US 20150250896A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0250896 A1 Zhao (43) Pub. Date: Sep. 10, 2015

(54) HYDROPHILIC LINKERS AND THEIR USES Publication Classification FOR CONUGATION OF DRUGS TO A CELL (51) Int. Cl BNDING MOLECULES A647/48 (2006.01) (71) Applicant: Yongxin R. ZHAO, Henan (CN) Ek E. 30.8 C07D 207/216 (2006.01) (72) Inventor: R. Yongxin Zhao, Lexington, MA (US) C07D 40/12 (2006.01) C07F 9/30 (2006.01) C07F 9/572 (2006.01) (73) Assignee: Hangzhou DAC Biotech Co., Ltd., (52) U.S. Cl. Hangzhou City, ZJ (CN) CPC ...... A61K47/48715 (2013.01); C07F 9/301 (2013.01); C07F 9/65583 (2013.01); C07F (21) Appl. No.: 14/432,073 9/5721 (2013.01); C07D 207/46 (2013.01); C07D 401/12 (2013.01); A61 K3I/454 (22) PCT Filed: Nov. 24, 2012 (2013.01)

(86). PCT No.: PCT/B2O12/0567OO Cell(57) binding- agent-drugABSTRACT conjugates comprising hydrophilic- S371 (c)(1), linkers, and methods of using Such linkers and conjugates are (2) Date: Mar. 27, 2015 provided. Patent Application Publication Sep. 10, 2015 Sheet 1 of 23 US 2015/0250896 A1

O HMDS OSiMe 2n O Br H-B-H HPC 3 2 COOEt essiop-\5. E B to NH 120 °C, 2h OsiMe3 J 50 °C, 2h eSiO OEt 120 oC, sh 1 2 3. 42% from 1 Bra-11a1'oet - Brn 11-1 or a 1-1 or ÓH 140 °C ÓEt ÓEt 4 5 6 - --Messio. 8 B1a-Br aus 20 cc, hP-1}^-'ot Br1-Y. OEt 3. OEt 7 s a OH OEt 8

S O. O. O. 1). 1N NaOH EtOCSSK ka. He He Moss-a-NM-n1'oet 2), pH 7.5 EtOH OH OEt PySSPy 9

(lisN S1 n^^ --\ou9 -->NHS/EDC ÉllsN S1 Y}-\--\'o-'o' OH OH OH OH O 10 11 Drug-SH, pH 4-7, then O O O ,noodles, or Proteiroteins "S-S-1-N-N-NOH OH H n = 1 - 20 12 Fig. 1. The synthesis of a di-phosphinic acid linker.

Br1Nile -/n aus,OEt -CH(OEt), > Br-al-M1'oetQ Q S-E-M1-\'oet OH OEt OE OEt is OEt OEt 14 2, 120 °C, 2h O O. O. O. EOCSSKw B1a-rB B^{-\-E-MB-n1'oetOH OEt OEt EtOH S 1). 1N NaOH O O. O. O.

Fig. 2. The synthesis of a tri-phosphinic acid linker Patent Application Publication Sep. 10, 2015 Sheet 2 of 23 US 2015/0250896 A1

Q 9 O O CH(OEt) O O O O Br-a-ra--M. 1Soet --> B^-\l-MB-n-'oet OH OEt OEt is OEt OEt OEt 19 2, 120 °C, 2h O EtOCSSK Br1N1 - Bru-N-Pro-YrOH oEt OEt OEt OEt EtOH S 1). 1N NaOH 1oss?\-ra-rá-B-B-B-4-1. OEt Her2), pH 7.5 OH OEt OEt OEt PySSPy

O O O I I O ÖH OH OH OH 23 O

Fig. 3. The synthesis of a tetra-phosphinic acid linker

Br1a--M.tallas OE n-i-M}^' of BizshO O Na2CO OEt OEt 13 OEt OEt - O --> DMF, 50 °C

O O 8 NHC O O NHSFEDC pAZS-\-\-rottO OEt OEt O -reflux o- Nr-uo"ÖH oH HerDMA 24 25 O O O-N Drug-SH Drug O O e s Cr-y-O OH OH O pH 4 ~7.5 Cr-Y- 26 Antibodies or Proteins Drug O NH mAb pH 5.5-8.5 -N1\-1\--/n O OH OH O n = 1 - 20 Drug-SH 28 9 O O O O O O s Antibodies H O OH OH O pH 6.5-8.5 O OH OH O n = 1 - 20

Fig. 4. The synthesis of a di-phosphinic acid linker.

Patent Application Publication Sep. 10, 2015 Sheet 4 of 23 US 2015/0250896 A1

Hs-a-o-O BY-1a-Br Bra-sa-to- 30% H.o2V2 Bra-9-n-to 51 DIPEA, 45°C 52 HOAc/KMnO(cat) 53

0.5 M NaOH { Drug-SH, then He -e as mAb or protein O Drug-YS 56 O

O O O O Hs-n-to- Br1n 1B BMs-n-o 30%KMO HOV2 B-1S-1 51 DIPEA, 45°C 57 nu 58 HOA O O O O O HSAC M Y O.S M NaOH SS NHS/EDC DIPEA AcS S O PySSPy,w pH 7.2 NS-S2N, MNS OH HeDCM O 59 O 60

O Drug-SH then s^^ mAh or proteina Drug O

O O Ms-\-y -eBr1N1Br, Br-1-s^^s^1/ 64 DIPEA, 45°C 65 30% HO R. HSAe it all it HOA - Br^1-a/n^1 HeDPEA Aesn1-a/n^1 / O O 66 O O 67

r O O O 0.5 MNaOH wa I NHS/EDC PySSPy,SMNne pH 7.2 (CaMaMa'ohNS-S NSEge

-e then mAb I'll S N or protein

Fig 6. The synthesis of mono-, and di-sulfonyl linkers Patent Application Publication Sep. 10, 2015 Sheet 5 of 23 US 2015/0250896 A1

O MSHw O Br1N1 Br O Br O1 Y - HS-sa-to- e Br1-sa/s-a-o- 51 DIPEA, 45°C 71 72 O O newN"- Brain/S-\'osiO 9 OS M NaOHNag s-Ma-onif Q NHS/EDC 18-crown-6 O 73 O 74

Drug-SH, then O O O -mAb or-o- protein Drug SMS-\-S^ O 0 n = 1 - 20

-e Br M S al O -30% H.O.> Br 1-allS O1 S1 DIPEA, 45°C 77 A. 78

HSA O O OS M NaOH C O O NHS/EDC -eDIPEA, 60°C As YS o? PySSPy, pH 7.2 NS-S a o- DCM D O O 80

Drug-SH,S thenT- Drug S S. Ms N mAb or protein O H 8a n = 1 - 20 B-1o-S-HS/N HS-s^1O o? BY-1N1 On AsaoO S1 DIPEA, 45°C 71 DIPEA, 45°C 82

O O O O 30% HO HSAc HOAc O 83 O O

0.5 M NaOH 1S O O O NHS/EDC PySSPy,-e pH 7.2 ClSNs-s NM-ul-\'oh - DM > O O 85

O O O Drug-SHES- fS Ys 19-v then mAh Drug N or proteino O 87O Hn

Fig. 7. The synthesis of mono-, and di-sulfonyl linkers Patent Application Publication Sep. 10, 2015 Sheet 6 of 23 US 2015/0250896 A1

(ho^-oh - HO-o-o-e-C'sO Tso-hoa-o-O 88 m = m1-30 aroAcSH Acs-hoa-o-O in0,5 N NaOH Bry S \-hoo-k 30%HOA H2O, yr. 91 B1a-Br 92

y O O O O 1N1-S EtOCSSK'e MY s^-Sa-ho-ohs^-in-ho OH -o-SSNs Br i-hoon EtOH O O m PySSPy, pH 7.2

O O O O O

-- (ys, Min-ho-oh SSPS- (ys, Min-ho-o- N s & 'm DMA S

O O O O Br1-in-ho-oh FNN-a-ho-ohEtN y NSFPSNHS/EDC O "97 O m. DMA O O O azyan O

Br^-sa-ho-o- YoDMF 7 Az O 100 O 101

30% HO O O 9 O O O. HOA, 60°Ca UN-NSy, r-i-hoon NHS/EDCDMA NYi-hoo O2 O 103 m = 1 - 30 O

y HS101al O O1a-'-HO CB/PPh.4 3 Br. Br He Br aul Y ( O l, - DCM as OY pp. Ytors O 104 m = 1--30 105 106

30% HO, SRP R ww S R O HOA > Bra-to-vaul-EtOCSSk O to Mo'ss-to-vy-\'o m-1 107 m 108

'6) R NHS EDC pHpsp- 7.2 SNSSS Vtory-longm-1 DMA 110 m = 1 - 30 Fig. 8. The synthesis of mono-Sulfonyl linkers containing polyethylene glycols. Patent Application Publication Sep. 10, 2015 Sheet 7 of 23 US 2015/0250896 A1

O SH O Br B Tso-foot- HS-EtN Hsn-sa-ho-o- BryllO C-S-foot 90 m = 1 -30 11 EtN, 50°C 12

30% HO,SCS Br R O EtOCSSK J.S Oif 9 O 0.5 M NaOH HOA o'oh to Mo su/~\hoon PySSPy, 113 Ö O 114 pH 7.2 ( VS Qal O NHS/EDC FN 's-?s-in-hoon's 5 O 115 C. A.A ... is ...... O Sinho'ohO O ES-N-\-shoot NHSEDC 117 ", DMA

O He NHS/EDC HOAc, 800C ONvvi{oons O DMA 121

O CBr/PPh B Br HSMSO M ( HO1n-NH DCM4f 13 OYi 138 p 104 m = 1--30 123 DIPEA 30% HO Q9 Q9 Q EtOCSSK S OW. V.O O -eHOAC Br-to SS-N-au-V// Y.EtOH S. M.O s/ton) SyllQ-M1 m-1 f m-1 125 126

0.5 M NaH2 O O. O. O '6)Pyssey, SN’s Svory-roll W pH 7.2 m-1 O 127

NHS/EDC/DMA

Fig. 9. The Synthesis of di-sulfonyl linkers containing polyethylene glycols. Patent Application Publication Sep. 10, 2015 Sheet 8 of 23 US 2015/0250896 A1

O O O O 1nSS/NS.2 O O O O. O HaskOBn Br.120 °C, 5h Bill Bruin?O OBnn'o-e- N-S^^*o-8 ÖBn 129 13 131

S O O O EtOCSSK 20%oV CHSOHWDCM 13 SV3 O OBn PySSPy, pH 7.2

O allO O 20% CHSOH 9 O O O OBn 136 M 8 OH 137 DMA

139 m= 1 - 20

O O NaH/DMF RO Q Q Azisi,15 O thenen so,9 AlSz.--SabaoO O OBn 140 20then CHSoHDCy pH 7, 100'C

O B-?tO NHS/EDC N1-\- OH - DMA e. O O OH 141

O O O Braal-S O HP(OSiMe3); (2) Br 9 EtOCSSK 8 143 1200Cthen Brt- NMRMS-1OH Ö 144of - - O S 0.5 M NaOH a O O O NHS/EDC MM-SMS-\oy is ()N S SVM M-n-oh DMA 145 pH 7.2 OH 146

N O H n = 1-20

Fig. 10. The synthesis of sulfonyl linkers containing a phosphinic acid. Patent Application Publication Sep. 10, 2015 Sheet 9 of 23 US 2015/0250896 A1

O. O O O Br1nSS/nB(S2 9 O O O O O likIn 129 on's9 B ^^so-130 N-S-MMoO OBn

HO-G)."Sf-(y-o-v-SM-no-e-E's Q 25%W CHSOHk NaCO/DMF O OBn CHCl O -(y-o-vis M-\'os-'-(y-o-vis/MolNHS EDQ o 0 O OH DMA O OH O 150 151

O O O O -Antibodies to OS-(S)- O1VS/N N mAb Drug?'NHNH,l or Proteins OI OH n = 1 - 20 pH 4-5

N H O ÖH H n = 1 - 20 153

9 O O O 9 O O O S it Drug. NHNH2 O A.)-cy O w - -o-vi?ion150 OH pH 4~5 Drun-H 154 -SM-\'onO OH

Drug N-N lis/-S O-N Nee-O----.DMA H O OH 155 O

Antibodies O }-Cy- O O O mAb or Proteins Drug'N- -Saha' H 6 OH Hn = 1 - 20 156 Fig. 11. The synthesis a hydrophilic linker containing contain a phosphinate, a Sulfonyl and a ketone groups that can be used for the conjugation of a drug to an or a protein via a hydrazone bond. Patent Application Publication Sep. 10, 2015 Sheet 10 of 23 US 2015/0250896 A1

20%HO HOAC

X----hoo-k EtOCSSK iXS CH-OH Q S

1). NaOH 2 O O 20%HC - a if O -e 2),PySSPy pH 7 -X---i-ho'?O Dioxane O 160 m = 1 ~ 30

O O NHS/EDC N s-s-s--i-hoon 83% O 161

2 N s-X---i-ho O-N Drug-SH"S- pH 4-7

y O. O. AntibodiesIntibodies Drugs-- 31a- inho No -N.) -eor Proteins 163 m = 1~ 30

O O mAb

Drus-s’(~~ in-hol n = 1 - 20 m = 1-30 164 Fig. 12. The synthesis of hydrophilic linkers that contain a disulfonyl, polyethylene glycols, a very hindered pyridyldisulfide group and a reactive carboxylic acid ester via substitution of 1,3-dibromo-3-methylbutane. The linker is used for the conjugation of an antibody or a protein via a hinder disulfide bond. Patent Application Publication Sep. 10, 2015 Sheet 11 of 23 US 2015/0250896 A1

O. O O O O Br1n-SS/nSS2 O O O O O H-i-kOBn BB's120 °C, 5h Bruss/B-\'o-O OBn N-S^^*o-8 ÖBn 129 130 131

O O O O O O NaN 25% CHSOH --> N^-in/n: o-e- out 13SV3 N^-in/n OH O OBn CHCl, O OH 165 166 Drug-22 Dru9. O al NHS/EDC - HO- e S -e pH 4-7 Nr1N/11 oh DMA NN O OH 167

Antibodies -e or Proteins

la 20%HC HN \-in/ni O Dioxane O OH 170

O

H O OH 172

Antibodies -o- or Proteins

Drug SM-vi-VN-\ 8 --5 l Ab H O175 ÖH

Fig. 13. . The Synthesis of hydrophilic linkers containing a phosphinate and a Sulfonyl groups that are used for the conjugation of drugs to a cell binding molecule either via a triazole or via a thiolether bond. Patent Application Publication Sep. 10, 2015 Sheet 12 of 23 US 2015/0250896 A1

H-B-HO HMDs HP OSiMes Br 1n 16Br( e 'ha-a-O CH(OCH) Ö NH 120°C, 2h OSiMe3 120 oc, sh 500 psi. 2 OH 176 120 oC

O HS al O Br1a11a1 n/NS 52 O B 1-1. ÖCH HAHe raM--MSn/s O1 3 177 EtN OCH 178

20%HO, HSAs KMnO/HOAc Bra-a--MSM-N-o-5 --Et OCH O 79

O O Q O O O O AeSQM-NMSM-o1 HOSnMe,N.S 85°C HS-M-NMSM-a-oh OCH O 180 CICHCHCI OH O 18

HOSnMe, 85°C O He O ( O O CICHCHCl then l NM B-MSM-n-oh He H/Tol. 120°C O O

Fig. 14. The synthesis of hydrophiliclinkers that contain a disulfonyl, phosphinate, a pyridyldisulfide group, a polyethyleneglycol (PEG) chain and a reactive carboxylic acid ester. The linkers are used for the conjugation of a cell binding molecule via cell thioether bond Patent Application Publication Sep. 10, 2015 Sheet 13 of 23 US 2015/0250896 A1

- a SH 187 3 188 EtN

O Br/N51\-Br O "so-s^1o-'o?-189 OBn 190 Bra/\s-ro-'okOCH 191 9 O -7 NH- 7 -- N-/Sys-sr-ro-'okO 1).(). HO/HOAHo Hoag NaCO/DMA O OBn 192 H 2). HC/Tol, 120°C

O O R" Drug-C=O C N-/- min11o-NHHCI g O OH 5 EDC/HCl(0.1 eq) 193 R" = H. CH, Et, Pr, Bu, Ph,

R" O O O Antibody + DTT DrugX2 ^^^n/\-N6 ÖH O -e-N-mAb-SH 194 O R O mAb D X2 MS^j\\-N O OH ; S n = 1 ~ 20 R" = H. CH, Et, Pr, Bu, Ph, 195

O O Antibody + DTT N-/\in\S^10-NHHC N-mAb-SH OH O 6 He

O f O O Ab R" "y^^^\-l Drug- =O & O ÖH sn-1-20 EDC/HCl(0.1 eq) 196 R" = H. CHEt, Pr, Bu, Ph,

yy O O O R mAb Drug eat W OMS^n/N-\-N 5 ÖH S O O n = 1 ~ 20 R" = H. CH, Et, Pr, Bu, Ph.

