GLYCOCONJUGATES

Abstract

This disclosure relates to glycoconjugates comprising glycosylated cell-binding agents conjugated to pyrrolobenzodiazepine (PBD) payloads. Glycoconjugates of particular interest include conjugates where the cell-binding agent is an antibody and the payload comprises a cytotoxic pyrrolobenzodiazepine (PBD) moiety, with the PBD moiety conjugated to the antibody via an oligosaccharide linker. The disclosure also relates to methods for preparing the glycoconjugates, along with methods for their use.

Claims

1. A glycoconjugate having the formula: ##STR00216## wherein: CBA is a Cell Binding Agent; GlcNAc is a N-acetyl-glucosamine moiety; Sug is a sugar moiety, wherein b=1 or 0; Gal is a galactose moiety; Sd(A.sup.P).sub.x is a sugar derivative comprising x conjugated payloads A, wherein x=1, 2, 3, or 4; wherein y=1 to 20; and wherein the payload A.sup.P is, comprises, or releases upon metabolism, a PBD compound such as a PBD dimer; optionally wherein GlcNAc, Gal, Sug, and/or Sd(A.sup.P).sub.x are D enantiomers.

2. The glycoconjugate of claim 1, wherein Sd(A.sup.P)x is a sialic acid derivative.

3. The glycoconjugate of claim 2, wherein the sialic acid derivative has the formula: ##STR00217## wherein: QQ is hydrogen or a conjugated payload; ZZ is hydroxyl or a conjugated payload; YY is hydroxyl or a conjugated payload; and/or XX is hydroxyl or a conjugated payload; and wherein at least one of QQ, ZZ, YY, and XX is a conjugated payload.

4. The glycoconjugate of any preceding claim having the the formula: ##STR00218## wherein: QQ is hydrogen or a conjugated payload; ZZ is hydroxyl or a conjugated payload; YY is hydroxyl or a conjugated payload; and/or XX is hydroxyl or a conjugated payload; and wherein at least one of QQ, ZZ, YY, and XX is a conjugated payload

5. The glycoconjugate of any one of claims 1 to 4 having the the formula: ##STR00219## wherein: QQ is hydrogen or a conjugated payload; ZZ is hydroxyl or a conjugated payload; YY is hydroxyl or a conjugated payload; and/or XX is hydroxyl or a conjugated payload; and wherein at least one of QQ, ZZ, YY, and XX is a conjugated payload.

6. The glycoconjugate of any preceding claim, wherein Sug is a fucose moiety.

7. The glycoconjugate of claim 6, wherein the fucose moiety has the structure: ##STR00220##

8. The glycoconjugate of any one of claims 3 to 7, having a conjugated payload at position QQ or ZZ, wherein x=1.

9. The glycoconjugate of any preceding claim having a conjugated payload at each of positions QQ and ZZ, wherein x=2; optionally wherein the payload at each of QQ and ZZ is the same.

10. The glycoconjugate of any preceding claim, wherein y=2.

11. The glycoconjugate of any preceding claim, wherein the CBA is a Fc fusion protein or an antibody.

12. The glycoconjugate of claim 11, wherein the GlcNAc moiety is conjugated to the Fc fusion protein or antibody at the asparagine 297 (Asn297) residue according to the EU index as set forth in Kabat.

13. The glycoconjugate of any preceding claim, wherein the payload is, comprises, or releases upon metabolism a PBD compound selected from the group consisting of: ##STR00221## ##STR00222##

14. The glycoconjugate of any one of claims 1 to 13, wherein the conjugated payload, A.sup.P, is a drug-linker comprising a drug moiety conjugated to the cell-binding agent via a linker moiety, the glycoconjugate having the formula: ##STR00223##

15. The glycoconjugate of claim 14, wherein each linker independently is a linker of formula Z1 or Z2: ##STR00224## wherein r=0 or 1, a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5, G.sup.LL is a linking moiety through which the linker is bound to the Sd moiety, the wavy line indicates where the linker is bound to the drug moiety, and one of X.sup.10, X.sup.11, X.sup.12, X.sup.13 and X.sup.14 may be selected from: ##STR00225## the remainder being a single bond.

16. The glycoconjugate of claim 15, wherein G.sup.LL is selected from: TABLE-US-00009 (G.sup.LL8-1) (G.sup.LL8-2) (G.sup.LL9-1) (G.sup.LL9-2) G.sup.LL10 G.sup.LL11 G.sup.LL12 G.sup.LL13 G.sup.LL14 where CBA indicates where the group is bound to Sd(A.sup.P).sub.x.

17. A method for the preparation of the glycoconjugate of any one of claims 1 to 16, the method comprising the steps of: (i) providing a glycosylated cell-binding agent; (ii) reacting the glycosylated cell-binding agent of (i) with a compound of the formula payload-G.sup.L, wherein the payload is as defined in any one of claims 1 to 16 and G.sup.L is linker group reactive with the functional group of the glycosylated cell-binding agent

18. The method of claim 17, wherein G.sup.L is selected from the group consisting of: TABLE-US-00010 (G.sup.L8) (G.sup.L9) (G.sup.L10) (G.sup.L11) (G.sup.L12) (G.sup.L13)

19. The method of either one of claim 17 or 18, wherein the provided glycosylated cell-binding agent is a glycosylated cell-binding agent having the formula: ##STR00241## wherein: CBA is a Cell Binding Agent; GlcNAc is a N-acetyl-glucosamine moiety; Sug is a sugar moiety, wherein b=1 or 0; Gal is a galactose moiety; Sd(A.sup.F).sub.x is a sugar derivative comprising x functional groups A.sup.F, wherein A.sup.F is independently selected from the group consisting of an azido group, an alkynyl group, and a keto group, and wherein x=1, 2, 3, or 4; and wherein y=1 to 20.

20. The method of claim 19, wherein Sd(A.sup.F)x is a sialic acid derivative having the formula: ##STR00242## wherein: QQ is hydrogen or a functional group A.sup.F; ZZ is hydroxyl or a functional group A.sup.F; YY is hydroxyl or a functional group A.sup.F; and/or XX is hydroxyl or a functional group A.sup.F; and wherein at least one of QQ, ZZ, YY, and XX is a functional group A.sup.F.

21. The method of any one of claims 17 to 20, wherein the glycosylated cell-binding agent is provided by a method comprising the steps of: (i) providing a Sd(A.sup.F).sub.x acceptor having the formula: ##STR00243## wherein CBA, GlcNAc, Sug, b, Gal, and y are defined as in of any one of claims 17 to 20; and (ii) contacting the Sd(A.sup.F).sub.x acceptor with a compound of the formula Sd(A.sup.F).sub.x-P* in the presence of a glycosyltransferase, wherein: Sd(A.sup.F).sub.x is as defined in any one of claims 17 to 20; and P* is a nucleoside phosphate moiety.

22. The method of any one of claims 88 to 92, wherein the nucleoside element of the nucleoside phosphate moiety is selected from the group consisting of: UDP, GDP, TDP, CDP, and CMP.

23. The method of either one of claim 21 or 22, wherein the Sd(A.sup.F).sub.x acceptor is provided by a process comprising the steps of: a) providing a Gal acceptor having the formula: ##STR00244## wherein CBA, GlcNAc, Sug, b, and y are defined as in claim 1; and b) contacting the Gal acceptor with a compound of the formula Gal-P* in the presence of a galactosyltransferase, wherein Gal is a Galactose moiety and P* is a nucleoside phosphate moiety;  optionally wherein, c) the Gal acceptor is produced by contacting with a glycosidase a oligoglycosylated cell-binding agent having the formula: ##STR00245##  wherein CHO is a carbohydrate moiety.

24. The method of any one of claims 21 to 23, wherein the Sd(A.sup.F).sub.x acceptor has only two terminal galactose moieties.

25. The method of any one of claims 21 to 24, wherein the glycosyltransferase is a sialyltransferase.

26. The method of claim 25, wherein the sialyltransferase is a polypeptide having sialyltransferase activity and comprising a sequence having at least 70% sequence identity SEQ ID NO. 1, SEQ ID NO. 4, or SEQ ID NO. 7.

27. The method of any one of claims 21 to 26, wherein the glycosidase is an endoglycosidase.

28. The method of claim 27, wherein the endoglycosidase is a polypeptide having endoglycosidase activity and comprising a sequence having at least 70% sequence identity SEQ ID NO. 3, SEQ ID NO. 6, or SEQ ID NO. 9.

29. The method of any one of claims 21 to 28, wherein the galactosyltransferase is a polypeptide having galactosyltransferase activity and comprising a sequence having at least 70% sequence identity SEQ ID NO. 2, SEQ ID NO. 5, or SEQ ID NO. 8.

30. A glycoconjugate of any one of claims 1 to 16 for use in a method of treatment.

31. A glycoconjugate of any one of claims 1 to 16 for use in a method of treating a proliferative disorder.

32. A method of treating a proliferative disorder, the method comprising administering an effective amount of a glycoconjugate of any one of claims 1 to 16 to a subject.

33. Use of a glycoconjugate of any one of claims 1 to 16 in the manufacture of a medicament for the treatment of a proliferative disorder.

34. The glycoconjugate, method, or use of any one of claims 31 to 33, wherein the proliferative disorder is cancer.

35. The glycoconjugate, method, or use of claim 34, wherein the cancer is selected from the group consisting of: histocytoma, glioma, astrocyoma, neuroblastoma, osteoma, lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, renal cancer, brain cancer, sarcoma, liposarcoma, osteosarcoma, Kaposi's sarcoma, melanoma, lymphomas, myeloma, and leukemias.

36. A method for the preparation of a glycoconjugate, the method comprising the steps of: a) providing an oligoglycosylated cell-binding agent having the formula: ##STR00246## wherein CBA is a Cell Binding Agent; GlcNAc is a N-acetyl-glucosamine moiety; Sug is a sugar moiety, wherein b=1 or 0; y=1 to 20; and CHO is a carbohydrate moiety; b) contacting the oligoglycosylated cell-binding agent with a glycosidase to produce a Gal acceptor having the formula: ##STR00247## c) contacting the Gal acceptor with a compound of the formula Gal-P* in the presence of a galactosyltransferase to produce a Sd(A.sup.F).sub.x acceptor having the formula: ##STR00248##  wherein Gal is a galactose moiety, and  P* is a nucleoside phosphate moiety; d) contacting the Sd(A.sup.F).sub.x acceptor with a compound of the formula Sd(A.sup.F).sub.x-P* in the presence of a glycosyltransferase to produce a glycosylated cell-binding agent having the formula: ##STR00249##  wherein: Sd(A.sup.F).sub.x is a sugar derivative comprising x functional groups A.sup.F, wherein x=1, 2, 3, or 4; e) reacting the glycosylated cell-binding agent with a compound of the formula payload-G.sup.L, wherein the payload is as according to any one of statements 39 to 52 and G.sup.L is a linker group reactive with the functional group A.sup.F of the glycosylated cell-binding agent,  to produce a glycoconjugate having the formula: ##STR00250##  wherein: Sd(A.sup.P).sub.x is a sugar derivative comprising x conjugated payloads A.sup.P, wherein x=1, 2, 3, or 4; wherein steps (b), (c), and (d) are performed in the same reaction volume.

