, in the cytotoxicity test in which the reaction was allowed to occur for the same time period, it was confirmed that, in the SNU-601 cell line exhibiting strong binding to the antiCLDN18.2 antibody and the strongest cytotoxicity, pyrrolobenzodiazepine-based antibody-drug conjugates (ADC1 and ADC2) exhibited strong cytotoxicity compared to auristatin-based antibody-drug conjugates (ADC3, ADC4, and ADCS). In the SNU-620 cell line to which the antibody was shown to bind strongly, it was confirmed that there was no significant difference in anticancer efficacy between the two types of antibody-drug conjugates. As a result of comparing the cytotoxicity of three types of auristatin-based antibody-drug conjugates in the pancreatic cancer cell line PATU-8988s, it was confirmed that all the three types of ADCs showed strong cytotoxicity.

[0450] In the normal cell lines HaCaT, Fa2N-4, HK-2, and Hs738.st/Int, ADC1 to ADC5 showed no cytotoxicity within the effective dose range, and exhibited remarkably reduced cytotoxicity compared to when a toxin was administered alone or when administered to cancer cell lines, through which it can be seen that the ADC according to the present disclosure exhibits cytotoxicity in a cancer cell-specific manner.

<Experimental Example 3> In Vivo Efficacy Evaluation

[0451] A gastric cancer cell line SNU-601 was cultured, and then 100 ?l of PBS containing 20,000 cells was mixed with 100 ?l of Matrigel, and the mixture was engrafted into Balb/c-nude mice. When the size of a tumor reached 150 mm.sup.3 to 200 mm.sup.3, each of the antibody-drug conjugates was intravenously administered as shown in Tables 5 and 6 below.

TABLE-US-00005 TABLE 5 Comparison in in vivo efficacy between ADC samples (first) Test sample Population Dose (mg/kg) Dose interval PBS 5 qdx1 ADC1 5 0.2 5 0.4 ADC3 5 0.5 5 1 5 2

TABLE-US-00006 TABLE 6 Comparison in in vivo efficacy between ADC samples (second) Test sample Population Dose (mg/kg) Dose interval PBS 5 qdx1 ADC3 5 1 ADC4 5 2 5 4 ADC5 5 2 5 4

[0452] As a result of the first experiment, it was confirmed that the pyrrolobenzodiazepine-based antibody-drug conjugate ADC1 exhibited tumor growth inhibitory efficacy at both 0.2 mg/kg and 0.4 mg/kg compared to the control. It was confirmed that the auristatin-based antibody-drug conjugates ADC3 exhibited tumor growth inhibitory efficacy at all of 0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg in a dose-dependent manner. The experimental results (first) of comparison in in vivo efficacy between ADC samples are the same as shown in FIG. 2. As a result of the second experiment, it was confirmed that the ADC3 1 mg/kg group exhibited tumor growth inhibitory efficacy similar to the first experimental results. It was confirmed that ADC4 and ADC5 exhibited tumor growth inhibitory efficacy at all the doses in a dose-dependent manner. The experimental results (second) of comparison in in vivo efficacy between ADC samples are the same as shown in FIG. 3.

CITED REFERENCES

[0453] Duncan, A. R., J. M. Woof, L. J. Partridge, D. R. Burton, and G. Winter. 1988. Localisation of the binding site for the human high-affinity Fc receptoron IgG. Nature 332:563-564. [0454] Lund, J., G. Winter, P. T. Jones, J. D. Pound, T. Tanaka, M. R. Walker, P. J. Artymiuk, Y. Arata, D. R. Burton, R. Jefferis, and J. M. Woof. 1991. Human Fc?RI and Fc?RII interact with distinct but overlapping sites on human IgG. J. Immunol. 147:2657-2662. [0455] Sarmay, G., J. Lund, Z. Rozsnayay, J. Gergeley, and R. Jefferis. 1992. Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc receptor. Mol. Immunol. 29:633-639.

INDUSTRIAL APPLICABILITY

[0456] The present disclosure can be effectively used in the pharmaceutical field through a novel antibody-drug conjugate targeting claudin 18 isoform 2, an active metabolite thereof, a method of producing same, use thereof, and a pharmaceutical composition including the same.