Fig. 15. The Synthesis of phosphinate and disulfonyl-containing linkers that contains an alkoxylamino and a maleimido Substituent, enabling ketone or aldehyde containing drug to link to an antibody via a thioether and an alkoxime bond. Patent Application Publication Sep. 10, 2015 Sheet 14 of 23 US 2015/0250896 A1

B1-B HSSSH 1n-Sat1ns-n-Br HONHBoe 197 EtN 198 EtN 9 O. S O A/NH O O O Brasa- Yo-No-K O 7 N/N MYo-'o? 199 H NaCO/DMA 's 200 H 1). HO/HOAc O O. Og Drug-C=OR" 2). HC/Tol, 120°C C. ^^i-ro-NH.HC EDC/HCl (0.1 eq) f) 201 R" = H. CH, Et, Pr, Bu, Ph,

O O Antibody + DTT S n^NC N-mAb-SH 6 O O -e 20 2

O O R )-N mAb Drug e ro-Asn-n/N S O O n = 1 ~20 R" = H. CH, Et, Pr, Bu, Ph. 203O

O O Antibody + DTT

I N-MiYro-NH.HCI -HeN- mAb-SH O O 2O1

O HN O Ab Drug--C=O O Sn= 1 - 20 EDC/HCl(0.1 eq) 204 R" = H. CH, Et, Pr, Bu, Ph, O O R )2N mAb Drug? ro-/- $n-in/N S 5 O CY n = 1 ~ 20 R" = H. CH, Et, Pr, Bu, Ph, 203

Fig. 16. The synthesis of disulfonyl-containing linkers that contains an alkoxylamino and a maleimido Substituent, enabling ketone or aldehyde-containing drug to link to an antibody via a thioether and an alkoxime bond. Patent Application Publication Sep. 10, 2015 Sheet 15 of 23 US 2015/0250896 A1

O SH O Br O B O1 HS 1n HS-1-s^n-to- HeBr1N1 Br1-sa/ns^1 o? 51 DIPEA, 45°C 52 DIPEA, 45°C 71 Ce(BrO) O O HSA O O O. HO/CHCN Br1-SaM-1soS. all Y DipAc AcS1N1 SMNM-1No 205 206 0.5 MNao S O 9 NHS/EDC Pyssey,He pH, NS-SY SMY^ on Hedom

HS O O1 a rg bla^s^-o-O 5 DIPEA, 45°C 76 HSA - As \ O O 0.5r MNaog Cn Oa O NSPS DIPEA, 60°C Psssp. pH 7.2 NS-S 211 oH

O O Drug-SH, then S mAb or protein Drug ---ar213 ~ 20

O 1n 1 O 1a O 51 DIPEA, 45°C S1 DIPEA, 45°C 82

Cc(BrO) O O O O O O HeHO/CHCN -na-a-ay/ HSA Aes N-al M^. 7 214 25

O O O.S M NaOH S O NHS/EDC - D M-N-oh He PySSPy, pH 7.2 NS-S 216 DMA

or protein 218

Fig. 17. The Synthesis of Sulfoxide-containing and disulfoxide-containing linkers that are used for the conjugation of drugs to a cell binding molecule via a disulfide bond. Patent Application Publication Sep. 10, 2015 Sheet 16 of 23 US 2015/0250896 A1

9 O O O A7 NH O O R O Q MeSnOH3. Br-i-S^'oS. Mill Y - - 7 \i-S-\'o-i? CICHCHQEl O 72 NaCO/DMA O O 219 1200C O 2 NHSEDC O o O Q / N^-SSM-9-1O oh DMA S-CN-N-SNMM-NYs O O O 220 O O 22 O O H COOH Fmoc-N OH NHS/EDC Fmoc N- ONN H.NNMNH, H 5 DM O -O-> 222 223 O NaHCO/HO/THF

H O H. O. OH rose','onH 5 O H.N-()He ' ros.'H -- 224 YNN,H EDC/DMA or225 M8H NH

H O OH --eEtNH ''2 ''-O-H 221 NMP O RM N-4 -NMP Re

O '227 N1,N- DIPEA H NH O O O O H. O. O / R" / Nra-SMS-1 N-N-()-\ u NH-Drug O II NH O H O b1 o-Q)-No, --DIPEA O O N^N- R = H, Me, Et, P 228 H NH B Ph IVle, l, 9 O O H. O. O Y1-a/n N N-NNH o1YN-Drugl mAb - DTT O H O RM O w N- mAb-SH O N-4 R" H-e 229 H NH O R O Q H O mAb N1-S-MS-N-N-N-N O Drug S O NH C E. H. O O1 YN1w O O N/nN- R'' 230 H NH n = 1-20

Fig. 18. The synthesis of a disulfonyl-containing linker (221) that used for conjugation of amine-containing cytotoxic drugs to an antibody via the Val-Cit-PABC linkage. Patent Application Publication Sep. 10, 2015 Sheet 17 of 23 US 2015/0250896 A1

": Q O OAc N O HO O O O Ab

N S H C----,O n = r?20

Y? H. O H. O. OAc O HdoH

Ab r. AtSM YN O N *N OH a O O S W Y- WW S H O n = 1-20 19-b). ADC structure of a tubulysin analog through a disulfonyl linker. OH H 92 OAc O O M N Nu- O NY N 2 ? N H S1\--Nu Ab 6 N S H OH HN w O O n = 1-20 19-c). ADC structure of a tubulysin analog through a Sulfonyl linker. mAb O Oif a QWYN/ O O N 1-SS H. (CH HO H HO O EN Suk ser S OCH CH. O. 'HC S Hso HC

H3C7 No O OCHOCHs (bh 2's"Ho1 O Yo HOH3CO OH HCNO HCO A7 n = 1 - 20 19-d). ADC structure of a calicheamicin analog through a hydrophilic linker (via hydrazone and disulfide linkage).

n = 1 - 20

19-e). ADC structure of a calicheamicin analog via a hydrophilic linker (disulfide linkage). Patent Application Publication Sep. 10, 2015 Sheet 18 of 23 US 2015/0250896 A1

mAb

3

N HCOZ7 ". NAN7 n = 1 - 20

O O a- o mAb lax S1 S N O

mAb h

n = 1-20 19-i). ADC structure of a Maytansinoid via a hydrophilic linker (via alkoxime linkage). Patent Application Publication Sep. 10, 2015 Sheet 19 of 23 US 2015/0250896 A1

Q-N O 9 O O. HN HN 1My H \-us--u N mAb N. NH2 O. O H

O O ON n = 1-20 19-j). ADC structure of an auristatin analog (monomethyl auristatin E (MMAE)) through a hydrophilic linker (thiOcther and citrulline-valine peptide linkages). 9 H O N O O O HN1- \-N S n1Ng N mAb HN 1N H H N-NH. O O O h' O H N N O CH, O 1- bcH, HC6 O1 OH n = 1-20 19-k). ADC structure of an auristatin analog (monomethyl auristatin F (MMAF)) through a hydrophilic linker (thioether and citrulline-valine peptide linkages). 9 H O HN 1-N Y1\-N S O O O mAb HN 1a O Yn-vi-\'N N-NH,' ÖH O O H O o-NN N N N ÖH, O /S ÖCH, O H3CO 0 oyoc/- 1-2 19-l). ADC structure of monomethyl auristatin F-OMe (MMAF-OMe) through a hydrophilic linker (thioether and citrulline-valine peptide linkages).

1-N w O O O Ab HN 'NICrusX--N-aux"

O O H

O Ra, O 1. N ocH O H3CO O O CCOCH / n = 1-20 19-m). ADC structure of monomethyl auristatin T-OMe (MMAT-OMe) through a hydrophilic linker (disulfide and citrulline-valine peptide linkage) of this patent. Patent Application Publication Sep. 10, 2015 Sheet 20 of 23 US 2015/0250896 A1

mAb n = 1 - 20

19-in). ADC structure of a doxorubicin compound via a hydrophilic linker (disulfide linkage).

mAb

n = 1 ~ 2.0

19-O). ADC structure of a doxorubicin compound via a hydrophilic linker (alkoxime linkage).

mAb

n = 1 - 20

19-p). ADC structure of a daunorubicin via a hydrophilic linker (alkoxime linkage). ^2 M -CIO O N mAb J.9 -- CC? H S O HOI H6 1)-N-Ng. , O n = 1-20

9-q). ADC structure of a CC-1065 analog through a hydrophilic linker (hydrazinecarboxylate linkage) of this patent. Patent Application Publication Sep. 10, 2015 Sheet 21 of 23 US 2015/0250896 A1

n = 1-20 19-r). ADC structure of a pyrrolobenzodiazepine dimer prodrug through a hydrophilic linker (hydrazide linkage) of this patent.

Ab

n = 1-20

19-s). ADC structure of a pyrrolobenzodiazepine (tomayimycin) dimer prodrug through a hydrophilic linker (disulfide bond linkage) of this patent.

n = 1-20

19-t). ADC structure of a pyrrolobenzodiazepine dimer prodrug through a hydrophilic linker (disulfide bond linkage) of this patent.

n = 1-20

19-u). ADC structure of a pyrrolobenzodiazepine dimer prodrug through a hydrophilic linker (alkoxime bond linkage) of this patent. Patent Application Publication Sep. 10, 2015 Sheet 22 of 23 US 2015/0250896 A1

HOS SOH O 3 N RS 3 M mAb H N Me MeO NO=N-o OS N O O n = 1-20 19-v). ADC Structure of a pyrrolobenzodiazepine dimer prodrug through a hydrophilic linker (alkoxime bond linkage) of this patent. loc-sir-I-Aa a A a y-No?^^S" . . . . /." mAb O O n = 1-20 19-w). ADC structure of a pyrrolobenzodiazepine dimer prodrug through a hydrophilic linker (alkoxime bond linkage) of this patent.

HOs

n = 1-20 19-X). ADC structure of an indolinohenzodiazepine dimer prodrug through a hydrophilic linker (alkoxime bond linkage) of this patent. /2. C O

S v. t HNCC Y-CH SeoO O

O O NH 3 n = 1-20 19-y). ADC Structure of a duocarmycin analog via a hydrophilic linker (peptide linkage). O O O NS-N g R O Ab S N-N1)--Nu HN 1V H O O RNH, S O HO O H OH N o''''''''s1 6 8 6. O n = 1-20 19-Z). ADC structure of an auristatin analog through a hydrophilic linker (thioether and citrulline-Valine peptide linkageS). Patent Application Publication Sep. 10, 2015 Sheet 23 of 23 US 2015/0250896 A1

Antiher2-TZ03 via a hydrophilic linker 10

-O Linkers/Antibody IV Drugs/Antibody

O 30 60 90 120 150 180 210 240 Time (min) Fig. 20. The use of a hydrophilic linker (86) in modifying a cell-binding agent (antiHer2 antibody) and following by production of a cell-binding agent-drug (TZ03) conjugate containing the hydrophilic linker. Cytotoxicity of antiCD22-TZ041 conjugates

with varying drug loads on Ramos cells

10-13 10-12 10-11 10-10 10-9 10-8 10-7 Concentration, M Fig. 21.5 days in Vito assays of the cytotoxicity of the antiCD22-TZ041 (tubulysin analog) conjugate with different drug load ratios via a hydrophilic linker (86). US 2015/0250896 A1 Sep. 10, 2015

HYDROPHILIC LINKERS AND THEIR USES after proteolytic degradation of the antibody inside the cell; FOR CONUGATION OF DRUGS TO A CELL and (d) disulfide-containing linkers, which are cleaved upon BNDING MOLECULES exposure to an intracellular thiol (Zhao, R.; Wilhelm, S. etal, 2011 J. Med. Chem. 36,5404). FIELD OF THE INVENTION 0003 Conjugates of cell-binding agents with drugs or modified chemical compounds via different types of linkers 0001. The present invention relates to the preparation of have been described (U.S. Pat. Nos. 4,680,338, 5,122.368, novel hydrophilic linkers used for the conjugation of a drug, 5,141,648, 5,208,020, 5,416,064; 5,475,092, 5,543,390, in particular, a cytotoxic agent to a biological molecule. The 5,563,250 5,585,499, 5,880,270, 6,214,345, 6,436,931, present invention also relates to methods of making cell 6,372,738, 6,340,701, 6,989,452, 7,129,261, 7,375,078, binding agent-drug (cytotoxic agent) conjugates comprising 7,498.302, 7,507,420, 7,691.962, 7,910,594, 7,968,586, either modification of drugs with these hydrophilic linkers 7,989.434, 7,994,135, 7,999,083, 8,153,768, 8.236,319, first, followed by reaction with cell-binding agents; or modi Zhao, R.; etal. (2011).J.Med. Chem. 36,5404: Doronina, S.; fication of cell-binding agents with these hydrophilic linkers et al. (2006) Bioconjug Chem, 17, 114; Hamann, P.; et al. first, followed by reaction with drugs. (2005) Bioconjug Chem. 16, 346). Typically, in these conju gates, the cell-binding agents are first modified with a bifunc BACKGROUND OF THE INVENTION tional agent such as SPDP (N-succinimidyl 3-(2-py ridyldithio)propionate), or SMCC (succinimidyl-4-(N- 0002 Nowadays the development on targeted maleimidomethyl)cyclohexane-1-carboxylate), or SPDB therapy requires not only to Supplement the conventional (N-succinimidyl 4-(2-pyridyldithio)butanoate), to introduce and radiotherapy while sparing healthy cells, an active disulfide or a maleimido moiety. Reaction with a greatly reducing or eliminating the often unpalatable side thiol-containing cytotoxic drug provides a conjugate in which effects, but also aim to overcome drug resistance (Türk, D. the cell-binding agent, such as a , and and Szakács, G., Curr Opin Drug Discov Devel. 2009, 12, drug are linked via disulfide bonds or thioether bonds. 246: Zhao, R. etal, J. Med. Chem. 2011, 56, 5404; Yauch, R. 0004. However, the use of the cell binding molecule-drug L. Settleman, J. Curr Opin Genet Dev. 2012, 22, 45). There conjugates, such as antibody-drug conjugates (ADCs), in are several systemic deliveries for targeted treatment of tumor developing therapies for a wide variety of has been that have been studied during the past three decades: Heat limited both by the availability of specific targeting agents activated targeted drug delivery: Tissue-selective drug deliv (carriers) as well as the conjugation methodologies which ery for cancer using carrier-mediated transport systems: result in the formation of protein aggregates when the amount Tumor-activated prodrug therapy for targeted delivery of che of the drugs that are conjugated to the carrier (i.e., the drug motherapy; Pressure-induced filtration of drug across vessels loading) is increased. Normally the tendency for cytotoxic to tumor; Promoting selective permeation of the anticancer drug conjugates to aggregate is especially problematic when agent into the tumor, Two-step targeting using a bispecific the conjugation reactions are performed with the hydropho antibody; Site-specific delivery and light-activation of anti bic linkers. Since higher drug loading increases the inherent cancer proteins. Many special formulations and carriers have potency of the conjugate, it is desirable to have as much drug been studied for target delivery of anticancer drugs, such as loaded on the carrier as is consistent with retaining the affinity Albumin-based drug carriers; Carbohydrate-enhanced che of the carrier protein. The presence of aggregated protein, motherapy; Proteins and peptides based drug carriers; Fatty which may be nonspecifically toxic and immunogenic, and acids as targeting vectors linked to active drugs; Microsphere therefore must be removed for therapeutic applications, carriers; Monoclonal antibodies as carriers; Vitamins, e.g. folates as carriers; Nanoparticle carriers; Liposome carriers, makes the scale-up process for the production of these con e.g. pegylated liposomes (enclosed in a polyethylene glycol jugates more difficult and decreases the yield of the products. bilayer); Polyethylene glycol (PEG) carriers: Single-chain 0005 Consequently, there is a critical need to improve -binding molecule carriers; Polymeric micelle carri methods for conjugating drugs/cytotoxic drugs to carriers ers; Lipoprotein-based drug carriers; Dendrimers; etc. An (cell binding molecules) that minimize the amount of aggre ideal drug delivery vehicle must be non-toxic, biocompatible, gation and thereby allow for as high a drug loading as possible non-immunogenic, and biodegradable (Scott, R. Crabbe, D; through application of a hydrophilic cross-linker. etal (2008) Expert Opin. Drug Deli. 5, 459) and avoid rec ognition by the host’s defense mechanisms (Saltzman, W.; SUMMARY OF THE INVENTION Torchilin, V. (2008). “Drug delivery systems’ Access Sci 0006. The present invention provides hydrophilic linkers ence. McGraw-Hill Co.). The link between the delivery containing phosphinate, Sulfonyl, and/or Sulfoxide groups to vehicles, in particular, antibodies and the cell-killing agent link drugs to a cell-binding agent (e.g., an antibody). The plays a critical role in the development of targeted drug deliv preferred formula of the cell binding molecule hydrophilic ery systems, as the nature of the linker significantly affects the linker-drug conjugates can be represented as: Cb-(-L-Drug), potency, selectivity and the pharmacokinetics of the resulting wherein Cb is a cell-binding agent, L is a hydrophilic linker, conjugates (Zhao, R.; Wilhelm, S. etal. (2011).J.Med. Chem. Drug is a drug molecule, and n is an integer from 1 to 20. The 36, 5404: Doronina, S.; Mendelsohn, B. et al. (2006) Bio advantages in applying the hydrophilic linker in the cell mol conjug Chem, 17, 114; Hamann, P.; Hinman, L., et al. (2005) ecule-drug conjugate are: a). reducing the aggregation of the Bioconjug Chem. 16, 346). Four types of linkers had been conjugates in water based media; b). enabling higher drug used for preparation of cell binding agent-drug conjugates per-cell binding molecule-ratio conjugate, resulting in higher that have entered the clinic: (a) acid-labile linkers, exploiting potency; c). being retained inside the target cell after the the acidic endosomal and lysosomal intracellular microenvi drug-linker released from the conjugates, which can combat ronment; (b) linkers cleavable by lysosomal proteases; (c) permeability-glycoprotein (Pgp)-expressing multidrug resis chemically stable thioether linkers that release a lysyl adduct tant (MDR) cells. US 2015/0250896 A1 Sep. 10, 2015