37. The method according to claim 36, wherein the Gal acceptor product of step (b) is not purified from the reaction volume before it is contacted with the galactosyltransferase of step (c); and wherein the Sd(A.sup.F).sub.x acceptor product of step (c) is not purified from the reaction volume before it is contacted with the glycosyltransferase of step (d).

38. The method according to either one of claim 36 or 37, wherein steps (b), (c), and (d) are performed at the same time.

39. The method according to any one of claims 36 to 38, wherein steps (b), (c), and (d) comprise an incubation of between 24 and 48 hours, such as about 36 hours.

40. The method according to any one of claims 36 to 39, wherein the method further comprises an additional step (d′) between steps (d) and (e), wherein step (d′) comprises the addition of further Sd(A.sup.F).sub.x-P* and/or glycosyltransferase.

41. The method according to claim 40, wherein the further Sd(A.sup.F).sub.x-P* and/or glycosyltransferase are added on completion of the incubation of step (d).

Description

BRIEF DESCRIPTION OF THE FIGURES

[2172] Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:

[2173] FIG. 1.

[2174] Synthesis of PL1603

[2175] FIG. 2.

[2176] Glycosylation remodelling and conjugation according to Approach 1. GlcNAc=A-acetyl-glucosamine, Man=mannose, Gal=galactose, Fuc=fucose, Sia=sialic acid, PBD/DBP=PL1603. Reaction conditions: [2177] (i) UDP-Galactose, galactosyltransferase, MOPS buffer (50 mM, 20 mM MnCl2, pH 7.2), 24 h; [2178] (ii) CMP-Neu5N.sub.3, ST6Gal1 sialyltransferase, 1% BSA, alkaline phosphatase, cacodylate buffer (50 mM, pH 7.6), 24 h; [2179] (iii) PL1603.

[2180] FIG. 3.

[2181] Glycosylation remodelling and conjugation according to Approach 2. GlcNAc=A-acetyl-glucosamine, Man=mannose, Gal=galactose, Fuc=fucose, Sia=sialic acid, PBD/DBP=PL1603. Reaction conditions: [2182] (i-1) EndoS; [2183] (i-2) EndoS/BtFucH; [2184] (ii) UDP-Galactose, β4GalT1, MOPS buffer (50 mM, 20 mM MnCl2, pH 7.2), 1% BSA, 1.3% alkaline phosphatase; [2185] (iii) CMP-Neu5N3, cacodylate buffer (50 mM, pH 7.6), 1% BSA, 1.3% alkaline phosphatase; [2186] (iv) PL1603.

[2187] FIG. 4.

[2188] HIC profile of Her-PL1603-App1 and Her-PL1603-App2

[2189] FIG. 5.

[2190] In vivo efficacy of Her-PL1603-App1 and Her-PL1603-App2 versus the benchmark Her2×ADC

[2191] FIG. 6.

[2192] Pharmacokinetics (PK) of Her-PL1603-App1 and Her-PL1603-App2 in rats

STATEMENTS OF INVENTION

[2193] 1. A glycoconjugate having the formula:

##STR00127##

[2194] wherein: [2195] CBA is a Cell Binding Agent; [2196] GlcNAc is a N-acetyl-glucosamine moiety; [2197] Sug is a sugar moiety, wherein b=1 or 0; [2198] Gal is a galactose moiety; [2199] Sd(A.sup.P).sub.x is a sugar derivative comprising x conjugated payloads A, wherein x=1, 2, 3, or 4; [2200] wherein y=1 to 20; and [2201] wherein the payload A.sup.P is, comprises, or releases upon metabolism, a PBD compound, such as a PBD dimer; [2202] optionally wherein GlcNAc, Gal, Sug, and/or Sd(A.sup.P).sub.x are D enantiomers.

[2203] 2. The glycoconjugate of statement 1, wherein the PBD compound is a compound of formula I:

##STR00128##

[2204] wherein: [2205] [C6] [2206] R.sup.6 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2207] R.sup.6′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2208] [C9] [2209] R.sup.9 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2210] R.sup.9′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2211] [Y] [2212] Y is selected from O, S, or NH; [2213] Y′ is selected from O, S, or NH; [2214] [C2] [2215] when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: [2216] (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, hydroxy, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; [2217] (ib) C.sub.1-5 saturated aliphatic alkyl; [2218] (ic) C.sub.3-6 saturated cycloalkyl;

##STR00129##

wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;

##STR00130##

wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

##STR00131##

where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; [2219] when there is a single bond present between C2 and C3, [2220] R.sup.2 is selected from H, OH, F, diF and

##STR00132##

where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; [2221] [C2′] [2222] when there is a double bond present between C2′ and C3′, R.sup.2′ is selected from the group consisting of: [2223] (iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, hydroxy, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; [2224] (iib) C.sub.1-5 saturated aliphatic alkyl; [2225] (iic) C.sub.3-6 saturated cycloalkyl;

##STR00133##

wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5;

##STR00134##

wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

##STR00135##

where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; [2226] when there is a single bond present between C2′ and C3′, [2227] R.sup.2′ is selected from H, OH, F, diF and

##STR00136##

where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; [2228] [C7] [2229] R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; [2230] R.sup.7′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; [2231] [R″] [2232] R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; [2233] [B10-C11] [2234] (a) R.sup.10 is H, and R.sup.11a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or [2235] (b) R.sup.10 and R.sup.11a form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or [2236] (c) R.sup.10 is H and R.sup.11a is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or [2237] (d) R.sup.10 is H and R.sup.11a is H; or [2238] (e) R.sup.11a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.10 is selected from:

##STR00137##  where R.sup.Z is selected from:

##STR00138## [2239] (z-ii) OC(═O)CH.sub.3; [2240] (z-iii) NO.sub.2; [2241] (z-iv) OMe; [2242] (z-v) glucuronide; [2243] (z-vi) —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, OCH.sub.2CH.sub.2OMe; [2244] (a) R.sup.20 is H, and R.sup.21a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or [2245] (b) R.sup.20 and R.sup.21a form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or [2246] (c) R.sup.20 is H and R.sup.21a is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or [2247] (d) R.sup.20 is H and R.sup.21a is H; or [2248] (e) R.sup.21a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.20 is selected from:

##STR00139## [2249]  where R.sup.Z is selected from:

##STR00140## [2250] (z-ii) OC(═O)CH.sub.3; [2251] (z-iii) NO.sub.2; [2252] (z-iv) OMe; [2253] (z-v) glucuronide; [2254] (z-vi) —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, and OCH.sub.2CH.sub.2OMe.

[2255] 3. The glycoconjugate of either one of statements 1 or 2, wherein b=0.

[2256] 4. The glycoconjugate of either one of statements 1 or 2, wherein b=1.

[2257] 5. The glycoconjugate of any preceding statement, wherein Sd(A.sup.P)x is a sialic acid derivative.

[2258] 6. The glycoconjugate of statement 5, wherein the sialic acid derivative has the formula:

##STR00141##

[2259] wherein: [2260] QQ is hydrogen or a conjugated payload; [2261] ZZ is hydroxyl or a conjugated payload; [2262] YY is hydroxyl or a conjugated payload; and/or [2263] XX is hydroxyl or a conjugated payload;

[2264] and wherein at least one of QQ, ZZ, YY, and XX is a conjugated payload.

[2265] 7. The glycoconjugate of any preceding statement having the the formula:

##STR00142##

[2266] wherein: [2267] QQ is hydrogen or a conjugated payload; [2268] ZZ is hydroxyl or a conjugated payload; [2269] YY is hydroxyl or a conjugated payload; and/or [2270] XX is hydroxyl or a conjugated payload;

[2271] and wherein at least one of QQ, ZZ, YY, and XX is a conjugated payload

[2272] 8. The glycoconjugate of statement 7, wherein the GlcNAc moiety is bonded to the CBA with an α-N-glycosidic linkage and has the formula:

##STR00143##

[2273] 9. The glycoconjugate of statement 7, wherein the cell-binding agent is a protein, or comprises a protein portion, and wherein the GlcNAc moiety is conjugated to the cell-binding agent through an asparagine side chain via an α-N-glycosidic bond with the formula:

##STR00144##

[2274] 10. The glycoconjugate of any one of statements 1 to 6 having the the formula:

##STR00145##

[2275] wherein: [2276] QQ is hydrogen or a conjugated payload; [2277] ZZ is hydroxyl or a conjugated payload; [2278] YY is hydroxyl or a conjugated payload; and/or [2279] XX is hydroxyl or a conjugated payload;

[2280] and wherein at least one of QQ, ZZ, YY, and XX is a conjugated payload.

[2281] 11. The glycoconjugate of statement 10, wherein the GlcNAc moiety is bonded to the CBA with an α-N-glycosidic linkage and has the formula:

##STR00146##

[2282] 12. The glycoconjugate of statement 10, wherein the cell-binding agent is a protein, or comprises a protein portion, and wherein the GlcNAc moiety is conjugated to the cell-binding agent through an asparagine side chain via an α-N-glycosidic bond with the formula:

##STR00147##

[2283] 13. The glycoconjugate of any preceding statement, wherein Sug is a fucose moiety.

[2284] 14. The glycoconjugate of any preceding statement, wherein Sug is a fucose moiety α1-6 linked to the GlcNAc moiety.

[2285] 15. The glycoconjugate of either one of statements 13 or 14, wherein the fucose moiety has the structure:

##STR00148##

[2286] 16. The glycoconjugate of any one of statements 6 to 15 having a conjugated payload at position QQ.

[2287] 17. The glycoconjugate of any one of statements 6 to 16 having a conjugated payload at position ZZ.

[2288] 18. The glycoconjugate of any preceding statement, wherein x=1.

[2289] 19. The glycoconjugate of any preceding statement having a conjugated payload at each of positions QQ and ZZ; [2290] optionally wherein QQ and ZZ are the same.

[2291] 20. The glycoconjugate of any one of statements 1 to 19, wherein x=2.

[2292] 21. The glycoconjugate of any preceding statement, wherein y=1, 2, 3, or 4.

[2293] 22. The glycoconjugate of any preceding statement, wherein y=2.

[2294] 23. The glycoconjugate of any preceding statement, wherein y=1 to 2, 1 to 3, 2 to 4, 3-6 or 4-8.