0007. In one aspect of the present invention, the hydro 0020 q is 1-20; Q, T, m, n, R. R. R. R. R. R. and M philic linker is represented by formula (I) whereinY can react are described the same previously in formula (I). with a cell-binding agent and Z can react with a cytotoxic 0021. In a further aspect, the present invention provides a drug: modified cell-binding agent of formula (III), in which the cell-binding agent, Cb, has reacted with the hydrophilic linker, which still has Z, a group capable of reacting with a (I) drug: Y-R-to-Rih Fr-Rih -z (III) 0008. Wherein: Cb R-to-R-hitt-R1-7 0009 Y represents a functional group that enables reac KY, g tion with a cell-binding agent; 0010 Q and T are either -P(=O)(OM)-, or - S(O) , or —S(O)—: 0022 Wherein the substituents are as defined above. 00.11 m and n are integer from 0 to 5, but not 0 at the same 0023. In an even further aspect, the present invention pro time; In addition, when m=1, n=0, Q is not —P(=O)(OM)-; vides a modified drug of formula (IV), in which the drug, or when n=1, m=0, T is not —P(=O)(OM)-; Drug, has reacted with the hydrophilic linker, which still has 0012 Z represents a functional group that enables linkage Y, a group capable of reacting with the cell-binding agent: of a cytotoxic drug via a disulfide, thioether, thioester, pep tide, hydrazone, ether, ester, carbamate, carbonate, amine (secondary, tertiary, or quartary), imine, cycloheteroalkyane, (IV) heteroaromatic, alkoxime or amide bond; Y-R+Q-R,+r-R-typng 0013 R. R. R. R. Rs and R, are the same or different and are H. linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, 0024 Wherein the substituents are as defined above. linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon 0025. The present invention further relates to a method of atoms of esters, ether, amide, or polyethyleneoxy unit of making a cell-binding molecule-drug conjugate of formula formula (OCH2CH2), wherein p is an integer from 0 to about (II), wherein the drug is linked to a cell-binding agent via the 1000, or combination thereof. hydrophilic linker. 0014) Additionally R. R. R. and Ra are respectively a chain of atoms selected from C, N, O, S, Si, and P that 0026. The present invention also relates to a method of covalently connects the cell-surface binding ligand, the phos making a modified cell-binding molecule of formula (III), phinate or Sulfonyl group, the conjugated drug and among wherein the cell-binding molecule is reacted with the hydro themselves (R. R. R. and Ra). The atoms used in forming philic linker. the hydrophilic linker may be combined in all chemically 0027. The present invention also relates to a method of relevant ways, such as forming alkylene, alkenylene, and making a modified drug of formula (IV), wherein the drug is alkynylene, ethers, polyoxyalkylene, esters, amines, imines, reacted with the hydrophilic linker. polyamines, hydrazines, hydrazones, amides, ureas, semicar bazides, carbazides, alkoxyamines, alkoxylamines, ure BRIEF DESCRIPTION OF THE DRAWINGS thanes, amino acids, peptides, acyloxylamines, hydroxamic 0028 FIG. 1 shows the synthesis of diphosphinate-con acids, or combination thereof. taining cross-linking reagents that contain a pyridyldisulfide 00.15 M is H, or Na, or K, or NRRR or a pharmaceu group and a reactive carboxylic acid ester, and the linker used tical salt. R. R. and R are described above. for the conjugation of an antibody. Ammonium phosphinate 0016. In another aspect, this invention provides a cell are first converted into bis(trimethylsilyl)phosphonite, fol binding agent-drug conjugate of formula (II), in which the lowed by Michael addition with an acrylate and then substi cell-binding agent, Cb, and the drug, Drug, have reacted at the tution reaction with excess amount of 1,2-dibromo ethane to two ends of the hydrophilic linker: form (2-bromoethyl)(3-ethoxy-3-oxopropyl)phosphinic acid (4). The bromoethyl phosphinic acid moiety (5) was then substituted with bis(trimethylsilyl)phosphonite (2) and 1.2- (II) dibromo ethane to generate (2-bromoethyl)(2-(ethoxy(3- W V ethoxy-3-oxopropyl)phosphoryl)ethyl)phosphinic acid (8), whose bromide group was then replaced by potassium ethyl Xanthogenate, followed by basic hydrolysis and a Substitution reaction with an excess of 2,2'-dithiobispyridine, as well as 0017 wherein: condensation reaction of the acid 10 with N-hydroxysuccim 0018 Cb represents a cell-binding agent; ide (NHS) in an acid medium using the carbodiimide cou 0019 Drug represents the drug linked to the cell-binding pling agent EDC to give the diphosphinate linker (11). The agent via the hydrophilic linker by a disulfide, thioether, linker 11 can be used for the conjugation of drugs to a cell thioester, peptide, hydraZone, ether, ester, carbamate, carbon binding molecule (such as antibody). ate, cycloheteroalkyane, heteroaromatic, alkoxime or amide 0029 FIG. 2 shows the synthesis of a triphosphinate-con bond; taining linker 18 that contains a pyridyldisulfide group and a US 2015/0250896 A1 Sep. 10, 2015

reactive carboxylic acid ester. The linker can be used for the 0045 FIG. 18 show the synthesis of a disulfonyl-contain conjugation of a drug to cell binding molecule via a disulfide ing linker (221) that used for conjugation of amine-contain bond. ing cytotoxic drugs to an antibody via the Val-Cit-PABC 0030 FIG.3 shows the synthesis of tetraphosphinate-con linkage. taining cross linkers 23 that contain a reactive carboxylic acid 0046 FIG. 19 (19-a-19-z) shows the antibody-drug con ester and a pyridyldisulfide group, enabling linkage of a drug jugate (ADC) structures of the typical cytotoxic agents (the to a cell binding molecule via a disulfide bond. analogs of tubulysins, calicheamicins, maytansinoids, aurist 0031 FIG. 4 shows the synthesis of diphosphinate-con atins, doxorubicin, daunorubicin, CC-1065, pyrrolobenzodi taining cross-linker 26 that contain a maleimido group and a azepine dimmers) via the hydrophilic linkers of this patent. reactive carboxylic acid ester enabling linkage of a drug to a 0047 FIG. 20 shows the use of a hydrophilic linker (86) in cell binding molecule via a thioether bond modifying a cell-binding agent (antiHer2 antibody) and fol 0032 FIG. 5 shows the synthesis of monophosphinate, lowing by production of a cell-binding agent-drug (TZ03) diphosphinate, triphosphinate and tetraphosphinate-contain conjugate containing the hydrophilic linker. ing linkers that contain a pyridyldisulfide group and a reactive 0048 FIG. 21 shows 5 days in Vito assays of the cytotox carboxylic acid ester via substitution of ethyl 2-bromoacetate icity of the antiCD22-TZ041 (tubulysin analog) conjugate with bis(trimethylsilyl)phosphonite (2). with different drug load ratios via a hydrophilic linker (86). 0033 FIG. 6 shows the synthesis of linking reagents that contain a vinylsulfonyl (55), a monosulfonyl (61) and disul DETAILED DESCRIPTION OF THE INVENTION fonyl (69) groups. Definitions 0034 FIG. 7 shows the synthesis of mono-, and di-sulfo nyl linkers 0049 Alkyl means an aliphatic hydrocarbon group 0035 FIG.8 shows the synthesis of mono-sulfonyl linkers which may be straight or branched having 1 to 8 carbonatoms containing polyethylene glycols. in the chain or cyclic. “Branched” means that one or much lower alkyl groups such as methyl, ethyl or propyl are 0036 FIG. 9 shows the synthesis of di-sulfonyl linkers attached to a linear alkyl chain. Exemplary alkyl groups containing polyethylene glycols. The linker can be used for include methyl, ethyl, n-propyl, i-propyl. n-butyl, t-butyl, the conjugation of an antibody or a protein via a disulfide n-pentyl, 3-pentyl, octyl, nonyl, decyl cyclopentyl, cyclo bond. hexyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethyl 0037 FIG. 10 shows the synthesis of a hydrophilic linker penty1, 2,3-dimethylpentyl, 3.3-dimethylpenty1, 2,3,4-trim containing a phosphinic acid and a Sulfonyl group. ethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl, 2,4- 0038 FIG. 11 shows the synthesis of a hydrophilic linker dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl, 2,4- containing contain a phosphinate, a Sulfonyl and a ketone dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, groups that can be used for the conjugation of a drug to an isoheptyl, n-octyl, and isooctyl. A C-C alkyl group can be antibody or a protein via a hydraZone bond. unsubstituted or Substituted with one or more groups includ 0039 FIG. 12 shows the synthesis of hydrophilic linkers ing, but not limited to. —C-Cs alkyl, —O—(C-C alkyl). that contain a disulfonyl, polyethylene glycols, a much hin -aryl, -C(O)R', OC(O)R', C(O)OR', C(O)NH2, dered pyridyldisulfide and a reactive carboxylic acid ester C(O)NHR', C(O)N(R'). NHC(O)R', S(O).R', groups via substitution of 1,3-dibromo-3-methylbutane. The —S(O)R', —OH, -halogen.(F, Cl, Br or I), —N, NH, linker is used for the conjugation of drugs to an antibody or a —NH(R'), N(R'), and —CN; where each R" is indepen protein via a hinder disulfide bond. dently selected from —C-Cs alkyl and aryl. 0040 FIG. 13 shows the synthesis of hydrophilic linkers 0050. A "C-C carbocycle” means a 3-, 4-, 5-, 6-, 7- or containing a phosphinate and a sulfonyl groups that are used 8-membered Saturated or unsaturated non-aromatic carbocy for the conjugation of drugs to a cell binding molecule either clic ring. Representative C-Cs carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclo via a triazole or via a thiolether bond. pentadienyl, cyclohexyl, cyclohexenyl, 1.3-cyclohexadienyl, 0041 FIG. 14, shows synthesis of hydrophilic linkers that 1,4-cyclohexadienyl, cycloheptyl, 1.3-cycloheptadienyl, 1.3, contain a disulfonyl, phosphinate, a pyridyldisulfide group, a 5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. A polyethylene glycol (PEG) chain and a reactive carboxylic C-Cs carbocycle group can be unsubstituted or Substituted acid ester. The linkers are used for the conjugation of a cell with one or more groups including, but not limited to, binding molecule via cell thioether bond —C-Cs alkyl, —O—(C-C alkyl), -aryl, —C(O)R'. - OC 0042 FIG. 15 shows synthesis of hydrophilic linkers that (O)R’, C(O)OR, C(O)NH, C(O)NHR', C(O)N(R) contain a disulfonyl, phosphinate, an alkoxylamino and a NHC(O)R', S(O).R', S(O)R', -OH, -halogen, maleimido Substituents, enabling ketone or aldehyde-con - N - NH, -NH(R), N(R'), and CN; where each R' taining drug to link to an antibody via a thioether and an is independently selected from —C-Cs alkyl and aryl. alkoxime bond. 0051. A "C-Cs carbocyclo” refers to a C-C carbocycle 0043 FIG. 16 shows the synthesis of disulfonyl-contain group defined above wherein one of hydrogen atoms on the ing linkers that contains an alkoxylamino and a maleimido carbocycle is replaced with a bond. Substituent, enabling ketone or aldehyde-containing drug to 0.052 “Heterocycle” refers to an aromatic or non-aromatic link to an antibody via a thioether and an alkoxime bond. C-C carbocycle in which one to four of the ring carbon 0044 FIG. 17 shows the synthesis of sulfoxide-containing atoms are independently replaced with a heteroatom from the and disulfoxide-containing linkers that are used for the con group of O, N, S Se, and P. Preferable heteroatoms are oxy jugation of drugs to a cell binding molecule via a disulfide gen, nitrogen and Sulphur. Suitable heterocyclics are also bond. disclosed in The Handbook of Chemistry and Physics, 76" US 2015/0250896 A1 Sep. 10, 2015

Edition, CRC Press, Inc., 1995-1996, p. 2-25 to 2-26, the lishing Company, Easton, Pa., 1985, p. 1418, the disclosure of disclosure of which is hereby incorporated by reference. which is hereby incorporated by reference. 0053 Preferred non aromatic heterocyclic include, but are 0060. The novel conjugates disclosed herein use hydro not limited to pyrrolidinyl, pyrazolidinyl, imidazolidinyl, philic cross-linkers. Examples of some Suitable cross-linkers oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydro-pyranyl. and their synthesis are shown in FIGS. 1 to 18. dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, The Hydrophilic Linkers tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydro 0061 The synthetic routes to produce hydrophilic cross pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydro-py linkers as well as the preparation of the conjugates of drugs to ridyl, dihydropyridyl, tetrahydropyrinidinyl, dihydrothiopy a cell binding molecules of the present invention are shown in ranyl, azepanyl, as well as the fused systems resulting from FIGS. 1.-19. The hydrophilic cross linkers possess three ele the condensation with a phenyl group. ments: a) Substituents that are either phosphinate, or Sulfonyl 0054 “Alkyl”, “cycloalkyl”, “alkenyl”, “alkynyl”, “aryl', or Sulfoxide groups, or mixed of these groups, b) a group, "heteroaryl', "heterocyclic' and the like refer also to the Such as a N-hydroxysuccimimide ester, maleimido group, corresponding “alkylene'. “cycloalkylene”, “alkenylene'. haloacetyl group, and hydrazide, capable of reaction with a “alkynylene', 'arylene'. “heteroarylene”, “heterocyclene’ cell-binding agent, and c) a group. Such as but not limited to, and the likes which are formed by the removal of two hydro a disulfide, maleimide, haloacetyl, aldehyde, ketone, azide, gen atoms. amine, alkoxylamino and hydrazide, capable of reaction with 0055) “Halogen atom” refers to fluorine, chlorine, bro a drug. The hydrophilic substituents can be introduced by mine or iodine atom; preferably bromine and chlorine atom. methods described herein. For example of the phosphinate 0056 “Pharmaceutically” or “pharmaceutically accept Substituents, they can be introduced by first treating a com able” refer to molecular entities and compositions that do not mercially available ammonium phosphinate with an acrylate produce an adverse, allergic or other untoward reaction when via Michael addition and followed by substitution of excess administered to an animal, or a human, as appropriate. amount of dibromo alkane to a phosphinate group. For 0057 "Pharmaceutically acceptable excipient' includes example of the sulfonyl and sulfoxide substituents, they can any carriers, diluents, adjuvants, or vehicles, such as preserv be introduced by first generation of thioether components, ing or antioxidant agents, fillers, disintegrating agents, wet followed by oxidization of these components. More detail ting agents, emulsifying agents, Suspending agents, Solvents, synthesis of the hydrophilic linkers and their uses for the dispersion media, coatings, antibacterial and antifungal preparation of cell binding ligand-drug conjugates of this agents, isotonic and absorption delaying agents and the like. invention are disclosed in the FIGS. 1.-19. The use of Such media and agents for pharmaceutical active 0062 Preferably, the hydrophilic linkers are compounds Substances is well known in the art. Except insofar as any of the formula (I) below: conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contem plated. Supplementary active ingredients can also be incor (I) porated into the compositions as Suitable therapeutic combi Y-R-to-R-hiti-R-E-z nations. Rs R6 0058 As used herein, “pharmaceutical salts' refer to derivatives of the disclosed compounds wherein the parent 0063 wherein: compound is modified by making acid or base salts thereof. 0064 Y represents a functional group that enables reac The pharmaceutically acceptable salts include the conven tion with a cell-binding agent; tional non-toxic salts or the quaternary ammonium salts of the 0065 Q and T are either -P(=O)(OM)-, or -S(O) , parent compound formed, for example, from non-toxic inor or —S(O)—: ganic or organic acids. For example, Such conventional non 0.066 m and n are integer from 0 to 5, but not 0 at the same toxic salts include those derived from inorganic acids such as time. In addition, when m=1, n=0, Q is not —P(=O)(OM)-; hydrochloric, hydrobromic, Sulfuric, Sulfamic, phosphoric, or when n=1, m=0, T is not —P(=O)(OM)-; nitric and the like; and the salts prepared from organic acids 0067 Z represents a functional group that enables linkage Such as acetic, propionic, Succinic, tartaric, citric, methane of a cytotoxic drug via a disulfide, thioether, thioester, pep Sulfonic, benzenesulfonic, glucoronic, glutamic, benzoic, tide, hydrazone, ether, ester, carbamate, carbonate, amine salicylic, toluenesulfonic, oxalic, fumaric, maleic, lactic and (secondary, tertiary, or quartary), imine, cycloheteroalkyane, the like. Further addition salts include ammonium salts such heteroaromatic, alkoxime or amide bond; as tromethamine, meglumine, epolamine, etc., metal salts 0068. R. R. R. R. R. and R. are the same or different Such as Sodium, potassium, calcium, Zinc or magnesium. and are H. linear alkyl having from 1-6 carbon atoms, 0059. The pharmaceutical salts of the present invention branched or cyclic alkyl having from 3 to 6 carbon atoms, can be synthesized from the parent compound which contains linear, branched or cyclic alkenyl or alkynyl, or 1-6 carbon a basic or acidic moiety by conventional chemical methods. atoms of esters, ether, amide, or polyethyleneoxy unit of Generally, such salts can be prepared via reaction the free formula (OCH2CH2), wherein p is an integer from 0 to about acidic or basic forms of these compounds with a stoichiomet 1000, or combination thereof. ric amount of the appropriate base or acid in water or in an 0069. In another embodiment, R. R. R. and R can be organic solvent, or in a mixture of the two. Generally, non respectively a chain of atoms selected from C, N, O, S, Si, and aqueous media like ether, ethyl acetate, ethanol, isopropanol, P that covalently connects the cell-surface binding ligand, the oracetonitrile are preferred. Lists of suitable salts are found in phosphinate or Sulfonyl or Sulfoxide group, the conjugated Remington's Pharmaceutical Sciences, 17" ed., Mack Pub drug and themselves (R, R2, R and Ra). The atoms used in US 2015/0250896 A1 Sep. 10, 2015 forming the hydrophilic linker may be combined in all chemi cyclohexane-1 carboxylate (“MCC), (4-acetyl)aminoben cally relevant ways. Such as forming alkylene, alkenylene, Zoate (“SIAB), 4-thio-butyrate (SPDB), 4-thio-2-hydrox and alkynylene, ethers, polyoxyalkylene, esters, amines, imi ysulfonyl-butyrate (2-Sulfo-SPDB), ethyleneoxy— nes, polyamines, hydrazines, hydrazones, amides, ureas, semicarbazides, carbazides, alkoxyamines, alkoxylamines, CHCHO as one or more repeating units (“EO” or urethanes, amino acids, acyloxylamines, hydroxamic acids, “PEO”). Additional linker components are known in the art and many others. In addition, it is to be understood that the and some are described herein. atoms forming the linker (L) may be either Saturated or unsat 0077. Example structures of these components containing urated, or may be radicals, or may be cyclized upon each other to form divalent cyclic structures, including cyclo alkanes, linkers are: cyclic ethers, cyclic amines, arylenes, heteroarylenes, and the like in the linker. O 0070) M is H, or Na, or K, or NRRR or a pharmaceu O tical salt. R. R. and R are described above. 0071 Examples of the functional group, Y, that enables a- S N-1N 1.--- N S reaction with a cell-binding agent include amine reacting O H O agents such as but not limited to N-hydroxysuccinmide esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluo rophenyl esters; thiol reactive agents such as but not limited to MC, 6-maleimidocaproyl containing) pyridyldisulfides, nitropyridyldisulfides, maleimides, halo acetates and carboxylic acid chlorides. 0072 Examples of the functional group, Z, which enables O linkage of a cytotoxic drug, include groups that enable link O age via an either disulfide, or thioether, thioester, peptide, hydrazone, ester, carbamate, carbanate, alkoxime or amide S bond. Such functional groups include, but are not limited to, A- | N-1 ng-1-1 N A thiol, disulfide, amino, carboxy, aldehydes, maleimido, halo O O acetyl, hydrazines, and hydroxy. O s O 0073. In preferred embodiments, R. R. R., and R4 are O linear alkyl having from 1-6 carbon atoms, or polyethyl eneoxy unit of formula (OCHCH.), p=1-100, S 0074 The synthesis of 2-dithio-pyridyl containing cross A- it a A linkers of formulae (I) is shown, for example, in FIGS. 1, 2, 3, IN-1a:n-OH O 5, 6, 7, 8, 9, 10, 12, 13, 14, and 17. The synthesis of male imido-containing cross linkers of the formula (I) is shown, for example, in FIGS. 4, 8, 9, 10.12, 14, and 18. The synthesis of (ME, maleimidoethyl containing). thioether-containing cross linkers of the formula (I) is shown, for example, in FIGS. 6, 8, 10, and 13. The synthesis of polyethylene glycol-containing hydrophilic cross linkers of O O O formula (I) is shown, for example, in FIGS. 8,9, and 14. The synthesis of azide-containing hydrophilic cross linkers of ---> formula (I) for Huisgen 1,3-dipolar cycloaddition of azides to Oin 1SNH 1^3,S alkynes is shown, for example, in FIG. 13. The synthesis of hydrophilic cross linkers of formula (I) bearing a hydrazide or ketone moieties enabling linkage via acid-labile bonds is shown, for example, in FIGS. 11, 16 and 18. The synthesis of hydrophilic cross linkers of formula (I) bearing an alkoxy lamino moiety enabling linkage via alkoxime bonds is shown, for example, in FIGS. 15 and 16. The synthesis of dipeptide containing cross linkers of the formula (I) is shown, for example, in FIG. 18. Cell-Binding Agent-Drug Conjugates 0075. The conjugates of the present invention can be rep O s resented by the following formula, Cb-(-L-Drug), wherein O Cb is a cell-binding agent, L is a hydrophilic linker, Drug is a drug molecule, and n is an integer from 1 to 20. O 0076. The hydrophilic linker L. may be composed of one or w more linker components. Exemplary linker components Clu N-1 OH , include 6-maleimidocaproyl (“MC), maleimidopropanoyl O (“MP), valine-citrulline (“val-cit’ or “vc'), alanine-pheny lalanine ("ala-phe' or “af), p-aminobenzyloxycarbonyl (“PAB), 4-thiopentanoate (“SPP), 4-(N-maleimidomethyl) (PAB, p-aminobenzyloxycarbonyl containing) US 2015/0250896 A1 Sep. 10, 2015

0078 Preferably, the conjugates have the following for O O O mula (II): H

H.N. N \ (II) HN OH et-to-ri-ry) g

0079 wherein: NN-1- 0080 Cb represents a cell-binding agent; HN N ~- I0081. Drug represents the drug linked to the cell-binding agent via the hydrophilic linkers of this invention by a disul fide, thioether, thioester, peptide, hydraZone, ether, ester, car bamate, carbonate, heterocyclic ring, amine, imine, alkoxime or amide bond; I0082 q is 1-20; Q, T, m, n, R. R. R. R. R. R. and M are described the same previously in formula (I). (valine-citrulline containing) I0083. As described in more detail below, the drug can be any of many Small molecule drugs, including, but not limited to, tubulysins, calicheamicins, auristatins, maytansinoids, CC-1065 analogs, morpholinos doxorubicins, taxanes, cryp f O --> tophycins, epothilones, and benzodiazepine dimers (e.g., - N dimmers of pyrrolobenzodiazepine (PBD) or tomayimycin), O H s indolinobenzodiazepines, imidazobenzothiadiazepines, or O oxazolidinobenzodiazepines). O I0084. To synthesize the conjugate, the cell-binding agent can be first modified with the hydrophilic linkers of the f/Sr. ry-N- present invention to introduce reactive disulfide groups, N OH maleimido, haloacetyl, azide, 1-yne, ketone, or hydrazide - N-1 groups. Synthesis of the cell-binding agent-drug conjugates O linked via disulfide bonds is achieved by a disulfide exchange between the disulfide bond in the modified cell-binding agent and a drug containing a free thiol group. Synthesis of the (MCC, 4-(N-maleimidomethyl)cyclohexane-1 carboxylate) cell-binding agent-drug conjugates linked via thioether is achieved by reaction of the maleimido or haloacetyl or eth ylsulfonyl modified cell-binding agent and a drug containing O O a free thiol group. Synthesis of conjugates bearing an acid labile hydrazone link can be achieved by reaction of a carbo Al-I N 1-3, nyl group with the hydrazide moiety in the linker, by methods OH known in the art (see, for example, P. Hamann et al., Hinman, L. M., et al., Cancer Res. 53, 3336-334, 1993; B. Laguzza et al., J. Med. Chem., 32: 548-555, 1959; P. Trail et al., Cancer S-N-1NN Res., 57; 100-105, 1997). WO H 1-3, I0085 Alternatively, the drug can be modified with the N hydrophilic linkers of the present invention to give a modified drug of formula (IV) bearing functionality capable of reacting ((4-acetyl)aminobenzoate containing) with a cell binding agent. For example athiol-containing drug can be reacted with the hydrophilic linker of formula (I) bearing a maleimdo or a haloacetyl or an ethylsulfonyl Sub stituent at neutral pH in aqueous buffer to give a drug con O nected to the hydrophilic linker via a thioether link. A thiol S containing drug can undergo disulfide exchange with a hydrophilic linker bearing a pyrdlyldithio moiety to give a v-u-OH & modified drug attached via a disulfide bond to the hydrophilic cross linker. A drug bearing a hydroxyl group or a thiol group O can be reacted with a hydrophilic linker bearing a halogen of this invention, in the presence of a mild base, to give a modi fied drug bearing an ether orthiolether link. A hydroxyl group wn--- l containing drug can be condensed with a hydrophilic cross n linker of formula (I) bearing a carboxyl group, in the presence of a dehydrating agent, Such as EDC or dicyclohexylcar (4-thio-2-hydroxysulfonyl-butyrate, 2-sulfo-SPDB) bodimide, to give an ester link. An amino group containing US 2015/0250896 A1 Sep. 10, 2015

drug can similarly undergo condensation with a carboxyl reaction is incubated at a temperature of from 4°C. to 40°C., group on the hydrophilic linker of formula (I) to give an amide preferably at ambient temperature. The progress of the reac bond. tion can be monitored by measuring the increase in the I0086. The conjugate may be purified by standard bio absorption at 320 nm or another appropriate wavelength. chemical means, such as gel filtration on a Sephadex G25 or After the reaction is complete, isolation of the modified cell Sephacryl S300 column, adsorption chromatography, and ion binding agent can be performed in a routine way, using for exchange or by dialysis. In some cases (e.g. folic acid, mel example gel filtration chromatography, or adsorptive chroma anocyte stimulating hormone, EGF etc) the cell-binding tography. agent-drug conjugates can be purified by chromatography 0091. The extent of modification can be assessed by mea Such as by HPLC, medium pressure column chromatography Suring the absorbance of the nitropyridine thione, dinitropy or ion exchange chromatography. ridine dithione, pyridine thione, carboxamidopyridine dithione and dicarboxamidopyridine dithione group released. Modified Cell-Binding Agents FIG. 20 shows the results from the modification of the cell 0087. The cell-binding agent modified by reaction with binding agent, the her2 antibody, with a chydrophilic cross linkers of the present invention are preferably represented by linker of the present invention. The time course of linker/ antibody (L/A) incorporation is shown, for example, along the formula (III) with the drugs/antibody (D/A) linked. The hydrophilic cross linkers described herein have diverse functional groups that (III) can react with any cell-binding agent that possesses a Suitable Cb-F-R-Q-R2- -T-R-R-Z Substituent. For example cell-binding agents bearing an amino or hydroxyl Substituent can react with cross linkers Rs Rs 1, bearing an N-hydroxysuccinimide (NHS) ester, cell-binding agents bearing a thiol Substituent can react with cross linkers 0088 wherein the substituents are as described above for bearing a maleimido or haloacetyl group. Additionally, cell the hydrophilic linkers and the cell-binding agent drug con binding agents bearing a carbonyl Substituent can react with jugates. cross linkers bearing a hydrazide or a hydroxylamine. One 0089. In preferred embodiments, Z is a disulfide substitu skilled in the art can readily determine which linker to use ent, a maleimido, haloacetyl group, or an N-hydroxysuccin based on the known reactivity of the available functional imide ester, and Cb linked with R is through thioether, group on the cell-binding agent. amide, or disulfide bond. The modified cell-binding agent can be prepared via a reaction of the cell-binding agent with the Modified Cytotoxic Drugs hydrophilic linkers by methods known in the art for other 0092. The cytotoxic drugs modified by reaction with cross-linkers (U.S. Pat. Nos. 5,846,545, 5,585,499, 5,475, cross-linkers of the present invention are preferably repre 092, 5,414,064, 5,208,020, and 4,563,304; Carlsson, J. et al. sented by the formula (IV): Biochem.J. (1978) 173,723-737(1978); Goff, D.A., BioCon jugate Chem. (1990), 1,381-386; L. Delprino et al. J. Pharm. Sci. (1993), 82, 506-512; S. Arpicco et al., Bioconjugate (IV) Chem (1997), 8,327-337). Y-R-E-R-hitt-Ribngv 0090 Advantageously, because the phosphinate groups Rs R6 and Sulfonyl groups on the hydrophilic linkers are soluble in water or require only a small percentage of organic solvent to maintain solubility in aqueous Solution, the reaction between 0093 wherein the substituents are as defined above. the cell-binding agent and the cross-linker can be conducted 0094. In preferred embodiments, Y is a disulfide substitu in aqueous solution. The cross-linking reagent is dissolved in ent, a maleimido, haloacetyl group, or an N-hydroxysuccin aqueous buffer, optionally containing a small amount (typi imide ester. cally <10% by volume) of a polar organic solvent that is 0.095 The modified drugs can be prepared by reacting the miscible with water, for example different alcohols, such as drug with the cross linkers of the present invention to give a methanol, ethanol, and propanol, acetone, acetonitrile, tet modified drug of formula (IV) bearing a functionality capable rahydrofuran (THF), 1,4-dioxane, dimethyl formamide of reacting with a cell binding agent. For example a thiol (DMF), dimethyl acetamide (DMA), or dimethylsulfoxide containing drug can be reacted with the cross linker of for (DMSO) at a high concentration, for example 1-100 mM, and mula (I) bearing a maleimdo Substituent at neutral pH in then an appropriate aliquot is added to the buffered aqueous aqueous buffer to give a drug connected to the hydrophilic Solution of the cell-binding agent. An appropriate aliquotisan linker via thioether linkage. A thiol-containing drug can amount of Solution that introduces 1-10 cross-linking groups undergo disulfide exchange with a hydrophilic linker bearing per cell-binding agent, preferably 1-5 groups, and the Volume a pyrdlyldithio moiety to give a modified drug attached via a to be added should not exceed 10%, preferably 5%, and most disulfide bond to the hydrophilic cross linker. A drug bearing preferably 0-3% of the volume of the cell-binding agent solu a hydroxyl group can be reacted with a cross linker bearing a tion. The aqueous Solutions for the cell-binding agents are halogen, in the presence of a mild base, to give a modified buffered between pH 6 and 9, preferably between 6.5 and 7.5 drug bearing an ether link. A hydroxyl group containing drug and can contain any non-nucleophilic buffer salts useful for can be condensed with a cross linker of formula (I) bearing a these pH ranges. Typical buffers include phosphate, trietha carboxyl group, in the presence of a dehydrating agent, such nolamine HCl, HEPES, and MOPS buffers, which can con as EDC or dicyclohexylcarbodimide (DCC), to give an ester tain additional components, such as cyclodextrins, sucrose link. A drug bearing a thiol group can be reacted with a cross and salts, for examples, NaCl and KCl. After the addition the linker bearing a malimido or a vinylsulfonyl, or a haloacetyl US 2015/0250896 A1 Sep. 10, 2015 group, to give a modified drug bearing thioether link. An clonal antibodies are preferred as a cell-surface binding agent amino group containing drug can similarly undergo conden if an appropriate one is available. And antibodies may be sation with a carboxyl group on the hydrophilic cross linker of murine, human, humanized, chimeric, or derived from other formula (I) to give an amide bond. The modified drug can be species. purified by standard methods such as column chromatogra 0.098 Production of antibodies used in the present inven phy over silica gel or alumina, crystallization, preparatory tion involves in vivo or in vitro procedures or combinations thin layer chromatography, ion exchange chromatography or thereof. Methods for producing polyclonal anti-receptor pep HPLC. tide antibodies are well-known in the art, such as in U.S. Pat. No. 4,493.795 (to Nestor et al). A monoclonal antibody is Cell-Binding Agents typically made by fusing myeloma cells with the spleen cells 0096. The cell-binding molecule that comprises the con from a mouse that has been immunized with the desired jugates and the modified cell-binding agents of the present antigen (Köhler, G.; Milstein, C. (1975). Nature 256: 495 invention may be of any kind presently known, or that become 497). The detailed procedures are described in Antibod known, molecule that binds to, complexes with or reacts with ies—A Laboratory Manual, Harlow and Lane, eds., Cold a moiety of a cell population sought to be therapeutically or Spring Harbor Laboratory Press, New York (1988), which is otherwise biologically modified. incorporated herein by reference. Particularly monoclonal 0097. The cell binding agents include, but are not limited antibodies are produced by immunizing mice, rats, hamsters to, large molecular weight proteins such as, for example, or any other mammal with the antigen of interest Such as the full-length antibodies (polyclonal antibodies, monoclonal intact target cell, isolated from the target cell, whole antibodies, dimers, multimers, multispecific antibodies (e.g., , attenuated whole virus, and viral proteins. Splenocytes bispecific antibodies); single chain antibodies; fragments of are typically fused with myeloma cells using polyethylene antibodies such as Fab, Fab'. F(ab'), F, Parham, J. Immu glycol (PEG) 6000. Fused hybrids are selected by their sen nol. 131, 2895-2902 (1983), fragments produced by a Fab sitivity to HAT (hypoxanthine-aminopterin-thymine) Hybri expression library, anti-idiotypic (anti-Id) antibodies, CDR's, domas producing a monoclonal antibody useful in practicing and epitope-binding fragments of any of the above which this invention are identified by their ability to immunoreact immuno-specifically bind to cancer cell antigens, viral anti specified receptors or inhibit receptor activity on target cells. gens, microbial antigens or a protein generated by the 0099. A monoclonal antibody used in the present inven that is capable of recognizing, binding to a tion can be produced by initiating a monoclonal hybridoma specific antigen or exhibiting the desired biological activity culture comprising a nutrient medium containing a hybri (Milleretal (2003).J. of Immunology 170:4854-4861); inter doma that secretes antibody molecules of the appropriate ferons (such as type I, II, III); peptides; lymphokines such as antigen specificity. The culture is maintained under condi IL-2, IL-3, IL-4, IL-6, GM-CSF, -gamma (IFN-Y); tions and for a time period sufficient for the hybridoma to hormones such as insulin, TRH (thyrotropin releasing hor secrete the antibody molecules into the medium. The anti mones), MSH (melanocyte-stimulating hormone), Steroid body-containing medium is then collected. The antibody hormones, such as androgens and estrogens, melanocyte molecules can then be further isolated by well-known tech stimulating hormone (MSH); growth factors and colony niques, such as using protein-A affinity chromatography; stimulating factors such as epidermal growth factors (EGF), anion, cation, hydrophobic, or size exclusive chromatogra granulocyte-macrophage colony-stimulating factor (GM phies (particularly by affinity for the specific antigen after CSF), transforming growth factors (TGF), such as TGFC. Protein A, and sizing column chromatography); centrifuga TGFB, insulin and insulin like growth factors (IGF-I, IGF-II) tion, differential solubility, or by any other standard technique G-CSF, M-CSF and GM-CSF Burgess, Immunology Today, for the purification of proteins. 5, 155-158 (1984); vaccinia growth factors (VGF): fibroblast 0100 Media useful for the preparation of these composi growth factors (FGFs); smaller molecular weight proteins, tions are both well-known in the art and commercially avail poly-peptide, peptides and peptide hormones, such as bomb able and include synthetic culture media. An exemplary Syn esin, gastrin, gastrin-releasing peptide; platelet-derived thetic medium is Dulbecco's minimal essential medium growth factors; and cytokines, such as interleu (DMEM: Dulbecco et al., Virol. 8,396 (1959)) supplemented kin-2 (IL-2), interleukin-6 (IL-6), leukemia inhibitory fac with 4.5 gm/l glucose, 20 mm glutamine, 20% fetal calf tors, granulocyte-macrophage colony-stimulating factor serum and with an anti-foaming agent, Such as polyoxyeth (GM-CSF); Vitamins. Such as folate; apoproteins and glyco ylene-polyoxypropylene block copolymer. proteins, such as transferrin O'Keefeet al., 260J. Biol. Chem. 0101. In addition, antibody-producing cell lines can also 932-937 (1985); sugar-binding proteins or lipoproteins, such be created by techniques other than fusion, Such as direct as lectins; cell nutrient-transport molecules; and Small transformation of B lymphocytes with oncogenic DNA, or molecular inhibitors. Such as prostate-specific membrane transfection with an oncovirus, Such as Epstein-Barr virus antigen (PSMA) inhibitors and small molecular tyrosine (EBV, also called human herpesvirus 4 (HHV-4)) or Kaposi's kinase inhibitors (TKI), non-peptides or any other cell bind sarcoma-associated herpesvirus (KSHV). See, U.S. Pat. Nos. ing molecule or Substance, Such as bioactive polymers (Dhar, 4,341,761; 4,399,121: 4,427,783; 4,444,887; 4,451.570; etal, Proc. Natl. Acad. Sci. 2008, 105, 17356-61); dendrimers 4,466,917; 4,472,500; 4,491,632; 4,493,890. A monoclonal (Lee, etal, Nat. Biotechnol. 2005, 23, 1517-26: Almutairi, et antibody may also be produced via an anti-receptorpeptide or al; Proc. Natl. Acad. Sci. 2009, 106, 685-90); nanoparticles peptides containing the carboxyl terminal as described well (Liong, et al. ACS Nano, 2008, 19, 1309-12; Medarova, et al. known in the art. See Niman et al., Proc. Natl. Acad. Sci. Nat. Med. 2007, 13,372-7: Javier, etal, Bioconjugate Chem. USA, 80: 4949-4953 (1983); Geysen et al., Proc. Natl. Acad. 2008, 19, 1309-12); liposomes (Medinai, et al. Curr. Phar. Sci. USA, 82: 178-182 (1985); Lei et al. Biochemistry Des. 2004, 10, 2981-9); viral capsides (Flenniken, et al. 34(20): 6675-6688, (1995). Typically, the anti-receptor pep Nanotechnol. 2009, 327, 71-93). In general mono tide or a peptide analog is used either alone or conjugated to US 2015/0250896 A1 Sep. 10, 2015 an immunogenic carrier, as the immunogen for producing antibodies (SZardenings, J Recept Signal Transduct Res. anti-receptor peptide monoclonal antibodies. 2003; 23(4):307-49). The use of peptides from such random 0102 There are also a number of other well-known tech peptide libraries can be similar to antibodies and antibody niques for making monoclonal antibodies as binding mol fragments. The binding molecules of peptides or proteins ecules in this invention. Particularly useful are methods of may be conjugated on or linked to a large molecules or mate making fully human antibodies. One method is phage display rials, such as, but is not limited, an albumin, a polymer, a technology which can be used to select a range of human liposome, a nano particle, as long as such attachment permits antibodies binding specifically to the antigen using methods the peptide or protein to retain its antigenbinding specificity. of affinity enrichment. Phage display has been thoroughly 0106 Examples of antibodies used for conjugation of described in the literature and the construction and screening drugs via the hydrophilic linkers of this prevention for treat of phage display libraries are well known in the art, see, e.g., ing cancer, , and infectious disease Dente etal, Gene. 148(1):7-13 (1994); Little etal, Biotechnol include, but are not limited to, (anti-GD2), Adv. 12(3):539-55 (1994); Clackson et al., Nature 352:264 (anti CA-125), Abciximab (anti CD41 ( alpha-IIb). 628 (1991); Huse et al., Science 246:1275-1281 (1989). (anti-TNF-C.), (anti-EpCAM, 0103 Monoclonal antibodies derived by hybridoma tech CD326), (anti-TNF-C.); Afutuzumab (anti nique from another species than human, Such as mouse, can CD20), Alacizumab pegol (anti-VEGFR2), ALD518 (anti be humanized to avoid human anti-mouse antibodies when IL-6), (Campath, MabCampath, anti-CD52), infused into humans. Among the more common methods of Altumomab (anti-CEA), Anatumomab (anti-TAG-72), humanization of antibodies are complementarity-determin (IMA-638, anti-IL-13), (anti ing region grafting and resurfacing. These methods have been HLA-DR), Arcitumomab (anti-CEA), (anti-L- extensively described, see e.g. U.S. Pat. Nos. 5,859.205 and selectin (CD62L), Atlizumab (, Actemra, 6,797,492; Liu et al. Immunol Rev. 222:9-27 (2008); Alma RoActemra, anti-IL-6 receptor), (anti-Rhesus gro et al. Front Biosci. 13:1619-33 (2008); Lazaretal, Mol factor), BapineuZumab (anti-beta amyloid), Immunol. 44(8): 1986-98 (2007); Li et al. Proc. Natl. Acad. (Simulect, antiCD25 (a chain of IL-2 receptor), Sci. USA. 103(10):3557-62 (2006) each incorporated herein (anti-phosphatidylserine), (LymphoScan, anti by reference. Fully human antibodies can also be prepared by CD22), (Benlysta, LymphoStat-B, anti-BAFF), immunizing transgenic mice, rabbits, monkeys, or other (anti-CD125), (anti-CCL11 mammals, carrying large portions of the human immunoglo (eotaxin-1)), (Scintimun, anti-CEA-related bulin heavy and light chains, with an immunogen. Examples antigen), (Avastin, anti-VEGF-A), Biciromab of Such mice are: the Xenomouse. (Abgenix, Inc.), the (FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti HuMAb-Mouse (Medarex/BMS), the VelociMouse (Regen CD44 v6), (BiTE, anti-CD19), Brentuximab eron), see also U.S. Pat. Nos. 6,596,541, 6,207,418, No. (cAC10, anti-CD30 TNFRSF8), (anti-IL-12, 6,150,584, No. 6,111,166, No. 6,075,181, No. 5,922,545, IL-23) (Ilaris, anti-IL-1), Cantuzumab (C242, Nos. 5,661,016, 5,545,806, 5,436,149 and 5,569,825. In anti-CanAg), Capromab, (Removab, anti-Ep human therapy, murine variable regions and human constant CAM, anti-CD3), CC49 (anti-TAG-72), (anti regions can also be fused to construct called "chimeric anti CD4), (Cimzia anti-TNF-C.), bodies' that are considerably less immunogenic in man than (Erbitux, IMC-C225, anti-EGFR), murine mAbs (Kipriyanov et al. Mol Biotechnol. 26:39-60 (anti-EpCAM), (anti-IGF-1), (2004); Houdebine, Curr Opin Biotechnol. 13:625-9 (2002) (anti-CD4), Clivatuzumab (anti-MUC1), each incorporated herein by reference). In addition, site-di (anti-TRAIL-R2), CR6261 (anti-Influenza A hemaggluti rected mutagenesis in the variable region of an antibody can nin), (anti-CD40), (Zenapax, anti result in an antibody with higher affinity and specificity for its CD25 (O. chain of IL-2 receptor)), (anti-CD38 antigen (Brannigan et al. Nat Rev Mol Cell Biol. 3:964–70, (cyclic ADP ribose hydrolase), Denosumab (Prolia, anti (2002)); Adams et al., J. Immunol Methods. 231:249-60 RANKL), (anti-B-lymphoma cell), Dorlimo (1999)) and exchanging constant regions of a mAb can mab, DorlixiZumab, (anti-GD3 ganglioside), improve its ability to mediate effector functions of binding (Soliris, anti-C5), Edobacomab (anti-endot and cytotoxicity. oxin), (Panorex, MAb17-1A, anti-EpCAM), 0104 Antibodies immunospecific for a malignant cell (Raptiva, anti-LFA-1 (CD11a), (My antigen can also be obtained commercially or produced by cograb, anti-Hsp90), (anti-SLAMF7), Elsilimo any method known to one of skill in the art such as, e.g., mab (anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), chemical synthesis or recombinant expression techniques. Epitumomab (anti-episialin), (anti-CD22), The nucleotide sequence encoding antibodies immunospe (anti-ITGB2 (CD18)), (Rexomun, cific for a malignant cell antigen can be obtained commer anti-HER2/neu, CD3), (Abegrin, anti-integrin cially, e.g., from the GenBank database or a database like it, Of3), (anti-hepatitis B surface antigen), the literature publications, or by routine cloning and sequenc Fanolesomab (NeutroSpec, anti-CD15), (anti ing. interferon receptor), (anti-folate receptor 1), 0105. Apart from an antibody, a peptide or protein that (anti-respiratory syncytial virus), bind/block/target or in some other way interact with the (anti-IL-22), (anti-IGF-1 receptor), Fontoli epitopes or corresponding receptors on a targeted cell can be Zumab (anti-IFN-Y), (anti-rabies virus glyco used as a binding molecule. These peptides or proteins could protein), (anti-TGF-B), (anti be any random peptide or proteins that have an affinity for the CD80), Gantenerumab (anti-beta amyloid), epitopes or corresponding receptors and they don't necessar (anti-CD 147 ()), Gemtuzumab (anti-CD33), Giren ily have to be of the immunoglobulin family. These peptides tuximab (anti-carbonic anhydrase 9), Glembatumumab can be isolated by similar techniques as for phage display (CR011, anti-GPNMB), (Simponi, anti-TNF US 2015/0250896 A1 Sep. 10, 2015