[2295] 24. The glycoconjugate of any preceding statement, wherein the CBA is a protein.

[2296] 25. The glycoconjugate of statement 24, wherein the protein is a therapeutic protein.

[2297] 26. The glycoconjugate of any preceding statement, wherein the CBA is a Fc fusion protein.

[2298] 27. The glycoconjugate of statement 26, wherein the Fc domain is of the IgG isotype.

[2299] 28. The glycoconjugate of either one of statements 26 or 27, wherein the Fc domain is of the IgG1 subclass.

[2300] 29. The glycoconjugate of any preceding statement, wherein the CBA is an antibody.

[2301] 30. The glycoconjugate of statement 29, wherein the antibody is monoclonal.

[2302] 31. The glycoconjugate of either one of statements 29 or 30, wherein the antibody is of the IgG isotype.

[2303] 32. The glycoconjugate of any one of statements 29 to 31, wherein the antibody is of the IgG1 subclass.

[2304] 33. The glycoconjugate of any one of statements 29 to 32, wherein the GlcNAc moiety is conjugated to the antibody at the asparagine 297 (Asn297) residue according to the EU index as set forth in Kabat.

[2305] 34. The glycoconjugate of any one of statements 29 to 33, wherein the antibody is an intact antibody.

[2306] 35. The glycoconjugate of any preceding statement, wherein the GlcNAc moiety is conjugated to the CBA via the GlcNAc C1 carbon.

[2307] 36. The glycoconjugate of any preceding statement, wherein the CBA-N-GlcNAc linkage is in the beta anomeric configuration.

[2308] 37. The glycoconjugate of any one of statements 24 to 36, wherein the GlcNAc moiety is α-linked to an asparagine residue in the protein backbone.

[2309] 38. The glycoconjugate of any preceding statement, wherein the CBA specifically binds a target antigen selected from the group comprising of: BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hIg, ETB, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20R-alpha, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PSMA, SST, ITGAV, ITGB6, CEACAM5, MET, MUC1, CA9, EGFRvIII, CD33, CD19, IL2RA, AXL, CD30, BCMA, CT Ags, CD174, CLEC14A, GRP78-HSPA5, CD70, Stem Cell specific antigens, ASG-5, ENPP3, PRR4, GCC-GUCY2C, Liv-1-SLC39A6, 5T4, CD56-NCMA1, CanAg, FOLR1, GPNMB, TIM-1-HAVCR1, RG-1, B7-H4-VTCN1, PTK7, CD37, CD138, CD74, Claudins, EGFR, Her3, RON-MST1R, EPHA2, CD20-MS4A1, Tenascin C-TNC, FAP, DKK-1, CD52, CS1-SLAMF7, Endoglin, Annexin A1, V-CAM (CD106), DLK-1, KAAG1, IL13RA2, Endosialin, CD48, LRRC15, SLAMF6, and PLAC1.

[2310] 39. The glycoconjugate of any preceding statement, wherein the payload is, comprises, or releases upon metabolism a PBD compound selected from the group consisting of:

##STR00149##

[2311] 40. The glycoconjugate of any preceding statement, wherein the payload is, comprises, or releases upon metabolism a PBD compound having the formula of RelD:

##STR00150##

[2312] 41. The glycoconjugate of any preceding statement, wherein the payload is, comprises, or releases upon metabolism a PBD compound having the formula of RelE:

##STR00151##

[2313] 42. The glycoconjugate of any preceding statement, wherein the payload is, comprises, or releases upon metabolism a PBD compound having the formula of RelF:

##STR00152##

[2314] 43. The glycoconjugate of any preceding statement, wherein the payload is, comprises, or releases upon metabolism a PBD compound having the formula of RelG:

##STR00153##

[2315] 44. The glycoconjugate of any preceding statement, wherein the payload is, comprises, or releases upon metabolism a PBD compound having the formula of RelH:

##STR00154##

[2316] 45. The glycoconjugate of any preceding statement, wherein the payload has a linker moiety linking the CBA and the remainder of the payload.

[2317] 46. The glycoconjugate of any one of statements 1 to 45, wherein the conjugated payload, A.sup.P, is a drug-linker comprising a drug moiety conjugated to the cell-binding agent via a linker moiety, the glycoconjugate having the formula:

##STR00155##

[2318] 47. The glycoconjugate of statement 46, wherein -[Sd(-Linker-Drug).sub.x] has the formula:

##STR00156##

[2319] wherein the wavy line indicates where the Sd moiety is bound to the Gal moiety.

[2320] 48. The glycoconjugate of statement 46, wherein -[Sd(-Linker-Drug).sub.x] has the formula:

##STR00157##

[2321] wherein the wavy line indicates where the Sd moiety is bound to the Gal moiety.

[2322] 49. The glycoconjugate of any one of statements 46 to 48, wherein each drug-linker independently has a formula selected from the group consisting of:

##STR00158## [2323] wherein: [2324] R.sup.6 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2325] R.sup.6′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2326] R.sup.9 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2327] R.sup.9′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; [2328] Y is selected from O, S, or NH; [2329] Y′ is selected from O, S, or NH; [2330] when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: [2331] (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, hydroxy, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; [2332] (ib) C.sub.1-5 saturated aliphatic alkyl; [2333] (ic) C.sub.3-6 saturated cycloalkyl;

##STR00159##

wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;

##STR00160##

wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

##STR00161##

where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; [2334] when there is a single bond present between C2 and C3, [2335] R.sup.2 is selected from H, OH, F, diF and

##STR00162##

where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;

[2336] when there is a double bond present between C2′ and C3′, R.sup.2′ is selected from the group consisting of: [2337] (iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, hydroxy, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene; [2338] (iib) C.sub.1-5 saturated aliphatic alkyl; [2339] (iic) C.sub.3-6 saturated cycloalkyl;

##STR00163##

wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.12 group is no more than 5;

##STR00164##

wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

##STR00165##

where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; [2340] when there is a single bond present between C2′ and C3′, [2341] R.sup.2′ is selected from H, OH, F, diF and

##STR00166##

where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; [2342] R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; [2343] R.sup.7′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; [2344] R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; [2345] (a) R.sup.10 is H, and R.sup.11a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or [2346] (b) R.sup.10 and R.sup.11a form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or [2347] (c) R.sup.10 is H and R.sup.11a is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or [2348] (d) R.sup.10 is H and R.sup.11a is H; or [2349] (e) R.sup.11a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl and R.sup.10 is selected from:

##STR00167## [2350]  where R.sup.Z is selected from:

##STR00168## [2351] (z-ii) OC(═O)CH.sub.3; [2352] (z-iii) NO.sub.2; [2353] (z-iv) OMe; [2354] (z-v) glucuronide; [2355] (z-vi) —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, OCH.sub.2CH.sub.2OMe; [2356] (a) R.sup.20 is H, and R.sup.21a is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or [2357] (b) R.sup.20 and R.sup.21a form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or [2358] (c) R.sup.20 is H and R.sup.21a is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or [2359] (d) R.sup.20 is H and R.sup.21a is H; or [2360] (e) R.sup.21a is OH or OR.sup.A where R.sup.A is C.sub.1-4 alkyl and R.sup.20 is selected from:

##STR00169##  where R.sup.Z is selected from:

##STR00170## [2361] (z-ii) OC(═O)CH.sub.3; [2362] (z-iii) NO.sub.2; [2363] (z-iv) OMe; [2364] (z-v) glucuronide; [2365] (z-vi) —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, OCH.sub.2CH.sub.2OMe; [2366] R.sup.L1 and R.sup.11b [2367] R.sup.L1 is a linker for connection the Sd moiety; [2368] R.sup.11b is OH or OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; [2369] R.sup.L2 [2370] R.sup.L2 is of formula IIIa, formula IIIb or formula IIIc:

##STR00171## [2371] where A is a C.sub.5-7 aryl group, and either [2372] (i) Q.sup.1 is a single bond, and Q.sup.2 is selected from a single bond and —Z—(CH.sub.2).sub.n—, where Z is selected from a single bond, O, S and NH and n is from 1 to 3; or [2373] (ii) Q.sup.1 is —CH═CH—, and Q.sup.2 is a single bond;

##STR00172##

[2374] where;

[2375] R.sup.C1, R.sup.C2 and R.sup.C3 are independently selected from H and unsubstituted C.sub.1-2 alkyl;

##STR00173## [2376] where Q.sup.L is selected from O—R.sup.L2′, S—R.sup.L2′ and NR.sup.N—C(═O)—R.sup.L2′, and R.sup.N is selected from H, methyl and ethyl [2377] X is selected from the group comprising: O—R.sup.L2′, S—R.sup.L2′, CO.sub.2—R.sup.L2′, CO—R.sup.L2′, NR.sup.N—C(═O)—R.sup.L2′, NHNH—R.sup.L2′, CONHNH—R.sup.L2′,

##STR00174##

wherein R.sup.N is selected from the group comprising H and C.sub.1-4 alkyl; [2378] R.sup.L2′ is a linker for connection to the Sd moiety; [2379] R.sup.L3 [2380] R.sup.L3 is selected from formulae A1, A2, A3, A4 and A5:

##STR00175## [2381] Z.sup.1 is a C.sub.1-3 alkylene group; [2382] Z.sup.2 is a C.sub.1-3 alkylene group; [2383] Z.sup.3 is a C.sub.1-3 alkylene group; [2384] Z.sup.4 is a C.sub.1-3 alkylene group; [2385] Z.sup.5 is a C.sub.1-3 alkylene group; [2386] n is an integer between 0 and 48; [2387] Q is:

##STR00176##

where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue, or a non-peptide moiety defined as PM in WO2015/095124; [2388] R.sup.L3′ is a linker for connection to the Sd moiety; [2389] R.sup.T [2390] R.sup.T is:

##STR00177## [2391] wherein [2392] T and T″ are independently selected from a single bond or a C.sub.1-9 alkylene, which chain may be interrupted by one or more heteroatoms e.g. O, S, N(H), NMe, provided that the number of atoms in the shortest chain of atoms between Y and Y′ is 3 to 12 atoms; [2393] Z.sup.6 is CH or N; [2394] R.sup.T′ is selected from formulae B1, B2, B3, B4, B5, B6, and B7:

##STR00178## ##STR00179## [2395] n is an integer selected in the range of 0 to 48; [2396] R.sup.B4 is a C.sub.1-6 alkylene group; [2397] Q is:

##STR00180##

where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue, or a non-peptide moiety defined as PM in WO2015/095124; [2398] R.sup.L4 is a linker for connection to the Sd moiety.