O), Gomiliximab (anti-CD23 (IgE receptor)), neu), (anti-CTLA-4), Tucotuzumab celmo (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indima leukin (anti-EpCAM), (anti-hepatitis B virus), cis-125, anti-CA-125), Imciromab (Myoscint, anti-cardiac (anti-Escherichia coli), (Stelara, myosin), (Remicade, anti-TNF-C), anti-IL-12, IL-23), (anti-AOC3 (VAP-1)), Ved (anti-CD51), (anti-CD25 (O. chain of IL-2 recep olizumab, (anti-integrin Caf37), (anti-CD20), tor)), Inotuzumab (anti-CD22), (anti-CD152), (anti-AOC3 (VAP-1), (Nuvion, (anti-CD30 (TNFRSF8)), (anti anti-CD3), Vitaxin (anti-vascular integrin avb3), Volocix CD4), (CEA-Cide, anti-CEA), imab (anti-integrin Os B). Votumumab (HumaSPECT, anti (anti-IL-13), (anti-NCA-90 (granulocyte anti tumor antigen CTA A16.88), (HuMax-EGFr. gen)), (anti-TGFbeta2). (anti (anti-EGFR), (HuMax-CD4, anti-CD4), TRAIL-R2), (anti-hepatitis B surface antigen), Ziralimumab (anti-CD147 (basigin)), Zolimomab (anti (anti-CD33), (anti-CD40), CD5), (Enbrel(R), (AmeviveR), Abata (anti-CD23 (IgE receptor), cept (OrenciaR), (Arcalyst), 14F7 anti-IRP-2 (anti-TRAIL-R1), (anti-T-cell receptor), Matu (Iron Regulatory Protein 2), 14G2a (anti-GD2 ganglioside, Zumab (anti-EGFR), (Bosatria, anti-IL-5), from Nat. Cancer Inst. for and solid tumors), J591 (anti-TGFbeta1), (anti-CD74), (anti-PSMA, Weill Cornell Medical School for prostate can (anti-TAG-72), (BEC-2, anti cers), 225.28S anti-HMW-MAA (High molecular weight GD3 ganglioside), (anti-Rhesus factor), Mot melanoma-associated antigen), Sorin Radiofarmaci S.R.L. avizumab (Numax, anti-respiratory syncytial virus), (Milan, Italy) for melanoma. COL-1 (anti-CEACAM3, Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolo CGM1, from Nat. Cancer Inst. USA for colorectal and gastric mab (anti-C242), Naptumomab (anti-5T4), cancers), CYT-356 (OncoltadR), for prostate cancers), (TySabri, anti-integrin C), (anti-endotoxin), HNK20 (OraVax Inc. for respiratory syncytial virus), Immu (anti-EGFR), (anti-TNF-C.), RAIT (from Immunomedics for NHL), Lym-1 (anti-HLA (Theracim, Theraloc, anti-EGFR), Nofetumo DR10, Peregrine Pharm. for Cancers), MAK-195Fanti-TNF mab. (anti-CD20), (Afolimomab, (; TNFA, TNF-alpha: TNFSF2), from anti-LFA-1 (CD11a)), (Arzerra, anti-CD20), Abbott/Knoll for toxic shock), MEDI-500 T10B9, (anti-PDGF-RC.), (Xolair, anti-IgE anti-CD3, TRO.B ( receptor alpha/beta), complex, from Fc region), Oportuzumab (anti-EpCAM), MedImmune Inc for Graft-versus-host disease, RING (OvaRex, anti-CA-125), (anti-CD3), Pagibaxi SCAN anti-TAG 72 (tumour associated glycoprotein 72), mab (anti-lipoteichoic acid), (Synagis, Abbo from Neoprobe Corp. for Breast, Colon and Rectal cancers. synagis, anti-respiratory syncytial virus), Avicidin (anti-EPCAM (epithelial cell adhesion molecule), (Vectibix, ABX-EGF, anti-EGFR), (anti anti-TACSTD1 (Tumor-associated calcium signal transducer Pseudomonas aeruginosa), (anti-IL-4), Pem 1), anti-GA733-2 (gastrointestinal tumor-associated protein tumomab (Theragyn, anti-MUC1), (Omnitarg, 2), anti-EGP-2 (epithelial glycoprotein 2); anti-KSA: KS1/4 2C4, anti-HER2/neu), (anti-C5), Pintumomab antigen; M4S; tumor antigen 17-1A; CD326, from NeoRX (anti-adenocarcinoma antigen), (anti-CD4), Pri Corp. for Colon, Ovarian, Prostate cancers and NHL; Lym tumumab (anti-vimentin), PRO 140 (anti-CCR5), Racotumo phoCide (Immunomedics, NJ), Smart ID10 (Protein Design mab (1E10, anti-(N-glycolylneuraminic acid (NeuGc, Labs). Oncolym (Techniclone Inc, CA), Allomune NGNA)-gangliosides GM3)), (anti-rabies virus (BioTransplant, CA), anti-VEGF (Genentech, CA); CEAcide glycoprotein), (anti-VEGFR2), Ranibizumab (Immunomedics, NJ), IMC-1C11 (ImClone Systems, NJ) (Lucentis, anti-VEGF-A), (anti-anthrax toxin, and Cetuximab (ImClone, NJ). protective antigen), (anti-cytomegalovirus gly 0107. Other antibodies as binding ligands include, but are coprotein B), (anti-IL-5), (anti not limited to, are antibodies against the following antigens: HGF), (MabThera, Rituxanmab, anti-CD20), Aminopeptidase N (CD13), Annexin A1, B7-H3 (CD276, (anti-IGF-1 receptor), (anti various cancers), CA125 (ovarian), CA15-3 (carcinomas), IFN-O.), (LeukArrest, anti-CD11, CD18), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcino (Antova, anti-CD154 (CD40L)), Satumomab mas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), (anti-TAG-72), (anti-cytomegalovirus), Sibrotu CA242 (colorectal), placental alkaline phosphatase (carcino Zumab (anti-FAP), (anti-IFN-O.), mas), prostate specific antigen (prostate), prostatic acid phos (anti-IL-6), (anti-CD2), (Smart) MI95 (anti phatase (prostate), epidermal growth factor (carcinomas), CD33), Solanezumab (anti-beta amyloid), Sonepcizumab CD2 (Hodgkin’s disease, NHL lymphoma, multiple (anti-sphingosine-1-phosphate), SontuZumab (anti-episia myeloma). CD3 epsilon (T cell lymphoma, lung, breast, gas lin), Stamulumab (anti-myostatin), Sulesomab (LeukoScan, tric, ovarian cancers, autoimmune diseases, malignant (anti-NCA-90 (granulocyte antigen), TacatuZumab (anti-al ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin’s pha-fetoprotein), Tadocizumab (anti-integrin CfB), Tali lymphoma), CD22 (leukemia, lymphoma, multiple Zumab (anti-IgE). Tanezumab (anti-NGF), Taplitumomab myeloma, SLE), CD30 (Hodgkin’s lymphoma), CD33 (leu (anti-CD19), (Aurexis, (anti-clumping factor kemia, autoimmune diseases), CD38 (multiple myeloma), A), Telimomab, (anti-tenascin C), Tenelix CD40 (lymphoma, multiple myeloma, leukemia (CLL)), imab (anti-CD40), (anti-CD3), TGN1412 (anti CD51 (Metastatic melanoma, sarcoma), CD52 (leukemia), CD28), Ticilimumab (Tremelimumab, (anti-CTLA-4), Tig CD56 (small cell lung cancers, , Merkel cell atuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), carcinoma, and the liquid tumor, multiple myeloma), CD66e Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 (cancers), CD70 (metastatic renal cell carcinoma and non receptor), (anti-CD154 (CD40L)), Tositumo Hodgkin lymphoma), CD74 (multiple myeloma), CD80 mab (anti-CD20), (Herceptin, (anti-HER2/ (lymphoma), CD98 (cancers), mucin (carcinomas), CD221 US 2015/0250896 A1 Sep. 10, 2015