[2399] 50. The glycoconjugate of any one of statements 46 to 48, wherein each linker independently is a linker of formula Z1 or Z2:

##STR00181##

[2400] wherein r=0 or 1, a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5, G.sup.LL is a linking moiety through which the linker is bound to the Sd moiety, the wavy line indicates where the linker is bound to the drug moiety, and one of X.sup.10, X.sup.11, X.sup.12, X.sup.13 and X.sup.14 may be selected from:

##STR00182##

[2401] the remainder being a single bond.

[2402] 51. The glycoconjugate of statements 50, wherein G.sup.LL is selected from:

TABLE-US-00003 (G.sup.LL8-1) [00183] G.sup.LL11 [00184] (G.sup.LL8-2) [00185] G.sup.LL12 [00186] (G.sup.LL9-1) [00187] G.sup.LL13 [00188] (G.sup.LL9-2) [00189] G.sup.LL14 [00190] G.sup.LL10 [00191]

[2403] where CBA indicates where the group is bound to Sd(A.sup.P).sub.x.

[2404] 52. The glycoconjugate of any one of statements 46 to 51, wherein all the drug-linkers conjugated to the cell binding-agent are the same.

[2405] 53. A method for the preparation of the glycoconjugate of any one of statements 1 to 52, the method comprising the steps of: [2406] (i) providing a glycosylated cell-binding agent; [2407] (ii) reacting the glycosylated cell-binding agent of (i) with a compound of the formula payload-G.sup.L, wherein the payload is as defined in any one of statements 1 to 52 and G.sup.L is linker group reactive with the functional group of the glycosylated cell-binding agent

[2408] 54. The method of statement 53, wherein G.sup.L is selected from the group consisting of:

TABLE-US-00004 (G.sup.L8) [00192] (G.sup.L9) [00193] (G.sup.L10) [00194] (G.sup.L11) [00195] (G.sup.L12) [00196] (G.sup.L13) [00197]

[2409] 55. The method of either one of statements 53 or 54, wherein the provided glycosylated cell-binding agent is a glycosylated cell-binding agent having the formula:

##STR00198##

[2410] wherein: [2411] CBA is a Cell Binding Agent; [2412] GlcNAc is a N-acetyl-glucosamine moiety; [2413] Sug is a sugar moiety, wherein b=1 or 0; [2414] Gal is a galactose moiety; [2415] Sd(A.sup.F).sub.x is a sugar derivative comprising x functional groups A.sup.F, wherein A.sup.F is independently selected from the group consisting of an azido group, an alkynyl group, and a keto group, and wherein x=1, 2, 3, or 4; and

[2416] wherein y=1 to 20.

[2417] 56. The method of statement 55, wherein b=0.

[2418] 57. The method of statement 55, wherein b=1.

[2419] 58. The method any one of statements 55 to 57, wherein Sd(A.sup.F).sub.x is a sialic acid derivative.

[2420] 59. The method of statement 58, wherein the sialic acid derivative has the formula:

##STR00199##

[2421] wherein: [2422] QQ is hydrogen or a functional group A.sup.F; [2423] ZZ is hydroxyl or a functional group A.sup.F; [2424] YY is hydroxyl or a functional group A.sup.F; and/or [2425] XX is hydroxyl or a functional group A.sup.F;

[2426] and wherein at least one of QQ, ZZ, YY, and XX is a functional group A.sup.F.

[2427] 60. The method of any one of statements 55 to 59 wherein the glycosylated cell-binding agent of has the formula:

##STR00200##

[2428] wherein: [2429] QQ is hydrogen or a functional group A.sup.F; [2430] ZZ is hydroxyl or a functional group A.sup.F; [2431] YY is hydroxyl or a functional group A.sup.F; and/or [2432] XX is hydroxyl or a functional group A.sup.F;

[2433] and wherein at least one of QQ, ZZ, YY, and XX is a functional group A.sup.F.

[2434] 61. The method of any one of statements 53 to 60 wherein the glycosylated cell-binding agent of has the formula:

##STR00201##

[2435] wherein: [2436] QQ is hydrogen or a functional group A.sup.F; [2437] ZZ is hydroxyl or a functional group A.sup.F; [2438] YY is hydroxyl or a functional group A.sup.F; and/or [2439] XX is hydroxyl or a functional group A.sup.F;

[2440] and wherein at least one of QQ, ZZ, YY, and XX is a functional group A.sup.F.

[2441] 62. The method of any one of statements 55 to 61, wherein Sug is a fucose moiety.

[2442] 63. The method of statement 62, wherein the fucose moiety has the structure:

##STR00202##

[2443] 64. The method of any one of statements 59 to 63, wherein the glycosylated cell-binding agent of has a functional group A.sup.F at position QQ.

[2444] 65. The method of any one of statements 59 to 64, wherein the glycosylated cell-binding agent of has a functional group A.sup.F at position ZZ.

[2445] 66. The method of any one of statements 55 to 65, wherein x=1.

[2446] 67. The method of any one of statements 59 to 66, wherein the glycosylated cell-binding agent of has a functional group A.sup.F at each of positions QQ and ZZ; [2447] optionally wherein each of QQ and ZZ has the same functional group A.sup.F.

[2448] 68. The method of any one of statements 55 to 67, wherein x=2.

[2449] 69. The method of any one of statements 55 to 68, wherein y=1, 2, 3, or 4.

[2450] 70. The method of any one of statements 55 to 69, wherein y=2.

[2451] 71. The method of any one of statements 55 to 70, wherein y=1 to 2, 1 to 3, 2 to 4, 3-6 or 4-8.

[2452] 72. The method of any one of statements 55 to 71, wherein a functional group A.sup.F is an azido group.

[2453] 73. The method of any one of statements 55 to 72, wherein a functional group A.sup.F is an alkynyl group.

[2454] 74. The method of any one of statements 55 to 73, wherein the CBA is a protein.

[2455] 75. The method of statement 74, wherein the protein is a therapeutic protein.

[2456] 76. The method of any one of statements 55 to 75, wherein the CBA is an antibody.

[2457] 77. The method of statement 76, wherein the antibody is monoclonal.

[2458] 78. The method of either one of statements 76 or 77, wherein the antibody is of the IgG isotype.

[2459] 79. The method of any one of statements 76 to 78, wherein the antibody is of the IgG1 subclass.

[2460] 80. The method of any one of statements 76 to 79, wherein the GlcNAc moiety is conjugated to the antibody at the asparagine 297 (Asn297) residue according to the EU index as set forth in Kabat.

[2461] 81. The method of any one of statements 76 to 80, wherein the antibody is an intact antibody.

[2462] 82. The method of any one of statements 55 to 81, wherein the GlcNAc moiety is conjugated to the CBA via the GlcNAc C1 carbon.

[2463] 83. The method of any one of statements 55 to 82, wherein the CBA-N-GlcNAc linkage is in the beta anomeric configuration.

[2464] 84. The method of any one of statements 53 to 83, wherein the GlcNAc moiety is α-linked to an asparagine residue in the protein backbone.

[2465] 85. The method of any one of statements 53 to 84, wherein the CBA specifically binds a target antigen selected from the group comprising of: BMPR1B, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hIg, ETB, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20R-alpha, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, PSMA, SST, ITGAV, ITGB6, CEACAM5, MET, MUC1, CA9, EGFRvIII, CD33, CD19, IL2RA, AXL, CD30, BCMA, CT Ags, CD174, CLEC14A, GRP78-HSPA5, CD70, Stem Cell specific antigens, ASG-5, ENPP3, PRR4, GCC-GUCY2C, Liv-1-SLC39A6, 5T4, CD56-NCMA1, CanAg, FOLR1, GPNMB, TIM-1-HAVCR1, RG-1, B7-H4-VTCN1, PTK7, CD37, CD138, CD74, Claudins, EGFR, Her3, RON-MST1R, EPHA2, CD20-MS4A1, Tenascin C-TNC, FAP, DKK-1, CD52, CS1-SLAMF7, Endoglin, Annexin A1, V-CAM (CD106), DLK-1, KAAG1, IL13RA2, Endosialin, CD48, LRRC15, SLAMF6, and PLAC1.

[2466] 86. A composition consisting of population of glycoconjugates according to any one of statements 1 to 52, wherein at least 75% of the molecules making up the population are the same glycoform.

[2467] 87. A composition according to statement 86, wherein at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99% of the molecules making up the population are the same glycoform.

[2468] 88. The method of any one of statements 55 to 85, wherein the glycosylated cell-binding agent is provided by a method comprising the steps of: [2469] (i) providing a Sd(A.sup.F).sub.x acceptor having the formula:

##STR00203##

[2470] wherein CBA, GlcNAc, Sug, b, Gal, and y are defined as in of any one of statements 55 to 85; and [2471] (ii) contacting the Sd(A.sup.F).sub.x acceptor with a compound of the formula Sd(A.sup.F).sub.x-P* in the presence of a glycosyltransferase, wherein: [2472] Sd(A.sup.F).sub.x is as defined in any one of statements 55 to 85; and [2473] P* is a nucleoside phosphate moiety.

[2474] 89. The method of statement 88, wherein the Sd(A.sup.F).sub.x acceptor has the formula:

##STR00204##

[2475] 90. The method of statement 86, wherein the GlcNAc moiety is connected to the CBA through a α-N-glycosidic bond to give a Sd(A.sup.F).sub.x acceptor having the formula:

##STR00205##

[2476] 91. The method of statement 88, wherein the cell-binding agent is a protein, or comprises a protein portion, and wherein the GlcNAc moiety is conjugated to the cell-binding agent through an asparagine side chain via an α-N-glycosidic bond to give a Sd(A.sup.F).sub.x acceptor having the formula:

##STR00206##

[2477] 92. The method of any one of statements 88 to 91, wherein the nucleoside element of the nucleoside phosphate moiety is one of adenosine, guanosine, uridine, cytidine, or thymidine.

[2478] 93. The method of any one of statements 88 to 92, wherein the nucleoside element of the nucleoside phosphate moiety is selected from the group consisting of: UDP, GDP, CDP, and CMP.

[2479] 94. The method of any one of statements 88 to 93, wherein the compound of the formula Sd(A.sup.F).sub.x-P* has the formula:

##STR00207##

[2480] wherein: [2481] P* is a nucleoside phosphate moiety; and [2482] QQ is hydrogen or a functional group A; [2483] ZZ is hydroxyl or a functional group A; [2484] YY is hydroxyl or a functional group A; and/or [2485] XX is hydroxyl or a functional group A;

[2486] and wherein at least one of QQ, ZZ, YY, and XX is a functional group A.

[2487] 95. The method of any one of statements 88 to 94, wherein the compound of the formula Sd(A.sup.F).sub.x-P* has the formula:

##STR00208##

[2488] 96. The method of any one of statements 88 to 95, wherein the Sd(A.sup.F).sub.x acceptor is provided by a process comprising the steps of: [2489] a) providing a Gal acceptor having the formula:

##STR00209## [2490] wherein CBA, GlcNAc, Sug, b, and y are defined as in statement 1;  and [2491] b) contacting the Gal acceptor with a compound of the formula Gal-P* in the presence of a galactosyltransferase, wherein Gal is a Galactose moiety and P* is a nucleoside phosphate moiety;  optionally wherein, [2492] c) the Gal acceptor is produced by contacting with a glycosidase a oligoglycosylated cell-binding agent having the formula:

##STR00210##  wherein CHO is a carbohydrate moiety.

[2493] 97. The method of any one of statements 88 to 95, wherein the Sd(A.sup.F).sub.x acceptor has only one terminal galactose moiety.

[2494] 98. The method of any one of statements 88 to 95, wherein the Sd(A.sup.F).sub.x acceptor has only two terminal galactose moieties.

[2495] 99. The method of any one of statements 88 to 95, wherein the Sd(A.sup.F).sub.x acceptor has only three terminal galactose moieties.

[2496] 100. The method of any one of statements 88 to 95, wherein the Sd(A.sup.F).sub.x acceptor has only four terminal galactose moieties.

[2497] 101. The method of any one of statements 88 to 100, wherein the glycosyltransferase is a sialyltransferase.

[2498] 102. The method of statement 101, wherein the sialyltransferase is α-(2,3)-sialyltransferase.

[2499] 103. The method of statement 102, wherein the sialyltransferase is Pasteurella multocida α-(2,3)-sialyltransferase.

[2500] 104. The method of statement 102, wherein the sialyltransferase is CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase (ST3Gal IV).

[2501] 105. The method of statement 101, wherein the sialyltransferase is α-(2,6)-sialyltransferase.

[2502] 106. The method of statement 105, wherein the sialyltransferase is a β-galactoside α-(2,6)-sialyltransferase 1 (ST6Gal 1).

[2503] 107. The method of statement 101, wherein the sialyltransferase is α-(2,8)-sialyltransferase.

[2504] 108. The method of any one of statements 101, 102, 104, and 105 to 107, wherein the sialyltransferase is a mammalian sialyltransferase.

[2505] 109. The method of statement 108, wherein the sialyltransferase is a human sialyltransferase.

[2506] 110. The method of statement 108, wherein the sialyltransferase is a rat sialyltransferase.

[2507] 111. The method of statement 101, wherein the sialyltransferase is a polypeptide having sialyltransferase activity and comprising a sequence having at least 70% sequence identity SEQ ID NO. 1, SEQ ID NO. 4, or SEQ ID NO. 7.

[2508] 112. The method of statement 101, wherein the sialyltransferase has the sequence set out in SEQ ID NO. 1, SEQ ID NO. 4, or SEQ ID NO. 7.

[2509] 113. The method of any one of statements 96 to 112, wherein the glycosidase is an endoglycosidase.

[2510] 114. The method of any one of statements 96 to 113, wherein the glycosidase is in the class EC3.2.1.96.

[2511] 115. The method of statement 113, wherein the endoglycosidase is endo-β-N-acetylglucosaminidase D, endo-β-N-acetylglucosaminidase H, endoglycosidase S, endo-β-N-acetylglucosaminidase M, endo-β-N-acetylglucosaminidase LL, endo-β-N-acetylglucosaminidase F1, endo-β-N-acetylglucosaminidase F2, or endo-β-N-acetylglucosaminidase F3.

[2512] 116. The method of any one of statements 96 to 112, wherein the glycosidase is a combination of two or more endoglycosidases.

[2513] 117. The method of any one of statements 96 to 112, wherein the glycosidase is a combination of two or more glycosidases in the class EC3.2.1.96.

[2514] 118. The method of either one of statements 116 or 117, wherein the glycosidase is: endoglycosidase D and endoglycosidase S; endoglycosidase S and endoglycosidase LL; endoglycosidase D and endoglycosidase LL; endoglycosidase D and endoglycosidase H; endoglycosidase S and endoglycosidase H; endoglycosidase F1 and endoglycosidase F2; endoglycosidase F1 and endoglycosidase F3; endoglycosidase F2 and endoglycosidase F3; endoglycosidase D, endoglycosidase S and endoglycosidase LL; endoglycosidase D, endoglycosidase S and endoglycosidase H; or endoglycosidase D, endoglycosidase S and endoglycosidase F1.

[2515] 119. The method of statement 113, wherein the endoglycosidase is Endo S as disclosed in Collin, M. and Olsén, A. (2001). The EMBO Journal. 20, 3046-3055.

[2516] 120. The method of statement 113, wherein the endoglycosidase is a polypeptide having endoglycosidase activity and comprising a sequence having at least 70% sequence identity SEQ ID NO. 3, SEQ ID NO. 6, or SEQ ID NO. 9.

[2517] 121. The method of statement 113, wherein the endoglycosidase has the sequence set out in SEQ ID NO. 3, SEQ ID NO. 6, or SEQ ID NO. 9.

[2518] 122. The method of any one of statements 96 to 121, wherein the galactosyltransferase is human beta-1,4-galactosyltransferase 1 (B4GalT1).

[2519] 123. The method of statement 122, wherein the galactosyltransferase is a polypeptide having galactosyltransferase activity and comprising a sequence having at least 70% sequence identity SEQ ID NO. 2, SEQ ID NO. 5, or SEQ ID NO. 8.

[2520] 124. The method of statement 122, wherein the galactosyltransferase has the sequence set out in SEQ ID NO. 2, SEQ ID NO. 5, or SEQ ID NO. 8.

[2521] 125. A glycoconjugate of any one of statements 1 to 52 for use in a method of treatment.

[2522] 126. A glycoconjugate of any one of statements 1 to 52 for use in a method of treating a proliferative disorder.

[2523] 127. A method of treating a proliferative disorder, the method comprising administering an effective amount of a glycoconjugate of any one of statements 1 to 52 to a subject.

[2524] 128. Use of a glycoconjugate of any one of statements 1 to 52 in the manufacture of a medicament for the treatment of a proliferative disorder.

[2525] 129. The glycoconjugate, method, or use of any one of statements 126 to 128, wherein the proliferative disorder is cancer.

[2526] 130. The glycoconjugate, method, or use of statement 129, wherein the cancer is selected from the group consisting of: histocytoma, glioma, astrocyoma, neuroblastoma, osteoma, lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, renal cancer, brain cancer, sarcoma, liposarcoma, osteosarcoma, Kaposi's sarcoma, melanoma, lymphomas, myeloma, and leukemias.

[2527] 131. A method for the preparation of a glycoconjugate, the method comprising the steps of: [2528] a) providing an oligoglycosylated cell-binding agent having the formula:

##STR00211## [2529] wherein CBA is a Cell Binding Agent; [2530] GlcNAc is a N-acetyl-glucosamine moiety; [2531] Sug is a sugar moiety, wherein b=1 or 0; [2532] y=1 to 20; and [2533] CHO is a carbohydrate moiety; [2534] b) contacting the oligoglycosylated cell-binding agent with a glycosidase to produce a Gal acceptor having the formula:

##STR00212## [2535] c) contacting the Gal acceptor with a compound of the formula Gal-P* in the presence of a galactosyltransferase to produce a Sd(A.sup.F).sub.x acceptor having the formula:

##STR00213##  wherein Gal is a galactose moiety, and  P* is a nucleoside phosphate moiety; [2536] d) contacting the Sd(A.sup.F).sub.x acceptor with a compound of the formula Sd(A.sup.F).sub.x-P* in the presence of a glycosyltransferase to produce a glycosylated cell-binding agent having the formula:

##STR00214##  wherein: [2537] Sd(A.sup.F).sub.x is a sugar derivative comprising x functional groups A.sup.F, wherein x=1, 2, 3, or 4; [2538] e) reacting the glycosylated cell-binding agent with a compound of the formula payload-G.sup.L, [2539] wherein the payload is as according to any one of statements 39 to 52 and G.sup.L is a linker group reactive with the functional group A.sup.F of the glycosylated cell-binding agent,  to produce a glycoconjugate having the formula:

##STR00215##  wherein: [2540] Sd(A.sup.P).sub.x is a sugar derivative comprising x conjugated payloads A.sup.P, wherein x=1, 2, 3, or 4.

[2541] 132. The method according to statement 131, wherein steps (b), (c), and (d) are performed in the same reaction volume.

[2542] 133. The method according to either one of statements 131 or 32, wherein the Gal acceptor product of step (b) is not purified from the reaction volume before it is contacted with the galactosyltransferase of step (c).

[2543] 134. The method according to any one of statements 131 to 133, wherein the Sd(A.sup.F).sub.x acceptor product of step (c) is not purified from the reaction volume before it is contacted with the glycosyltransferase of step (d).

[2544] 135. The method according to any one of statements 131 to 134, wherein steps (b), (c), and (d) are performed at the same time.

[2545] 136. The method according to any one of statements 131 to 135, wherein steps (b), (c), and (d) comprise an incubation of between 24 and 48 hours, such as about 36 hours.

[2546] 137. The method according to statement 136, wherein the incubation is at about 37° C.

[2547] 138. The method according to any one of statements 131 to 137, wherein the method further comprises an additional step (d′) between steps (d) and (e), wherein step (d′) comprises the addition of further Sd(A.sup.F).sub.x-P* and/or glycosyltransferase.

[2548] 139. The method according to statement 138, wherein the further Sd(A.sup.F).sub.x-P* and/or glycosyltransferase are added to the products of step (d).

[2549] 140. The method according to either one of statements 138 or 139, wherein the further Sd(A.sup.F).sub.x-P* and/or glycosyltransferase are added on completion of the incubation of step (d).

[2550] 141. The method according to any one of statements 138 to 140, wherein step (d′) comprises an incubation of between 24 and 48 hours, such as about 36 hours.

[2551] 142. The method according to statement 141, wherein the incubation is at about 37° C.

EXAMPLES

Example 1: Synthesis of Drug-Linker Payload, PL1603

[2552] The PL1603 drug-linker was synthesised for use as a payload suitable for conjugation to the glycosylated antibody intermediates described herein.

[2553] Synthesis Method

[2554] See FIG. 1.

[2555] SG3305 (600 mg, 1.0 eq), Endo-BCN-PGE4-acid (1.2 eq) and EDCI-HCl (1.2 eq) were taken up in DCM (15 vol, 2% MeOH) and stirred at 0-5° C. (The synthesis of Endo-BCN-PEG4 is described in, for example, WO2016/053107 at page 142. Endo-BCN-PEG4 is also commercially available from, for example, Broadpharm®. The synthesis of SG3305 is described in, for example, Tiberghein et al., ACS Med Chem Lett 2016 7(11) 983-987 [DOI: 10.1021/acsmedchemlett.6b00062])

[2556] Upon completion of reaction, the reaction was quenched with purified water (10 vol). The mixture was partitioned and the organic layer was washed with brine, dried over sodium sulphate and concentrated under reduced pressure to give crude PL1603 (650 mg, 93.2%, 73.57% HPLC purity).