(solid tumors), CD227 (breast, ovarian cancers), CD262 CD152, CD154, CD156, CD158, CD163, CD166, CD168, (NSCLC and other cancers), CD309 (ovarian cancers), CD184, CDw186, CD195, CD202 (a, b), CD209, CD235a, CD326 (solid tumors), CEACAM3 (colorectal, gastric can CD271, CD303, CD304), Annexin A1, Nucleolin, Endoglin cers), CEACAM5 (carcinoembryonic antigen; CEA, CD66e) (CD105), ROBO4, Amino-peptidase N, -like-4 (DLL4), (breast, colorectal and lung cancers), DLL4(A-like-4), EGFR VEGFR-2 (CD309), CXCR49CD184), Tie2, B7-H3, WT1, (Epidermal Growth Factor Receptor, various cancers), MUC1, LMP2, HPV E6 E7, EGFRVIII, HER-2/neu, Idio CTLA4 (melanoma), CXCR4 (CD184, Heme-oncology, type, MAGE A3, p53 nonmutant, NY-ESO-1, GD2, CEA, solid tumors), Endoglin (CD105, solid tumors), EPCAM MelanA/MART1, Ras mutant, gp100, p53 mutant, Protein ase3 (PR1), bcr-abl, Tyrosinase, Survivin, hTERT, Sarcoma (epithelial cell adhesion molecule, bladder, head, neck, colon, translocation breakpoints, EphA2, PAP ML-IAP, AFP. NHL prostate, and ovarian cancers), ERBB2 (Epidermal EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Growth Factor Receptor 2: lung, breast, prostate cancers), ALK. Androgen receptor, Cyclin B1, Polysialic acid, MYCN, FCGR1 (autoimmune diseases), FOLR (folate receptor, ova RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, rian cancers), GD2 ganglioside (cancers), G-28 (a cell Surface MAGE A1, sILe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, antigengly Volipid, melanoma), GD3 idiotype (cancers), Heat GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Car shock proteins (cancers), HER1 (lung, stomach cancers), bonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL. B cell leukemia), human chorionic LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legu gonadotropin (carcinoma), IGF1R (insulin-like growth factor main, Tie2, Page4, VEGFR2, MAD-CT-1, FAP. PDGFR-B, 1 receptor, Solid tumors, blood cancers), IL-2 receptor (inter MAD-CT-2, Fos-related antigen 1. leukin 2 receptor, T-cell leukemia and lymphomas), IL-6R 0108. In another specific embodiment, the cell-binding ( receptor, multiple myeloma, RA, Castleman’s drug conjugates via the hydrophilic likers of this invention are disease, IL6 dependent tumors), (CVB3, C531, used for the treatment of cancers. The cancers include, but are C.6B4, Ollf3, C.5 B5, CVB5, for various cancers), MAGE-1 not limited, Adrenocortical Carcinoma, Anal Cancer, Bladder (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcino Cancer, Brain Tumor (Adult, Brain Stem Glioma, Childhood, mas), MAGE 4 (carcinomas), anti-transferrin receptor (car Cerebellar Astrocytoma, Cerebral Astrocytoma, Ependy cinomas), p97 (melanoma), MS4A1 (membrane-spanning moma, Medulloblastoma, Supratentorial Primitive Neuroec 4-domains subfamily A member 1, Non-Hodgkin’s B cell todermal and Pineal Tumors, Visual Pathway and Hypotha lymphoma, leukemia), MUC1 or MUC1-KLH (breast, ova lamic Glioma), Breast Cancer, Carcinoid Tumor, rian, cervix, bronchus and gastrointestinal cancer), MUC16 Gastrointestinal, Carcinoma of Unknown Primary, Cervical (CA125) (Ovarian cancers), CEA (colorectal), gp100 (mela Cancer, Colon Cancer, Endometrial Cancer, Esophageal Can noma), MART1 (melanoma), MPG (melanoma), MS4A1 cer, Extrahepatic Bile Duct Cancer, Ewings Family of (membrane-spanning 4-domains subfamily A. Small cell lung Tumors (PNET), Extracranial Germ Cell Tumor, Eye Cancer, cancers, NHL), Nucleolin, Neu oncogene product (carcino Intraocular Melanoma, Gallbladder Cancer, Gastric Cancer mas), P21 (carcinomas), Paratope of anti-(N-glycolyl (Stomach), Germ Cell Tumor, Extragonadal, Gestational Tro neuraminic acid, Breast, Melanoma cancers), PLAP-like tes phoblastic Tumor, Head and Neck Cancer, Hypopharyngeal ticular alkaline phosphatase (ovarian, testicular cancers), Cancer, Islet Cell Carcinoma, Kidney Cancer (renal cell can PSMA (prostate tumors), PSA (prostate), ROBO4, TAG 72 cer), Laryngeal Cancer, Leukemia (Acute Lymphoblastic, (tumour associated glycoprotein 72, AML, gastric, colorec Acute Myeloid, Chronic Lymphocytic, Chronic Myelog tal, ovarian cancers), T cell transmembrane protein (cancers), enous, Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer, Tie (CD202b), TNFRSF10B (tumor ncrosis factor receptor Lung Cancer (Non-Small Cell, Small Cell, Lymphoma superfamily member 10B, cancers), TNFRSF13B (tumor (AIDS-Related, Central Nervous System, Cutaneous T-Cell, necrosis factor receptor superfamily member 13B. multiple Hodgkin’s Disease. Non-Hodgkin’s Disease, Malignant myeloma, NHL, other cancers, RA and SLE), TPBG (tropho Mesothelioma, Melanoma, Merkel Cell Carcinoma, Meta blast glycoprotein, Renal cell carcinoma), TRAIL-R1 (Tu satic Squamous Neck Cancer with Occult Primary, Multiple mor necrosis apoprosis Inducing ligand Receptor 1, lym Myeloma, and Other Plasma Cell Neoplasms, Fun phoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, goides, Myelodysplastic Syndrome, Myeloproliferative Dis Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various orders, Nasopharyngeal Cancer, , Oral Can cancers). Some other tumor associated antigens recognized cer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer by antibodies have been reviewed (Gerber, et al., mabs 1:3, (Epithelial, Germ Cell Tumor, Low Malignant Potential 247-253 (2009); Novellino et al., Cancer Immunol Immu Tumor), Pancreatic Cancer (Exocrine, Islet Cell Carcinoma), nother. 54(3), 187-207 (2005). Franke, et al., Cancer Biother Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Can Radiopharm. 2000, 15, 459-76). Examples of these antigens cer, Penile Cancer, Pheochromocytoma Cancer, Pituitary that antibodies against are: Many other Cluster of Differen Cancer, Plasma Cell Neoplasm, Prostate Cancer Rhab tiations (CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, domyosarcoma, Rectal Cancer, Renal Cell Cancer (kidney CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, cancer), Renal Pelvis and Ureter (Transitional Cell), Salivary CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, Gland Cancer, Sezary Syndrome, Skin Cancer, Skin Cancer CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41, (Cutaneous T-Cell Lymphoma, Kaposi’s Sarcoma, Mela CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, noma), Small Intestine Cancer, Soft Tissue Sarcoma, Stom CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, ach Cancer, Testicular Cancer, Thymoma (Malignant), Thy CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, roid Cancer, Urethral Cancer, Uterine Cancer (Sarcoma), CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, Unusual Cancer of Childhood, Vaginal Cancer, Vulvar Can CD91, CD95, CD96, CD100, CD103, CD105, CD106, cer, Wilms Tumor. CD109, CD117, CD120, CD127, CD133, CD134, CD135, 0109. In another specific embodiment, the cell-binding CD138, CD141, CD142, CD143, CD144, CD147, CD151, drug conjugates via the hydrophilic likers of this invention are US 2015/0250896 A1 Sep. 10, 2015