[2557] Crude PL1603 (400 mg) was purified by RP-HPLC (C18, MeCN:H.sub.2O) and product containing fractions were combined and lyophilised to give PL1603 as a white solid (130 mg, 33%, 94.61% HPLC purity).

Example 2: Antibody Remodelling and Conjugation, Approach 1

[2558] Antibody Remodelling

[2559] The N-linked oligosaccharides on the Herceptin antibody were remodelled according to the methods described in Li et al. 2014 (Angew Chem Int Ed Engl., 2014, Jul. 7; 53(28):7179-82).

[2560] See FIG. 2, steps (i) and (ii).

[2561] Conjugation

[2562] 9.2 mg/ml of the remodelled Herceptin in 50 mM Cacodylate buffer pH 7.6 was conjugated by the addition of 20 molar equivalents of PL1603 (10 mM stock in DMA, structure provided in FIG. 1) and DMA to a final Co solvent level of 20% v/v. The conjugation reaction was incubated in a 20° C. water bath for 66 hours. A DAR by PLRP of 1.4 was achieved (batch SON160-16).

[2563] The resulting glycoconjugate is herein termed ‘Her-PL1603-App1’.

[2564] See FIG. 2, step (iii).

Example 3: Antibody Remodelling and Conjugation, Approach 2

[2565] Rationale for New Approach

[2566] In Approach 1, the activity of the recombinant sialyltransferase ST6Gal1 to the α(1,3)- and α(1,6)-arm of the biantennary N-glycan of the Fc region of antibodies can be differential by controlling the ratio of CMP-sialic acid and antibody. This can result in ADCs having DAR2 or DAR4. However, the careful controlling of the reaction stoichiometry that is required impacts on product reproducibility between batches.

[2567] Accordingly, alternative oligosaccharide structures were investigated with the aim of identifying oligosaccharide structures that were both obtainable using available synthetic methods and offered advantageous glycoconjugate properties.

[2568] A key discovery was the unexpected ability of wild-type human β4GalT1 galactosyl-transferase to transfer a galactose residue onto a α1-6 fucosylated GlcNAc residue. This reaction does not occur in nature. Moreover, it was found that the resulting galactosylated oligosaccharide could be further modified with the addition of an azido-modified sialic acid by the ST6Gal1 sialyltransferase.

[2569] Antibody Remodelling

[2570] The N-linked oligosaccharides on the Herceptin antibody were remodelled according to the following method:

[2571] Endo S. Treatment.

[2572] Trimming of IgG glycan was undergone using Endo S cloned from Streptococcus pyogenes and overexpressed as a fusion to the chitin binding domain in E. coli. (New England BioLabs). To the IgG antibody (10 mg/mL) in 30 mM histidine, 200 mM sorbitol and 0.02% tween-20, Endo S (0.13 mL, 100 kU/mL) in 10 mM Tris, 25 mM NaCl, 2.5 mM EDTA, 2.5 mM CaCl2, 25 mM sodium acetate was added. The resulting solution was incubated for approximately 48 hours at 37° C. followed by Protein A Sepharose Column (GE Healthcare) purification, buffer exchanging and concentrated into 1.2 mL of 50 mM MOPS containing 20 mM MnCl.sub.2.

[2573] Galactosylation of the IgG

[2574] Galactosylation of IgG bearing truncated N-glycan was achieved by addition of β-1,4-galactosyl transferase (200 μg/mL) to the Endo S treatment resulting material in 50 mM MOPS, 20 mM MnCl2, 10 mM UDP-galactose, pH 7.2, 80 μg/mL BSA, 85 U/mL calf intestine alkaline phosphatase and incubation at 37° C. for 70 h. To ensure complete galactosylation, an additional aliquot of UDP-galactose and galactosyl transferase were added to the reaction and incubated at 37° C. for an additional 24 h. The galactosylated IgG was purified using a Protein A Sepharose Column and the solution was exchanged in 50 mM cacodylate, pH 7.6 using an Amicon 10 kDa cutoff spin concentrator (Millipore).

[2575] Synthesis of CMP-Neu5N3 and CMP-Neu9N3

[2576] Sialic acid aldolase (0.2 U/μL, 5 μL), and CMP-sialic acid synthetase (0.2 U/μL, 5 μL) were added to a mixture of N-azidoacetyl-D-mannosamine (5 mg, 0.019 mmol) in tris-HCl buffer (100 mM, pH 8.9, 20 mM MgCl.sub.2, 1.9 mL), containing sodium pyruvate (10.5 mg, 0.095 mmol) and CTP (10 mg, 0.019 mmol). The tube was incubated at 37° C., and progress of the reaction was monitored by TLC (EtOH:aq. NH.sub.4HCO.sub.3 (1 M) 7:3, v:v), which after 5 hour indicated completion of the reaction. EtOH (3 mL) was added, and the precipitate was removed by centrifugation and the supernatant was concentrated under reduced pressure. The residue was redissolved in distilled water (500 μL) followed by lyophilization to provide a crude material that was applied to a Biogel fine P-2 column (50*1 cm, eluted with 0.1 M NH.sub.4HCO.sub.3 at 4° C. in dark). The product was detected by TLC, and appropriate fractions were combined and lyophilized to provide CMP-Neu5N.sub.3 as an amorphous white solid (10.1 mg, 81%).

[2577] .sup.1H NMR (300 MHz, d.sub.2o) δ 7.82 (d, J=7.5 Hz, 1H, H-6, cyt), 5.97 (d, J=7.6 Hz, 1H, H-5, cyt), 5.84 (d, J=4.2 Hz, 1H, H-1, rib), 4.18 (dd, J=7.4, 4.4 Hz, 2H, H-2+H-3, rib), 4.14-4.04 (m, 4H, H-4+H-5 rib, H-6 Neu), 4.04-3.97 (m, 1H, H-4), 3.95 (s, 2H, N.sub.3CH.sub.2CO), 3.87 (t, J=10.2 Hz, 1H, H-5), 3.82-3.74 (m, 1H, H-8), 3.72 (m, 1H, H-9a), 3.49 (d, J=11.8 Hz, 1H, H-9b), 3.30 (d, J=9.5 Hz, 1H, H-7), 2.36 (dd, J=13.3, 4.6 Hz, 1H, H-3 eq), 1.51 (td, J=12.0, 5.6 Hz, 1H, H-3ax). HRMS (ESI): m/z calcd for C.sub.20H.sub.30N.sub.7O.sub.16P [M−H].sup.−: 654.1414; found: 653.9477.

[2578] CMP-Neu9N.sub.3 was prepared following the reported procedure. CTP (126 mg, 0.24 mmol) was added to a solution of 5-Acetamido-9-azido-3,5,9-tri-deoxy-D-glycero-D-galacto-2-nonulosonic acid (50 mg, 0.15 mmol) in a Tris-HCl buffer (0.1 M, 9 mL, pH 8.9) containing MgCl.sub.2 (20 mM). The recombinant CMP-sialic acid synthetase from N. meningitis (4.0 U) and the inorganic pyrophosphatase from S. cerevisiae (2.0 U) were added and the reaction mixture was incubated at 37° C. with shaking. The progress of the reaction was monitored by TLC (isopropanol: 20 mM NH.sub.4OH, 4:1, v:v), which after 3 h indicated completion of the reaction. Ethanol (80 mL) was added and the mixture was kept on ice for 2 h prior to centrifugation. The supernatant was decanted and the pellet (mostly inorganic salts) was re-suspended in EtOH (30 mL), cooled on ice for 1 h and centrifuged. The combined ethanol extracts were concentrated in vacuo providing crude material (168 mg). Ethanol (1.8 mL) was slowly added to the material dissolved in H.sub.2O (0.2 mL) and precipitation occurred immediately. The mixture was kept on ice for 2 h.

[2579] Next, the supernatant was removed after centrifugation and the white pellet was dried and purified on a column of extra-fine Biogel P-2 eluted with 0.1 M NH.sub.4HCO.sub.3 at 4° C. The appropriate fractions were detected by UV and TLC (as above), collected, concentrated in vacuo (bath temperature <25° C.) and lyophilized to afford CMP-Neu9N3 (60 mg, 62%). .sup.1H NMR (D.sub.2O containing 0.1 M NH.sub.4HCO.sub.3, 600 MHz): δ 7.82 (d, 1H, J.sub.5,6=7.8 Hz, H-6, cyt), 5.97 (d, 1H, J.sub.5,6=7.8 Hz, H-5, cyt), 5.82 (d, 1H, J.sub.1,2=4.8 Hz, H-1 rib), 4.17 (t, 1H, J=4.8), 4.13 (t, 1H, J=4.8 Hz), 4.08 (m, 3H), 3.99 (d, 1H, J=12.0 Hz), 3.90 (m, 2H), 3.78 (t, 1H), 3.49 (dd, 1H, J=2.4, 13.2 Hz, H-9a), 3.35 (dd, 1H, J=6.0, 13.2 Hz, H-9b), 3.31 (dd, 1H, J=9.6 Hz), 2.33 (dd, 1H, J.sub.3eq,4=4.8 Hz, J.sub.3eq,3ax=13.2 Hz, H-3 eq), 1.90 (s, 3H, Me), 1.55 (ddd, 1H, J.sub.3ax,P=6.0 Hz, J.sub.3ax,3eq=13.2 Hz, J.sub.3ax,4=12.0 Hz, H-3ax); .sup.13C NMR (D.sub.2O, containing 0.1 M NH.sub.4HCO.sub.3, 600 MHz): δ 174.2, 170.4, 166.0, 160.7, 141.4, 96.5, 88.8, 82.9, 74.2, 71.5, 69.3, 69.1, 67.4, 64.9, 53.0, 51.7, 41.0, 22.0; ESI-MS: calcd for C.sub.20H.sub.28N.sub.7O.sub.15P.sup.2− [M+H].sup.−: m/z: 638.1470; found 638.1421.

[2580] Sialylation of the IgG

[2581] The sialylation of galactosylated IgG was performed in 50 mM cacodylate, 14 mg/mL of IgG, 8 mM CMP-Neu5N.sub.3, 90 μg/ml BSA, 90 U/mL calf intestine alkaline phosphatase and 0.4 mg/mL GFP-ST6Gal I at pH 7.6 and incubated at 37° C. for 4 days followed by Protein A Sepharose Column purification and buffer exchanging to 50 mM cacodylate. The extent of sialylation was monitored by LC-MS as described previously using a Shimadzu LCMS-IT-TOF Mass Spectrometer. Following every 48 hours incubation, the sample was buffer exchanged with 50 mM cacodylate, pH7.6 using an Amicon 10 kDa cutoff spin concentrator to remove CMP, an inhibitor of ST6Gal I and an additional aliquot of CMP-Neu5N.sub.3 and α2-6 sialyltransferase were added back to this washed preparation.