used in accordance with the compositions and methods for berg syndrome, Parsonnage-Turner syndrome, Pars planitis, the treatment or prevention of an autoimmune disease. The Pemphigus, Pemphigus vulgaris, Pernicious anaemia, autoimmune diseases include, but are not limited, Achlorhy Perivenous encephalomyelitis, POEMS syndrome, Pol dra Autoimmune Active Chronic Hepatitis, Acute Dissemi yarteritis nodosa, Polymyalgia rheumatica, Polymyositis, nated Encephalomyelitis, Acute hemorrhagic leukoencepha Primary biliary cirrhosis, Primary Sclerosing cholangitis, litis, Addison's Disease, Agammaglobulinemia, Alopecia Progressive inflammatory neuropathy, Psoriasis, Psoriatic areata, Amyotrophic Lateral Sclerosis, Ankylosing Spondyli Arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Ras tis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, mussen's encephalitis, Raynaud phenomenon, Relapsing Antisynthetase syndrome, Arthritis, Atopic allergy, Atopic polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid Dermatitis, Autoimmune Aplastic Anemia, Autoimmune car fever, Sarcoidosis, Schizophrenia, Schmidt syndrome, diomyopathy, Autoimmune hemolytic anemia, Autoimmune Schnitzler syndrome, Scleritis, Scleroderma, Sjögren's syn hepatitis, Autoimmune inner ear disease, Autoimmune lym drome, Spondyloarthropathy, Sticky blood syndrome, Stills phoproliferative syndrome, Autoimmune peripheral neur Disease, Stiff person syndrome, Subacute bacterial opathy, Autoimmune pancreatitis, Autoimmune polyendo endocarditis, Susac's syndrome, Sweet syndrome, Sydenham crine syndrome Types I, II, & III, Autoimmune progesterone Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Tem dermatitis, Autoimmune thrombocytopenic purpura, poral arteritis (giant cell arteritis), Tolosa-Hunt syndrome, Autoimmune uveitis, Balo disease/Balo concentric Sclerosis, Bechets Syndrome, Berger's disease, Bickerstaffs encepha Transverse Myelitis, Ulcerative Colitis (a type of idiopathic litis, Blau syndrome, Bullous Pemphigoid, Castleman's dis inflammatory bowel diseases). Undifferentiated connective ease, Chagas disease, Chronic Fatigue Immune Dysfunction tissue disease. Undifferentiated spondyloarthropathy, Vascu Syndrome, Chronic inflammatory demyelinating polyneur litis, Vitiligo, Wegener's granulomatosis, Wilson's Syn opathy, Chronic recurrent multifocal ostomyelitis, Chronic drome, Wiskott-Aldrich syndrome lyme disease, Chronic obstructive pulmonary disease, Churg 0110. In another specific embodiment, a binding molecule Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, used for the conjugate via the hydrophilic linkers of this Cogan syndrome, Cold agglutinin disease, Complement invention for the treatment or prevention of an autoimmune component 2 deficiency, Cranial arteritis, CREST syndrome, disease includes, but are not limited to, anti-elastin antibody; Crohns Disease (a type of idiopathic inflammatory bowel Abys against epithelial cells antibody; Anti-Basement Mem diseases), Cushing's Syndrome, Cutaneous leukocytoclastic brane CollagenType IV Protein antibody; Anti-Nuclear Anti angiitis, Dego's disease, Dercum’s disease, Dermatitis her body; Antids DNA; Antiss DNA, Anti Cardiolipin Antibody petiformis, Dermatomyositis, Diabetes mellitus type 1. Dif IgM, IgG; anti-celiac antibody; Anti Phospholipid Antibody fuse cutaneous systemic sclerosis, Dressler's syndrome, Dis IgK, IgG, Anti SM Antibody; Anti Mitochondrial Antibody; coid lupus erythematosus, Eczema, Endometriosis, Thyroid Antibody; Microsomal Antibody, T-cells antibody; Enthesitis-related arthritis, Eosinophilic fasciitis, Epider Thyroglobulin Antibody, Anti SCL-70; Anti-Jo; Anti-U.Sub. molysis bullosa acquisita, Erythema nodosum, Essential 1RNP; Anti-La/SSB; Anti SSA; Anti SSB; Anti Perital Cells mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia Antibody; Anti Histones: Anti RNP; C-ANCA; P-ANCA: ossificans progressiva, Fibromyalgia, Fibromyositis, Fibros Anti centromere; Anti-Fibrillarin, and Anti GBMAntibody, ing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant Anti-ganglioside antibody; Anti-Desmogein 3 antibody; cell arteritis, Glomerulonephritis, Goodpasture's syndrome, Anti-p62 antibody; Anti-sp100 antibody; Anti-Mitochondrial Graves disease, Guillain-Barré syndrome, Hashimoto's (M2) antibody; Rheumatoid factorantibody; Anti-MCV anti encephalitis, Hashimoto's thyroiditis, Haemolytic anaemia, body; Anti-topoisomerase antibody; Anti-neutrophil cyto Henoch-Schonlein purpura, Herpes gestationis, Hidradenitis plasmic(cANCA) antibody; Suppurativa, Hughes syndrome (See Antiphospholipid syn 0111. In certain preferred embodiments, the binding mol drome), Hypogammaglobulinemia, Idiopathic Inflammatory ecule for the conjugate in the present invention, can bind to Demyelinating Diseases, Idiopathic pulmonary fibrosis, Idio both a receptor or a receptor complex expressed on an acti pathic thrombocytopenic purpura (See Autoimmune throm vated lymphocyte which is associated with an autoimmune bocytopenic purpura), IgA nephropathy (Also Berger's dis disease. The receptor or receptor complex can comprise an ease), Inclusion body myositis, Inflammatory demyelinating immunoglobulin gene Superfamily member (e.g. CD2, CD3. polyneuopathy, Interstitial cystitis, Irritable Bowel Syn CD4, CD8, CD19, CD20, CD22, CD28, CD30, CD37, CD38, drome, Juvenile idiopathic arthritis, Juvenile rheumatoid CD56, CD70, CD79, CD90, CD125, CD152/CTLA-4, PD-1, arthritis, Kawasaki's Disease, Lambert-Eaton myasthenic or ICOS), a TNF receptor superfamily member (e.g. CD27. syndrome, Leukocytoclastic vasculitis, Lichen planus, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, INF-R1, Lichen Sclerosus, Linear IgA disease (LAD), Lou Gehrig's TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/ Disease (Also Amyotrophic lateral Sclerosis), Lupoid hepa TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO titis, Lupus erythematosus, Majeed syndrome, Méniere's dis 3), an integrin, a cytokine receptor, a chemokine receptor, a ease, Microscopic polyangiitis, Miller-Fisher syndrome, major histocompatibility protein, a lectin (C-type, S-type, or Mixed Connective Tissue Disease, Morphea, Mucha-Haber I-type), or a complement control protein. mann disease, Muckle-Wells syndrome, Multiple Myeloma, 0112. In another specific embodiment, useful binding Multiple Sclerosis, Myasthenia gravis, Myositis, Narcolepsy, ligands that are immunospecific for a viral or a microbial Neuromyelitis optica (Devic's Disease), Neuromyotonia, antigen are humanized or human monoclonal antibodies. As Occular cicatricial pemphigoid, OpSoclonus myoclonus Syn used herein, the term “viral antigen’ includes, but is not drome, Ord thyroiditis, Palindromic rheumatism, PANDAS limited to, any viral peptide, polypeptide protein (e.g. HIV (Pediatric Autoimmune Neuropsychiatric Disorders Associ gp120, HIV nef, RSV F glycoprotein, influenza virus ated with Streptococcus), Paraneoplastic cerebellar degen neuramimidase, influenza virus hemagglutinin, HTLV tax, eration, Paroxysmal nocturnal hemoglobinuria, Parry Rom herpes simplex virus glycoprotein (e.g. gB, gC, gD, and gE) US 2015/0250896 A1 Sep. 10, 2015 and hepatitis B surface antigen) that is capable of eliciting an Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleo immune response. As used herein, the term “microbial anti sis (Mono), Influenza, Isosporiasis, Kawasaki disease, gen’ includes, but is not limited to, any microbial peptide, Keratitis, Kingella kingae , Kuru, Lassa fever, polypeptide, protein, Saccharide, polysaccharide, or lipid Legionellosis (Legionnaires disease), Legionellosis (Pon molecule (e.g., a bacterial, fungi, pathogenic protozoa, or tiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listerio polypeptide including, e.g., LPS and capsular polysac sis, Lyme disease (Lyme borreliosis), Lymphatic filariasis charide 5/8) that is capable of eliciting an immune response. (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Examples of antibodies available 1 for the viral or microbial Marburg hemorrhagic fever, Measles, Melioidosis (Whit infection include, but are not limited to, Palivizumab which is more's disease), Meningitis, Meningococcal disease, Metag a humanized anti-respiratory syncytial virus monoclonal onimiasis, , Molluscum contagiosum, antibody for the treatment of RSV infection: PRO542 which Mumps, Murine typhus (Endemic typhus), Mycoplasma is a CD4 fusion antibody for the treatment of HIV infection; pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis Ostavir which is a human antibody for the treatment of hepa (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob titis B virus; PROTVIR which is a humanized IgG. Sub.1 disease (VCJD, invoD), Nocardiosis. Onchocerciasis (River antibody for the treatment of cytomegalovirus; and anti-LPS blindness), (South American blasto antibodies. mycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis 0113. The cell binding molecules—drug conjugates via pubis (Pubic lice, Crab lice), Pelvic inflammatory disease, the hydrophilic linkers of this invention can be used in the Pertussis (Whooping cough), Plague, Pneumococcal infec treatment of infectious diseases. These infectious diseases tion, , Pneumonia, Poliomyelitis, include, but are not limited to, Acinetobacter , Acti Prevotella infection, Primary amoebic meningoencephalitis, nomycosis, African sleeping sickness (African trypanoso Progressive multifocal leukoencephalopathy, Psittacosis, Q miasis), AIDS (Acquired immune deficiency syndrome), fever, Rabies, Rat-bite fever, Respiratory syncytial virus Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium infection, , Rhinovirus infection, Rickettsial haemolyticum infection, Argentine hemorrhagic fever, infection, Rickettsialpox, Rift Valley fever, Rocky mountain Ascariasis, , Astrovirus infection, Babesiosis, spotted fever, Rotavirus infection, Rubella, Salmonellosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial SARS (Severe Acute Respiratory Syndrome), Scabies, Schis vaginosis, Bacteroides infection, Balantidiasis, Baylisascaris tosomiasis, Sepsis, Shigellosis (Bacillary dysentery), infection, BK virus infection, Black , Blastocystis Shingles (Herpes Zoster), Smallpox (Variola), , hominis infection, , Bolivian hemorrhagic Staphylococcal food poisoning, Staphylococcal infection, fever, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia Strongyloidiasis, Syphilis, Taeniasis, Tetanus (Lockjaw), infection, Buruli ulcer, Calicivirus infection (Norovirus and (Barber's itch), (Ringworm of the Sapovirus), Campylobacteriosis, (Moniliasis; Scalp), (Ringworm of the Body), Thrush), Cat-scratch disease, Cellulitis, Chagas Disease (Jock itch), (Ringworm of the Hand), Tinea (American trypanosomiasis), Chancroid, Chickenpox, nigra, Tinea pedis (Athlete's foot), Tinea unguium (Onycho Chlamydia, Chlamydophila pneumoniae infection, Cholera, mycosis), (Pityriasis versicolor), Toxocari , Clonorchiasis, Clostridium difficile asis (Ocular Larva Migrans), Toxocariasis (Visceral Larva infection, , Colorado tick fever, Com Migrans), Toxoplasmosis, Trichinellosis, Trichomoniasis, mon cold (Acute viral rhinopharyngitis; Acute coryza), Trichuriasis (Whipworm infection), Tuberculosis, Tulare Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic mia, Ureaplasma urealyticum infection, Venezuelan equine fever, , Cryptosporidiosis, Cutaneous larva encephalitis, Venezuelan hemorrhagic fever, Viral pneumo migrans, Cyclosporiasis, Cysticercosis, Cytomegalovirus nia, West Nile Fever, (Tinea blanca), Yersinia infection, Dengue fever, Dientamoebiasis, Diphtheria, pseudotuberculosis infection, Yersiniosis, Yellow fever, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic . fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm 0114. The cell binding molecules, which are more prof infection), Enterococcus infection, Enterovirus infection, fered to be antibodies described in this patent that are against Epidemic typhus, Erythema infectiosum (Fifth disease), pathogenic strains include, but are not limit, Acinetobacter EXanthem Subitum, Fasciolopsiasis, Fasciolosis, Fatal famil baumannii, Actinomyces israelii, Actinomyces gerencseriae ial insomnia, Filariasis, Food poisoning by Clostridium per and Propionibacterium propionicus, Trypanosoma brucei, fringens, Free-living amebic infection, Fusobacterium infec HIV (Human immunodeficiency virus), Entamoeba his tion, Gas gangrene (Clostridial myonecrosis), , tolytica, Anaplasma genus, Bacillus anthracis, Arcanobacte Gerstmann-Sträussler-Scheinker syndrome, Giardiasis, rium haemolyticum, Junin virus, Ascaris lumbricoides, Glanders, Gnathostomiasis, Gonorrhea, Granuloma ingui genus, Astroviridae family, Babesia genus, Bacil male (Donovanosis), Group A Streptococcal infection, Group lus cereus, multiple , Bacteroides genus, Balantidium B Streptococcal infection, Haemophilus influenzae infection, coli, Baylisascaris genus, BK virus, , Blasto Hand, foot and mouth disease (HFMD), Hantavirus Pulmo cystis hominis, Blastomyces dermatitides, Machupo virus, nary Syndrome, Helicobacter pylori infection, Hemolytic Borrelia genus, Clostridium botulinum, Sabia, Brucella uremic syndrome, Hemorrhagic fever with renal syndrome, genus, usually Burkholderia cepacia and other Burkholderia Hepatitis A, Hepatitis B. Hepatitis C, Hepatitis D. Hepatitis E. species, Mycobacterium ulcerans, Caliciviridae family, Herpes simplex. , Hookworm infection, Campylobacter genus, usually and other Human bocavirus infection, Human ewingii ehrlichiosis, Candida species, Bartonella hemselae, Group A Streptococ Human granulocytic anaplasmosis, Human metapneumovi cus and Staphylococcus, Trypanosoma Cruzi, Haemophilus rus infection, Human monocytic ehrlichiosis, Human papil ducreyi, Varicella Zoster virus (VZV), Chlamydia trachoma lomavirus infection, Human parainfluenza virus infection, tis, Chlamydophila pneumoniae, Vibrio cholerae, Fonsecaea US 2015/0250896 A1 Sep. 10, 2015 14 pedrosoi, Clonorchis sinensis, Clostridium difficile, Coccid chophyton genus, tonsurans, Trichophyton ioides immitis and posadasii, Colorado tick genus, , , fever virus, rhinoviruses, coronaviruses, CJD prion, and Trichophyton mentagrophytes, Trichophyton rubrum, Crimean-Congo hemorrhagic fever virus, Cryptococcus neo , Trichophyton genus, genus, formans, Cryptosporidium genus, Ancylostoma braziliense; Toxocara canis or Toxocara Cati, Toxoplasma gondii, Tri multiple parasites, Cyclospora Cayetanensis, Taenia solium, chinella spiralis, Trichomonas vaginalis, Trichuris trichiura, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 Mycobacterium tuberculosis, Francisella tularensis, Urea and DEN-4)—Flaviviruses, Dientamoeba fragilis, Coryne plasma urealyticum, Venezuelan equine encephalitis virus, bacterium diphtheriae, Diphyllobothrium, Dracunculus Vibrio Colerae, Guanarito virus, West Nile virus, Trichos medimensis, Ebolavirus, Echinococcus genus, Ehrlichia poron beigelii, Yersinia pseudotuberculosis, Yersinia entero colitica, Yellow fever virus, order (Mucormyco genus, Enterobius vermicularis, Enterococcus genus, sis) and order (Entomophthoramycosis), Enterovirus genus, Rickettsia prowazekii, Parvovirus B19, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Human herpesvirus 6 and Human herpesvirus 7. Fasciolopsis Aeromonas hydrophila, Edwardsiella tarda, Yersinia pestis, buski, Fasciola hepatica and Fasciola gigantica, EH prion, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Sal Filarioidea Superfamily, Clostridium perfingens, Fusobac monella typhimurium, Treponema pertenue, Treponema car terium genus, Clostridium perfiringens; other Clostridium ateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira species, , GSS prion, Giardia intesti icterohemorrhagiae, Pneumocystis carinii, Brucella abortus, nalis, Burkholderia mallei, Gnathostoma spinigerum and Brucella suis, Brucella melitensis, Mycoplasma spp., Rick Gnathostoma hispidum, Neisseria gonorrhoeae, Klebsiella ettsia prowazeki, Rickettsia tsutsugumushi, Clamydia spp.; granulomatis, Streptococcus pyogenes, Streptococcus aga pathogenic fungi (Aspergillus filmigatus, Candida albicans, lactiae, Haemophilus influenzae, Enteroviruses, mainly Cox ); protozoa (Entomoeba histolytica, sackie A virus and Enterovirus 71, Sin Nombre virus, Heli cobacter pylori, Escherichia coli O157:H7, Bunyaviridae Trichomonas tenas, Trichomonas hominis, Tryoanosoma family, Hepatitis A Virus, Hepatitis B Virus, Hepatitis C gambiense, Trypanosoma rhodesiense, Leishmania dono Virus, Hepatitis D Virus, Hepatitis E Virus, Herpes simplex vani, Leishmania tropica, Leishmania braziliensis, Pneu virus 1, Herpes simplex virus 2. Histoplasma capsulatum, mocystis pneumonia, Plasmodium vivax, Plasmodium falci Ancylostoma duodenale and Necator americanus, Hemophi parum, Plasmodium malaria); or Helminiths (Schistosoma lus influenzae, Human bocavirus, Ehrlichia ewingii, Ana japonicum, Schistosoma mansoni, Schistosoma haemato plasma phagocytophilum, Human metapneumovirus, Ehrli bium, and hookworms). chia chafeensis, Human papillomavirus, Human 0115 Other antibodies as cell binding ligands used in this parainfluenza viruses, Hymenolepis nana and Hymenolepis invention for treatment of viral disease include, but are not diminuta, Epstein-Barr Virus, Orthomyxoviridae family, limited to, antibodies againstantigens of pathogenic viruses, Isospora belli, Kingella kingae, Klebsiella pneumoniae, including as examples and not by limitation: Poxyiridae, Her Klebsiella Ozaenas, Klebsiella rhinoscleromotis, Kuru prion, pesviridae, Adenoviridae, Papovaviridae, Enteroviridae, Lassa virus, Legionella pneumophila, Legionella pneumo Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, phila, Leishmania genus, Mycobacterium leprae and Myco influenza viruses, parainfluenza viruses, mumps, measles, bacterium lepromatosis, Leptospira genus, Listeria monocy respiratory syncytial virus, rubella, Arboviridae, Rhabdoviri togenes, Borrelia burgdorferi and other Borrelia species, dae, Arenaviridae, Non-A/Non-B Hepatitis virus, Rhinoviri Wuchereria bancrofti and Brugia malayi, Lymphocytic chori dae, Coronaviridae, Rotoviridae. Oncovirus such as, HBV omeningitis virus (LCMV), Plasmodium genus, Marburg (Hepatocellular carcinoma), HPV (Cervical cancer, Anal virus, Measles virus, Burkholderia pseudomalilei, Neisseria cancer), Kaposi's sarcoma-associated herpesvirus (Kaposi's meningitides, Metagonimus yokagawai, phy sarcoma), Epstein-Barr virus (Nasopharyngeal carcinoma, lum, Molluscum contagiosum virus (MCV), Mumps virus, Burkitt's lymphoma, Primary central nervous system lym Rickettsia typhi, Mycoplasma pneumoniae, numerous species phoma), MCPyV (Merkel cell cancer), SV40 (Simian virus of bacteria (Actinomycetoma) and fungi (), 40), HCV (Hepatocellular carcinoma), HTLV-I (Adult T-cell parasitic dipterous fly larvae, Chlamydia trachomatis and leukemia/lymphoma). Immune disorders caused virus: Neisseria gonorrhoeae, VCJD prion, Nocardia asteroides and such as Human Immunodeficiency Virus (AIDS); Central other Nocardia species. Onchocerca volvulus, Paracoccidio nervous system virus: such as, JCV (Progressive multifocal ides brasiliensis, Paragonimus westermani and other Para leukoencephalopathy), MeV (Subacute Sclerosing panen gonimus species, Pasteurella genus, Pediculus humanus capi cephalitis), LCV (Lymphocytic choriomeningitis), Arbovirus tis, Pediculus humanus corporis, Phthirus pubis, Bordetella encephalitis, Orthomyxoviridae (probable) (Encephalitis pertussis, Yersinia pestis, Streptococcus pneumoniae, Pneu lethargica), RV (Rabies), Chandipura virus, Herpesviral men mocystis jirovecii, Poliovirus, PrevOtella genus, Naegleria ingitis, Ramsay Hunt syndrome type II; Poliovirus (Poliomy fowleri, JC virus, Chlamydophila psittaci, Coxiella burnetii, elitis, Post-polio syndrome), HTLV-I (Tropical spastic para Rabies virus, Streptobacillus moniliformis and Spirillum paresis); Cytomegalovirus (Cytomegalovirus retinitis, HSV minus, Respiratory syncytial virus, , (Herpetic keratitis)); Cardiovascular virus such as CBV Rhinovirus, Rickettsia genus, Rickettsia akari, Rift Valley (Pericarditis, Myocarditis); Respiratory system/acute viral fever virus, Rickettsia rickettsii, Rotavirus, Rubella virus, nasopharyngitis/viral pneumonia: Epstein-Barr virus (EBV Salmonella genus, SARS coronavirus, Sarcoptes scabiei, infection/Infectious mononucleosis), Cytomegalovirus; Schistosoma genus, Shigella genus, Varicella Zoster virus, SARS coronavirus (Severe acute respiratory syndrome) Orth Variola major or Variola minor, , Staphy omyxoviridae: Influenzavirus A/B/C (Influenza/Avian influ lococcus genus, Staphylococcus genus, Staphylococcus enza), Paramyxovirus: Human parainfluenza viruses (Parain aureus, Streptococcus pyogenes, Strongyloides Stercoralis, fluenza), RSV (Human respiratory syncytial virus), hMPV: Treponema pallidum, Taenia genus, Clostridium tetani, Tri Digestive system virus MuV (Mumps), Cytomegalovirus US 2015/0250896 A1 Sep. 10, 2015