[2582] Conjugation

[2583] 10 mg/ml of the remodelled Herceptin in 50 mM Cacodylate buffer pH 7.6 was conjugated by the addition of 7.5 molar equivalents of PL1603 (10 mM stock in DMA, structure provided in FIG. 3) and DMA to a final Co solvent level of 20% v/v. The conjugation reaction was incubated in a 20° C. water bath overnight. A DAR of 1.8 was achieved (batch SON180-19) with purification by PLRP.

[2584] The conjugation reaction in approach 2 is notable for being considerably faster than the corresponding approach 1. This goes against initial expectations, which were that the termini of the longer glycans would be more accessible and so easier to modify. In fact, the data shows that the shorter glycans of approach 2 were more readily modifiable.

[2585] The resulting glycoconjugate is herein termed ‘Her-PL1603-App2’.

[2586] See FIG. 3, step (iv).

Example 4: Glycoconjugate Properties

[2587] Physical Properties

[2588] Her-PL1603-App1 and Her-PL1603-App2 were analysed by hydrophobic Interaction Chromatography. This was carried out using column a MabPac HIC-Butyl, 5 μm, 4.6×100 mm column (Thermo, #882558, lot 01425138, serial nb 001303) with a MabPac HIC-Butyl, 5 μm, 4.6×10 mm Guard cartridge: (Thermo, #882559, lot 1425011). With a Mobile Phase A of 1.5 M (NH.sub.4).sub.2SO.sub.4, 25 mM NaPO.sub.4 (pH 7.4) and a Mobile Phase B of 80% 25 mM NaPO.sub.4 (pH 7.4), 20% CH.sub.3CN. The assay was run at 0.8 ml per minute and a column temperature of 25° C. A 10 μl sample load at 1 mg/ml was used for the analysis.

[2589] The HIC showed a distinct difference between the two ADCs, with Her-PL1603-App1 separating into multiple hydrophobic species, whilst Her-PL1603-App2 eluted as one more hydrophilic peak (see FIG. 4).

[2590] The increased hydrophilicity of Her-PL1603-App2 over Her-PL1603-App1 is prima facie surprising in view of the fact that Her-PL1603-App2 has considerably fewer sugar residues than Her-PL1603-App1 (compare FIGS. 2 and 3).

[2591] Sugar residues are very hydrophilic moieties, so the expectation is that increasing their number would increase overall molecule hydrophilicity. The fact that this is not the case here suggests a more complex interaction is occurring between the antibody, oligosaccharide, and drug-linker elements of the ADC.

[2592] In-Vitro Binding to Her2

[2593] Her2 is the cognate antigen of the Herceptin antibody. Binding of Her-PL1603-App1 and Her-PL1603-App2 was determined by ELISA. Maxisorp ELISA plates were coated with 0.5 μg/mL recombinant human Her2 at room temperature, before blocking with 3% BSA. Sample titrations were prepared in assay buffer (0.1% BSA/0.05% tween) between 66.6 and 0.016 nM in quartering dilutions. Samples were then incubated on the antigen coated plate for 1 hour. A mouse anti-human antibody conjugated to HRP was used for detection (Sanquin M1328) and incubated for 1 hour before washing and adding the detection agent, TMB for 10 minutes before stopping the reaction with HCl. Binding absorbance data was acquired on the Spectramax plate reader at 450 nm.

[2594] For comparison, Her2 binding was also assessed for ‘Her-C220’ [an unconjugated version of Herceptin in which 3 of the 4 interchain cysteines have been substituted for either V (in the heavy chain) or S (in the light chain)] and B12 [an unconjugated monoclonal antibody against the HIV-1 protein; used here as a control].

TABLE-US-00005 ADC/antibody IC50 (nM) Her-PL1603-App1 0.23 Her-PL1603-App2 0.38 Her2-C220 0.17 B12 No Binding

[2595] The two ADCs bound to Her2 with similar affinity.

[2596] In-Vitro Cytotoxicity

[2597] The in vitro cytotoxicity of Her-PL1603-App1 and Her-PL1603-App2 against Her2+ve N87 cells was determined. A “thaw and go” cytotox assay was used to determine the cytoxicity, N87 cells were taken from cryogenic storage and seeded to 5×10.sup.4 cells/mL (5×10.sup.3 cells/well) on an EDGE plate then incubated for a minimum of 2 hours at 37° C./5% CO.sub.2/absolute humidity. An 11 point, 1 in 4 serial titration of the test and control samples was prepared in duplicate from 500 nM to 0.4768 pM with a final negative control. The titrated samples were added to the EDGE plate containing cells and incubated for 5 days at 37° C./5% CO.sub.2/absolute humidity. Celltiter Aqueous One solution was added and the plate was incubated at 37° C./5% CO.sub.2/absolute humidity for a final time, before measuring absorbance at 490 nm using the SpectraMax plate reader.

[2598] For comparison, cytotoxicity was also assessed for ‘Her2×ADC’ [Her2-C220 conjugated to the PBD drug-linker SG3249 (Tesirine) at the C220 residue] and B12-C220-SG3249 [the B12 antibody conjugated to the PBD drug-linker SG3249 (Tesirine) at the C220 residue].

TABLE-US-00006 ADC IC50 (pM) Her-PL1603-App1 24.7 Her-PL1603-App2 21.7 Her2xADC 17.9 B12-C220-SG3249 2994

[2599] Her-PL1603-App1 and Her-PL1603-App2 were found to have similar cytotoxicity to each other and also to the benchmark Her2×ADC. Significantly less cell kill was observed with the non-Her2-binding B12 control ADC.

[2600] In-Vivo Efficacy

[2601] The in vivo efficacy of the Her-PL1603-App1 and Her-PL1603-App2 conjugates was measured in the breast cancer Her2+ve BT474 xenograft model. For comparison, in vivo efficacy was also assessed for ‘Her2×ADC’.

[2602] Female severe combined immunodeficient mice (Fox Chase SCID®, CB17/lcr-Prkdcscid/lcrlcoCrl, Charles River) were ten weeks old with a body weight (BW) range of 16.1 to 21.8 g on Day 1 of the study. On the day of tumor implant, each test mouse received a 1 mm.sup.3 BT474 fragment implanted subcutaneously in the right flank, and tumor growth was monitored as the average size approached the target range of 100 to 150 mm.sup.3. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:

Tumor Volume (mm3)=w.sup.2×l/2

[2603] where w=width and l=length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.

[2604] Thirty-six days after tumor implantation, designated as Day 1 of the study, the animals were sorted into groups each consisting of ten mice with individual tumor volumes of 75 to 172 mm.sup.3 and group mean tumor volumes of 119-121 mm.sup.3. On Day 1 of the study, drugs were administered intravenously (i.v.) in a single injection (qd×1) via tail vein injection. The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal. Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 1000 mm3 or at the end of the study (Day 59), whichever came first.

[2605] Results are shown in FIG. 5.

[2606] The minimal efficacious dose (MED) of Her2×ADC and Her-PL1603-App1 was >0.6 mg/kg, while the MED for Her-PL1603-App2 was determined to be 0.3 mg/kg.

[2607] Tolerability: Rat Toxicology Study

[2608] Her2×ADC, Her-PL1603-App1 and Her-PL1603-App2 were evaluated in a single intravenous dose rat tolerability study.

[2609] Male sprague-dawley rats (n=3/group) were dosed at 4 mg/kg with Her-PL1603-App1 or 2 & 4 mg/kg with Her-PL1603-App2 on day 1, with necropsy on day 21 following dosing. Bodyweights and food consumption were monitored frequently with in-life sampling for clinical pathology (blood on days 8 and 21) and repeated sampling for pharmacokinetics. At necropsy, macroscopic observations were taken with selected organs weighed and retained for possible histopathology.

[2610] Her2×ADC was evaluated at 1.5 mg/kg, single intravenous injection to male Sprague Dawley rats was associated with reduced overall body weight gain (overall bodyweight gain was 39% lower), associated with reduced food consumption. White blood cell numbers were reduced on day 8 (−61%), with evidence of recovery by day 21. At necropsy, reduced thymus, spleen, testes and prostate/seminal vesicle weights and increased adrenal gland weight were observed.

[2611] Her-PL1603-App1 was poorly tolerated at 4 mg/kg, resulting in early euthanasia 11 days after dosing in 2 out of 3 animals. Bodyweight gain was markedly reduced in these animals, with none of the expected weight gain after dosing. Several haematology parameters were markedly reduced on day 8 (reticulocytes (−93%), white blood cells (−86%) and platelets (−66%)), with no evidence of recovery.

[2612] Her-PL1603-App2 was clinically well tolerated at 2 & 4 mg/kg. Bodyweight gain was dose-dependently reduced (overall bodyweight gain was 55% lower at 4 mg/kg), consistent with reduced food consumption. Several haematology parameters were reduced on day 8 (reticulocytes (−52%), white blood cells (−68%) and platelets (−22%)), with evidence of recovery by day 21. At necropsy, dose-dependent reductions in thymus, liver and spleen weights and increased lung weights were noted, with two animals presenting with pale kidneys at 4 mg/kg.

[2613] The maximum tolerated dose (MTD) for Her2×ADC was 1.5 mg/kg (the highest dose tested).

[2614] The maximum tolerated dose (MTD) for Her-PL1603-App1 was lower than 4 mg/kg.

[2615] The maximum tolerated dose (MTD) for Her-PL1603-App2 was 4 mg/kg.

[2616] Therapeutic Index

[2617] The Therapeutic Index (TI) of the ADCs may be calculated by first determining the Human equivalent dose of the MED and MTD and then dividing the HED of the MTD by the HED of the MED, as shown below:

TABLE-US-00007 mg/kg MTD HED of HED of MED Rat MED MTD rat TI Her2xADC >0.6 1.5 >0.050 0.22    <4 Her-PL1603-App1 >0.6 <4.0 >0.050 <0.60 <<12 Her-PL1603-App2 0.3 4.0 0.025 0.60     24 MED: Minimal effective dose in mice HED: Human equivalent dose MTD: Maximal Tolerable dose TI: Therapeutic Index

[2618] Her-PL1603-App2 exhibits a Therapeutic Index of at least twice that of Her-PL1603-App1.

[2619] Her-PL1603-App2 exhibits a Therapeutic Index of about 6 times that of Her2×ADC.

[2620] Pharmacokinetics (PK) of Her-PL1603-App1 and Her-PL1603-App2 in Rats

[2621] Plasma samples of rats dosed with a single dose of 2 and or 4 mg/kg of Her-PL1603-App1 and Her-PL1603-App2 and samples were taken 1, 3, 6, 48, 72, 168, 336 and 480 h after dosing. The samples were analysed for total human IgG and PBD conjugated IgG as described in Zammarchi Blood vol 131 (10), 1094-1105 2018.

[2622] FIG. 6A shows comparable PK profiles of Her2-PL1603-App1 for total IgG and PBD-IgG at 4 mg, which shows the conjugate is highly stable.

[2623] FIG. 6B shows comparable PK profiles of Her2-PL1603-App2 for total IgG and PBD-IgG both at 2 and 4 mg, which shows the conjugate is highly stable.

[2624] Further Comments on Properties

[2625] A further advantage of ‘Approach 2’ as described above in Examples 1-4 is that it is easier to control the DAR at 2. In earlier approaches employing an intact glycan, it was more difficult to control the DAR at 2, necessitating careful control of reaction conditions.

[2626] In addition, Approach 2 abolishes Fc(gamma) receptor activity which is an advantage for a number of ADC applications.

Example 5: One-Pot Remodelling

[2627] The enzymes used were produced in CHO cell as recombinant proteins at Evitria (https://www.evitria.com) and purified in-house at ADC-Therapeutics (Sequences shown below as SEQ ID NO. 4 (ST6Gal1), SEQ ID NO. 5 (Beta4Gal), and SEQ ID NO. 6 (EndoS)). Other reagents were: [2628] UDP-Galactose (Merck Life Sciences UK Limited. Cat: U4500-25MG) [2629] CMP-Sialic acid (Merck Life Sciences UK Limited Cat: 5052230001) [2630] CIAP (Calf Intestinal Alkaline Phosphatase) (ThermoFisher Cat: 18009019)

[2631] Initial experiments focused on a one pot reaction, ensuring that the Endo S and Beta 4GAL (β-1,4-galactosyl transferase) resulted in full galactosylation. To this end three aliquots were set-up under the following conditions: [2632] 1. 2 mls of antibody, buffer swapped in 50 Kd Mw Amicon into 50 mM MOPS/20 mM MgCl2 and adjusted to 10 mg/ml (˜1.8 mls) [2633] 2. STGA6 & Beta-Gal buffer swapped into 50 mM MOPS/20 mM MgCl2 and quantified by OD280 [2634] 3. 3×500 ul Aliquots of the Ab created (5 mgs of Ab in total) [2635] 4. To all Aliquots 25 ug of Endo S added (0.5%) [2636] 5. To all Aliquots 5 ul of CIAP per 1 mL Ab @10 mg/mL (ie 2.5 uL) [2637] 6. To all Aliquots add 50 ul of 300 mM UDP-Galactose=10 mM (dissolved in 150 ul MgCl2/MOPS buffer) per 1 mL Ab @10 mg/mL added (50 ul/ml) (Merck Life Sciences UK Limited. Cat: U4500-25MG) [2638] 7. To all Aliquots beta4Gal was added to 2.5% (125 ug/5 mg of Ab) [2639] 8. To Aliquots 2&3 add a further 2.5 ul CIAP [2640] 9. Add to aliquot 2&3 add CMP-Sialic acid to 4 mM (from 25 mM solution) ie 80 ul in 500 ul (Merck Life Sciences UK Limited Cat: 5052230001) [2641] 10. Add to aliquot 2&3 ST6GAL1 to 5% ie 250 ugs in 500 ul [2642] 11. Samples incubated for 36 hours at 37° C. [2643] 12. A further 5 ul CIP Aliquot, 50 ul UDP-Galactose (dissolve in 150 ul of MOPS/MgCl2) & 33 ul Beta4 Gal added to Aliquot 3 [2644] 13. All aliquots incubated for a further 24 hrs [2645] 14. Samples purified up using Protein A spin columns (Thermo) and quantified by OD280.

[2646] The three aliquots were analysed by LCMS and showed that the Aliquot 1, Endo S and Beta4Gal treated, had 100% galactosylation, Aliquot 2, Endo S/Beta4Gal/ST6Gal1, showed 92% Galactosylation with 34% sialylation and Aliquot 3, Endo S/Beta4Gal/ST6Gal1 plus additional Beta4Gal/UDP-Galactose showing 100% galactosylation and 35% sialylation.

[2647] The data indicated that additional Beta4Gal/UDP-Galactose addition was not needed and that the first two steps of the process could be a one-pot reaction.

[2648] The degree of sialylation of 35% was not considered sufficient, and potentially caused by the “poisoning” of the reaction by CMP. To attempt to overcome this a second set of reactions were set-up where a similar protocol was followed, except after 36 hours, one sample had additional ST6Gal1 and CMP-Sialic added and left for an additional for 36 hours at 37° C. The control sample Endo S/Beta4Gal, was incubated for the same length of time as the Endo S/Beta4Gal/ST6Gal1+A ST6Gal1/CMP-Sialic acid sample. The samples were again purified via Protein A chromatography and analysed by LCMS.

[2649] As before the, the one pot reaction of Endo S and Beta4Gal resulted in 100% galactosylation, the reaction with the additional ST6Gal1 and CMP-Sialic acid showed 88% sialylation, compared to the previous 35% incorporation, showing that potential “poisoning” by the CMP can be overcome by the addition of additional ST6Gal1 and CMP-Sialic acid and that the process is able to be run as a one-pot process without additional clean-up between stages.

TABLE-US-00008 SEQUENCE LISTING PART OF THE DESCRIPTION SEQ ID NO. 1: MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQS VSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKY KVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWG RCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRF LKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWEL WDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMG AYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC SEQ ID NO. 2: MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQ GGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVP HTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIII PFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTC FVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFP NNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKE TMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS SEQ ID NO. 3: MDKHLLVKRTLGCVCAATLMGAALATHHDSLNTVKAEEKTVQVQKGLPSIDSLHYLSENSK KEFKEELSKAGQESQKVKEILAKAQQADKQAQELAKMKIPEKIPMKPLHGPLYGGYFRTWH DKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYSLFWKELATKHVPKLNKQGTRVIRTIP WRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDVEHDSIPKVDKK EDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTYMADKNPLIERGAPYINLLLVQVYGSQ GEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYMIGFSFYEENAQEGNLWYDINSRKDE DKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAIDRDGVAHQPKKYAKQKEFKDATDN IFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALREAVMAQVGTRKGDLERFNGTLRLDN PAIQSLEGLNKFKKLAQLDLIGLSRITKLDRSVLPANMKPGKDTLETVLETYKKDNKEEPATIP PVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLEKVDISGNKLDLAPGTENRQIFDTMLS TISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLPVANEKVDLQSQLLFGTVTNQGTLIN SEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYTVKVTDSTLGTTTDKTLATDKEETYK VDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVIGGSADPVNARKVFDGQLGSETDNI SLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTNKPIQEASLQIFNIKDYNLDNLLENPN KFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWRVVFDTKGDRYSSPVVPELQILGYPL PNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMALETSLNSKIFDVTAINANAGVLKDCI EKRQLLKK SEQ ID NO. 4: HHHHHHWSHPQFEKGGGSGGGSGGSSAWSHPQFEKFQVLKSLGKLAMGSDSQSVSSSS TQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYK GPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVV SSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSL YNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQ EISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPL LYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC SEQ ID NO. 5: HHHHHHHHHHRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQ PRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELV AKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVIN QAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKF GFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVG RCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS SEQ ID NO. 6: EEKTVQVQKGLPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAK MKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYS LFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDE YVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTY MADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYM IGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAI DRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALRE AVMAQVGTRKGDLERFNGTLRLDNPAIQSLEGLNKFKKLAQLDLIGLSRITKLDRSVLPANM KPGKDTLETVLETYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLE KVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLP VANEKVDLQSQLLFGTVTNQGTLINSEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYT VKVTDSTLGTTTDKTLATDKEETYKVDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVI GGSADPVNARKVFDGQLGSETDNISLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTN KPIQEASLQIFNIKDYNLDNLLENPNKFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWR VVFDTKGDRYSSPVVPELQILGYPLPNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMA LETSLNSKIFDVTAINANAGVLKDCIEKRQLLKKHHHHHHHHHH SEQ ID NO. 7: FQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSK NLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSE WEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVG TKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRK LHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKT DVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC SEQ ID NO. 8: RDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVV DSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGG RYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLL NVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGG VSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKK NEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS SEQ ID NO. 9: EEKTVQVQKGLPSIDSLHYLSENSKKEFKEELSKAGQESQKVKEILAKAQQADKQAQELAK MKIPEKIPMKPLHGPLYGGYFRTWHDKTSDPTEKDKVNSMGELPKEVDLAFIFHDWTKDYS LFWKELATKHVPKLNKQGTRVIRTIPWRFLAGGDNSGIAEDTSKYPNTPEGNKALAKAIVDE YVYKYNLDGLDVDVEHDSIPKVDKKEDTAGVERSIQVFEEIGKLIGPKGVDKSRLFIMDSTY MADKNPLIERGAPYINLLLVQVYGSQGEKGGWEPVSNRPEKTMEERWQGYSKYIRPEQYM IGFSFYEENAQEGNLWYDINSRKDEDKANGINTDITGTRAERYARWQPKTGGVKGGIFSYAI DRDGVAHQPKKYAKQKEFKDATDNIFHSDYSVSKALKTVMLKDKSYDLIDEKDFPDKALRE AVMAQVGTRKGDLERFNGTLRLDNPAIQSLEGLNKFKKLAQLDLIGLSRITKLDRSVLPANM KPGKDTLETVLETYKKDNKEEPATIPPVSLKVSGLTGLKELDLSGFDRETLAGLDAATLTSLE KVDISGNKLDLAPGTENRQIFDTMLSTISNHVGSNEQTVKFDKQKPTGHYPDTYGKTSLRLP VANEKVDLQSQLLFGTVTNQGTLINSEADYKAYQNHKIAGRSFVDSNYHYNNFKVSYENYT VKVTDSTLGTTTDKTLATDKEETYKVDFFSPADKTKAVHTAKVIVGDEKTMMVNLAEGATVI GGSADPVNARKVFDGQLGSETDNISLGWDSKQSIIFKLKEDGLIKHWRFFNDSARNPETTN KPIQEASLQIFNIKDYNLDNLLENPNKFDDEKYWITVDTYSAQGERATAFSNTLNNITSKYWR VVFDTKGDRYSSPVVPELQILGYPLPNADTIMKTVTTAKELSQQKDKFSQKMLDELKIKEMA LETSLNSKIFDVTAINANAGVLKDCIEKRQLLKK