(Cytomegalovirus esophagitis); Adenovirus (Adenovirus the type of disease, the species to which the patient belongs, infection); Rotavirus, Norovirus, Astrovirus, Coronavirus: the diseased State of the patient, the route of administration, HBV (Hepatitis B virus), CBV. HAV (Hepatitis. A virus), all factors which dictate the required dose amounts, delivery HCV (Hepatitis C virus), HDV (Hepatitis D virus), HEV and regimen to be administered. (Hepatitis E virus), HGV (Hepatitis G virus); Urogenital I0120 In general terms, the conjugates via the linkers of virus such as, BK virus, MuV (Mumps). this invention may be provided in an aqueous physiological 0116. According to a further object, the present invention buffer solution containing 0.1 to 10% w/v conjugates for also concerns pharmaceutical compositions comprising the parenteral administration. Typical dose ranges are from 1 conjugate via the hydrophilic linkers of the invention together ug/kg to 0.1 g/kg of body weight per day; a preferred dose with a pharmaceutically acceptable carrier for treatment of range is from 0.01 mg/kg to 20 mg/kg of body weight per day cancer and autoimmune disorders. The method for treatment or an equivalent dose in a human child. The preferred dosage of cancer and autoimmune disorders can be practiced in vitro, of drug to be administered is likely to depend on such vari in vivo, or ex vivo. Examples of in vitro uses include treat ables as the type and extent of progression of the disease or ments of cell cultures in order to kill all cells except for disorder, the overall health status of the particular patient, the desired variants that do not express the target antigen; or to relative biological efficacy of the compound selected, the kill variants that express undesired antigen. Examples of ex formulation of the compound, the route of administration Vivo uses include treatments of hematopoietic stem cells (intravenous, intramuscular, or other), the pharmacokinetic (HSC) prior to the performance of the transplantation properties of the compound by the chosen delivery route, and (HSCT) into the same patient in order to kill diseased or the speed (bolus or continuous infusion) and schedule of malignant cells. For instance, clinical ex vivo treatment to administrations (number of repetitions in a given period of remove tumour cells or lymphoid cells from bone marrow time). prior to autologous transplantation in cancer treatment or in I0121 The conjugates via the linkers of the present inven treatment of autoimmune disease, or to remove T cells and tion are also capable of being administered in unit dose forms, other lymphoid cells from allogeneic bone marrow or tissue wherein the term “unit dose” means a single dose which is prior to transplant in order to prevent graft-Versus-host dis capable of being administered to a patient, and which can be ease, can be carried out as follows. Bone marrow is harvested readily handled and packaged, remaining as a physically and from the patient or other individual and then incubated in chemically stable unit dose comprising either the active con medium containing serum to which is added the conjugate of jugate itself, or as a pharmaceutically acceptable composi the invention, concentrations range from about 1 pM to 0.1 tion, as described hereinafter. As such, typical total daily dose mM, for about 30 minutes to about 48 hours at about 37° C. ranges are from 0.01 to 100 mg/kg of body weight. By way of The exact conditions of concentration and time of incubation general guidance, unit doses for humans range from 1 mg to (-dose) are readily determined by the skilled clinicians. After 3000 mg per day. Preferably the unit dose range is from 1 to incubation the bone marrow cells are washed with medium 500 mg administered one to four times a day, and even more containing serum and returned to the patient by i.v. infusion preferably from 10 mg to 500 mg. once a day. Conjugates according to known methods. In circumstances where the provided herein can be formulated into pharmaceutical com patient receives other treatment such as a course of ablative positions by admixture with one or more pharmaceutically chemotherapy or total-body irradiation between the time of acceptable excipients. Such unit dose compositions may be harvest of the marrow and reinfusion of the treated cells, the prepared for use by oral administration, particularly in the treated marrow cells are stored frozen in liquid nitrogen using form of tablets, simple capsules or Soft gel capsules; or intra standard medical equipment. nasally, particularly in the form of powders, nasal drops, or 0117 For clinical in vivo use, the conjugate via the linkers aerosols; or dermally, for example, topically in ointments, of the invention will be supplied as solutions or as a lyo creams, lotions, gels or sprays, or via trans-dermal patches. philized solid that can be redissolved in sterile water for injection. Examples of suitable protocols of conjugate admin DrugS/Cytotoxic Agents istration are as follows. Conjugates are given weekly for 8 0.122 Drugs that can be conjugated to a cell-binding mol weeks as an i.v. bolus. Bolus doses are given in 50 to 500 ml ecule in the present invention are Small molecule drugs of normal saline to which human serum albumin (e.g. 0.5 to 1 including cytotoxic agents, which can be linked to or after mL of a concentrated solution of human serum albumin, 100 they are modified for linkage to the cell-binding agent. A mg/mL) can be added. Dosages will be about 50 ug to 20 “small molecule drug is broadly used herein to refer to an mg/kg of body weight per week, i.V. (range of 10 ug to 200 organic, inorganic, or organometallic compound that may mg/kg per injection). 8 weeks after treatment, the patient may have a molecular weight of for example 100 to 1800, more receive a second course of treatment. Specific clinical proto suitably from 120 to 1400. Small molecule drugs are well cols with regard to route of administration, excipients, dilu characterized in the art, such as in WO05058367A2, and in ents, dosages, times, etc., can be determined by the skilled U.S. Pat. No. 4,956.303, among others and are incorporated clinicians. in their entirety by reference. The drugs include known drugs 0118. Examples of medical conditions that can be treated and those that may become known drugs. according to the in vivo or ex vivo methods of killing selected I0123 Drugs that are known include, but not limited to, 1). cell populations include malignancy of any types of cancer, Chemotherapeutic agents: a). Alkylating agents: Such as autoimmune diseases, graft rejections, and infections (viral, Nitrogen mustards: chlorambucil, chlornaphazine, cyclo bacterial or parasite). phosphamide, dacarbazine, estramustine, ifosfamide, 0119 The amount of a conjugate which is required to mechlorethamine, mechlorethamine oxide hydrochloride, achieve the desired biological effect, will vary depending mannomustine, mitobronitol, melphalan, mitolactol, pipo upon a number of factors, including the chemical character broman, novembichin, phenesterine, prednimustine, istics, the potency, and the bioavailability of the conjugates, thiotepa, trofosfamide, uracil mustard; CC-1065 (including US 2015/0250896 A1 Sep. 10, 2015 its adoZelesin, carzelesin and bizelesin synthetic analogues); dynemicin; esperamicin, kedarcidin, C-1027, maduropeptin, duocarmycin (including the synthetic analogues, KW-2189 as well as neocarzinostatin chromophore and related chro and CBI-TMI); benzodiazepine dimers (e.g., dimmers of pyr moprotein enediyne antiobiotic chromomophores), aclacino rolobenzodiazepine (PBD) or tomayimycin, indolinobenzodi mysins, actinomycin, authramycin, aZaserine, bleomycins, azepines, imidazobenzothiadiazepines, or oxazolidinobenzo cactinomycin, carabicin, carminomycin, carzinophilin; chro diazepines); Nitrosoureas: (carmustine, lomustine, momycins, dactinomycin, daunorubicin, detorubicin, chlorozotocin, fotemustine, nimustine, ranimustine); Alkyl 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino Sulphonates: (buSulfan, treosulfan, improSulfan and piposul doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, fan); TriaZenes: (dacarbazine); Platinum containing com idarubicin, marcellomycin, nitomycins, , pounds: (carboplatin, cisplatin, oxaliplatin); aziridines. Such nogalamycin, olivomycins, peplomycin, potfiromycin, puro as benzodopa, carboquone, meturedopa, and uredopa; ethyl mycin, quelamycin, rodorubicin, streptonigrin, Streptozocin, enimines and methylamelamines including altretamine, tri tubercidin, ubenimex, Zinostatin, Zorubicin: f). Others: such ethylenemelamine, trietylenephosphoramide, triethyleneth as Polyketides (acetogenins), especially bullatacin and bul iophosphaoramide and trimethylolomelamine; b). Plant latacinone; gemcitabine, epoxomicins (e. g. carfilzomib), Alkaloids: Such as Vinca alkaloids: (Vincristine, vinblastine, bortezomib, , , , tose vindesine, Vinorelbine, navelbin); Taxoids: (paclitaxel, doc dostat, Zybrestat, PLX4032, STA-9090, Stimuvax, allovec etaxol) and their analogs, Maytansinoids (DM1, DM2, DM3, tin-7, Xegeva, Provenge, Yervoy, Isoprenylation inhibitors DM4, maytansine and ansamitocins) and their analogs, cryp (such as Lovastatin), Dopaminergic neurotoxins (such as tophycins (particularly cryptophycin 1 and cryptophycin 8): 1-methyl-4-phenylpyridinium ion), Cell cycle inhibitors epothilones, eleutherobin, discodermolide, bryostatins, (such as staurosporine), Actinomycins (such as Actinomycin dolostatins, auristatins, tubulysins, cephalostatins; pancrat D, dactinomycin), Bleomycins (such as bleomycin A2, bleo istatin; a sarcodictyin; spongistatin; c). DNA Topoisomerase mycin B2, peplomycin), Anthracyclines (such as daunorubi Inhibitors: Such as Epipodophyllins: (9-aminocamptothecin, cin, doxorubicin (adriamycin), idarubicin, epirubicin, piraru camptothecin, crisinatol, daunomycin, etoposide, etoposide bicin, Zorubicin, mitoxantrone, MDR inhibitors (such as phosphate, irinotecan, mitoxantrone, novantrone, retinoic Verapamil), Cat ATPase inhibitors (such as thapsigargin), acids (retinols), teniposide, topotecan, 9-nitrocamptothecin Histone deacetylase inhibitors (Vorinostat, Romidepsin, (RFS 2000)); mitomycins: (mitomycin C); d). Anti-metabo Panobinostat, Valproic acid, Mocetinostat (MGCD0103), lites: such as {|Anti-folate: DHFR inhibitors: (, Belinostat, PCI-24781, Entinostat, SB939, Resminostat, trimetrexate, denopterin, pteropterin, aminopterin (4-ami Givinostat, AR-42, CUDC-101, sulforaphane, Trichostatin nopteroic acid) or the other folic acid analogues); IMP dehy A) , Thapsigargin, Celecoxib, glitaZones, epigallocatechin drogenase Inhibitors: (mycophenolic acid, tiazofurin, ribavi gallate, Disulfiram, Salinosporamide A.; Anti-adrenals, such rin, EICAR); Ribonucleotide reductase Inhibitors: as aminoglutethimide, mitotane, triloStane; aceglatone; (hydroxyurea, deferoxamine); Pyrimidine analogs: Uracil aldophosphamide glycoside; aminolevulinic acid; amsa analogs: (ancitabine, azacitidine, 6-azauridine, capecitabine crine; arabinoside, bestrabucil; bisantrene; ediatraxate; defo (Xeloda), carmofur, cytarabine, dideoxyuridine, doxifluri famine; demecolcine; diaziquone; eflornithine (DFMO), dine, enocitabine, 5-Fluorouracil, floxuridine, ratitrexed (To elfomithine; elliptinium acetate, etoglucid, gallium nitrate; mudex)); Cytosine analogs: (cytarabine, cytosine arabino gacytosine, hydroxyurea; ibandronate, lentinan; lonidamine; side, fludarabine); Purine analogs: (, fludarabine, mitoguaZone, mitoxantrone; mopidamol; nitracrine; pen mercaptopurine, thiamiprine, thioguanine); folic acid to statin: phenamet; pirarubicin, podophyllinic acid; 2-ethyl replenisher, such as frolinic acid; e). Hormonal therapies: hydrazide; procarbazine: PSKR); razoxane: rhizoxin; sizofi such as Receptor antagonists: Anti-estrogen: (megestrol, ran; Spirogermanium; tenuaZonic acid; triaziquone; 2.2.2"- raloxifene, tamoxifen); LHRH agonists: (goscrclin, leupro trichlorotriethylamine; trichothecenes (especially T-2 toxin, lide acetate); Anti-androgens: (bicalutamide, flutamide, Verrucarin A, roridin A and anguidine); urethane, siRNA, calusterone, dromoStanolone propionate, epitiostanol, goser antisense drugs, and a nucleolytic enzyme. elin, leuprolide, mepitioStane, nilutamide, testolactone, trilostane and other androgens inhibitors); Retinoids/Del 0.124 2). An anti-autoimmune disease agent includes, but toids: Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, is not limited to, cyclosporine, cyclosporine A, aminocaproic cholecalciferol, ergocalciferol); Photodynamic therapies: acid, azathioprine, bromocriptine, chlorambucil, chloro (verteporfin, phthalocyanine, photosensitizer PC4. quine, cyclophosphamide, corticosteroids (e.g. amcinonide, demethoxy-hypocrellin A); Cytokines: (Interferon-alpha, betamethasone, budesonide, hydrocortisone, flunisolide, flu Interferon-gamma, tumor necrosis factor (TNFs), human pro ticasone propionate, fluocortolone danazol, dexamethasone, teins containing a TNF domain)}: f). Kinase inhibitors, such Triamcinolone acetonide, beclometasone dipropionate), as BIBW 2992 (anti-EGFR/Erb2), imatinib, gefitinib, pegap DHEA, enanercept, hydroxychloroquine, infliximab, tanib, Sorafenib, dasatinib, Sunitinib, erlotinib, nilotinib, meloxicam, methotrexate, mofetil, mycophenylate, pred lapatinib, axitinib, pazopanib. Vandetanib, E7080 (anti nisone, , . VEGFR2), mubritinib, ponatinib (AP24534), bafetinib 0.125 3). An anti-infectious disease agent includes, but is (INNO-406), bosutinib (SKI-606), cabozantinib, vismode not limited to, a). Aminoglycosides: amikacin, astromicin, gib, iniparib, ruxolitinib, CYT387, axitinib, tivoZanib, sor gentamicin (netilmicin, Sisomicin, isepamicin), hygromycin afenib, bevacizumab, cetuximab, Trastuzumab, Ranibi B. kanamycin (amikacin, arbekacin, bekanamycin, dibeka Zumab, Panitumumab, ispinesib; g). antibiotics, such as the cin, tobramycin), neomycin (framycetin, paromomycin, enediyne antibiotics (e.g. calicheamicins, especially cali ribostamycin), netilmicin, spectinomycin, Streptomycin, cheamicin. Y1, 61, C.1.and B1, see, e.g., J. Med Chem., 39 tobramycin, Verdamicin; b). Amphenicols: azidamfenicol, (11), 2103-2117 (1996), Angew Chem Intl. Ed. Engl.33:183 chloramphenicol, florfenicol, thiamphenicol; c). Ansamy 186 (1994); dynemicin, including dynemicin A and deoxy cins: geldanamycin, herbimycin; d). Carbapenems: biap US 2015/0250896 A1 Sep. 10, 2015 enem, doripenem, ertapenem, imipenem/cilastatin, mero 0.126 4). Anti-viral drugs: a). Entry/fusion inhibitors: penem, panipenem; e). Cephems: carbacephem (loracarbef), , , . (), PRO cefacetrile, cefaclor, cefradine, cefadroxil, cefalonium, cefa 140, CD4 (ibalizumab); b). Integrase inhibitors: , loridine, cefalotin or cefalothin, cefalexin, cefaloglycin, cefa , globoidnan A; c). Maturation inhibitors: beviri mandole, cefapirin, cefatrizine, cefazaflur, cefazedone, cefa mat, Vivecon; d). Neuraminidase inhibitors: oseltamivir, Zan Zolin, cefbuperaZone, cefcapene, cefdaloxime, cefepime, amivir, peramivir; e). Nucleosides & nucleotides: , cefninox, cefoxitin, cefprozil, cefroxadine, ceftezole, aciclovir, adefovir, , , brivudine, cido cefuroxime, cefixime, cefdinir, cefditoren, cefepime, cefe fovir, clevudine, , (ddI), elvucit abine, emitricitabine (FTC), entecavir, famciclovir, fluorou tamet, cefimenoxime, cefodizime, cefonicid, cefoperaZone, racil (5-FU), 3'-fluoro-substituted 2',3'-dideoxynucleoside ceforanide, cefotaxime, cefotiam, cefoZopran, cephalexin, analogues (e.g. 3'-fluoro-2',3'-dideoxythymidine (FLT) and cefpimizole, cefpiramide, cefpirome, cefpodoxime, cef 3'-fluoro-2',3'-dideoxyguanosine (FLG), fomivirsen, ganci prozil, cefauinome, cefsulodin, ceftazidime, cefteram, cefti clovir, idoxuridine, (3TC), 1-nucleosides (e.g. buten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, B-1-thymidine and B-1-2'-deoxycytidine), penciclovir, cefuroxime, cefuZonam, cephamycin (cefoxitin, cefotetan, , ribavirin, , (d4T), taribavirin (vi cefnetazole), oxacephem (flomoxef, latamoxe?): f). Glyco ramidine), telbivudine, tenofovir, trifluridine Valaciclovir, peptides: bleomycin, Vancomycin (oritavancin, telavancin), valganciclovir, (ddC), (AZT): f). Non teicoplanin (dalbavancin), ramoplanin; g). Glycylcyclines: e. nucleosides: amantadine, ateviridine, , diarylpy g. tigecycline:g). B-Lactamase inhibitors: penam (Sulbactam, rimidines (, ), , docosanol, taZobactam), clavam (clavulanic acid); i). Lincosamides: , , (phosphonoformic acid), imi clindamycin, lincomycin; ). Lipopeptides: daptomycin, quimod, interferon alfa, , , methisaZone, A54145, calcium-dependent antibiotics (CDA); k). Mac , NOV-205, peginterferon alfa, podophyllotoxin, rolides: azithromycin, cethromycin, clarithromycin, dirithro rifampicin, rimantadine, residuimod (R-848), tromantadine; mycin, erythromycin, flurithromycin, josamycin, ketolide g). Protease inhibitors: , , boceprevir, (telithromycin, cethromycin), midecamycin, miocamycin, , , , , , ple oleandomycin, rifamycins (rifampicin, rifampin, rifabutin, conaril, , , telaprevir (VX-950), ; rifapentine), rokitamycin, roXithromycin, spectinomycin, h). Other types of anti-virus drugs: , arbidol, calano spiramycin, tacrolimus (FK506), troleandomycin, tellithro lide a, , cyanovirin-n, diarylpyrimidines, epigallo mycin; 1). Monobactams: aztreonam, tigemonam, m). catechin gallate (EGCG), foscarnet, , taribavirin Oxazolidinones: linezolid; n). Penicillins: amoxicillin, ampi cillin (pivampicillin, hetacillin, bacampicillin, metampicillin, (viramidine), hydroxyurea, KP-1461, , plecon talampicillin), azidocillin, azlocillin, benzylpenicillin, ben aril, portmanteau inhibitors, ribavirin, . Zathine benzylpenicillin, benzathine phenoxymethylpenicil I0127. 5). The drugs used for conjugates via a hydrophilic lin, clometocillin, procaine benzylpenicillin, carbenicillin linker of the present invention also include radioisotopes. (carindacillin), cloxacillin, dicloxacillin, epicillin, fluclox Examples of radioisotopes () are H, C, C, acillin, mecillinam (pivmecillinam), mezlocillin, meticillin, 18F 32P 35S, 6.Cu, Ga, 86Y. 99Tc, 11 In, 123, 1241, 125I, 13 II, nafcillin, oxacillin, penamecillin, penicillin, pheneticillin, 'Xe, 77Lu, ''At, or 'Bi. Radioisotope labeled antibod phenoxymethylpenicillin, piperacillin, propicillin, Sulbeni ies are useful in receptor targeted imaging experiments or can cillin, temocillin, ticarcillin; o). Polypeptides: bacitracin, be for targeted treatment such as with the antibody-drug colistin, polymyxin B; p). Quinolones: alatrofloxacin, balof conjugates of the invention (Wu et al (2005) Nature Biotech loxacin, ciprofloxacin, clinafloxacin, danofloxacin, difloxa nology 23(9): 1137-1146). The cell binding molecules, e.g. cin, enoxacin, enrofloxacin, floxin, garenoxacin, gatifloxa an antibody can be labeled with ligand reagents through the cin, gemifloxacin, grepafloxacin, kano trovafloxacin, hydrophilic linkers of the present patent that bind, chelate or levofloxacin, lomefloxacin, marbofloxacin, moxifloxacin, otherwise complex a radioisotope metal, using the techniques nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, pefloxa described in Current Protocols in Immunology, Volumes 1 cin, trovafloxacin, grepafloxacin, sitafloxacin, sparfloxacin, and 2, Coligen etal, Ed. Wiley-Interscience, New York, N.Y., temafloxacin, to Sufloxacin, trovafloxacin: q). Strepto Pubs. (1991). Chelating ligands which may complex a metal gramins: pristinamycin, quinupristin?dalfopristin); r). Sul ion include DOTA, DOTP, DOTMA, DTPA and TETA (Mac fonamides: mafenide, prontosil, Sulfacetamide, Sulfame rocyclics, Dallas, Tex.). thizole, Sulfanilimide, SulfaSalazine, Sulfisoxazole, I0128 6). The pharmaceutically acceptable salts, acids or trimethoprim, trimethoprim-sulfamethoxazole (co-trimox derivatives of any of the above drugs. azole); S). Steroid antibacterials: e.g. fusidic acid; t). Tetracy I0129. Preferred cytotoxic agents that conjugated to a cell clines: doxycycline, chlortetracycline, clomocycline, deme binding molecule via a hydrophilic linker of this patent are clocycline, lymecycline, meclocycline, metacycline, tubulysins, maytansinoids, taxanoids (taxanes), CC-1065 minocycline, oxytetracycline, penimepicycline, rollitetracy analogs, daunorubicin and doxorubicin compounds, benzo cline, tetracycline, glycylcyclines (e.g. tigecycline); u). Other diazepine dimers (e.g., dimers of pyrrolobenzodiazepine types of antibiotics: annonacin, arsphenamine, bactoprenol (PBD), tomayimycin, anthramycin, indolinobenzodiazepines, inhibitors (Bacitracin), DADAL/AR inhibitors (cycloserine), imidazobenzothiadiazepines, or oxazolidinobenzodiaz dictyostatin, discodermolide, eleutherobin, epothilone, epines), calicheamicins and the enediyne antibiotics, actino ethambutol, etoposide, faropenem, fusidic acid, furazoli mycin, azaserines, bleomycins, epirubicin, tamoxifen, idaru done, isoniazid, laulimalide, metronidazole, mupirocin, bicin, dolastatins/auristatins (e.g. monomethyl auristatin E, mycolactone, NAM synthesis inhibitors (e.g. fosfomycin), MMAE, MMAF, auristatin PYE, auristatin TP, Auristatins nitrofurantoin, paclitaxel, platensimycin, pyrazinamide, 2-AQ, 6-AQ, EB (AEB), and EFP (AEFP)), duocarmycins, quinupristin?dalfopristin, rifampicin (rifampin), taZobactam thiotepa, Vincristine, hemiasterlins, esperamicins, and their tinidazole, uvaricin; analogues and derivatives thereof. US 2015/0250896 A1 Sep. 10, 2015

0130 Tubulysins that are preferred for conjugation in the are exampled in. KC. Nicolaou et al., J. Am. Chem. Soc. 117. present invention are well known in the art and can be isolated 2409-2420, (1995); Ojima etal, J. Med. Chem. 39:3889-3896 from natural sources according to known methods or pre (1996): 40:267-278 (1997): 45,5620-5623 (2002); Ojima et pared synthetically according to known methods (e.g. Bala al., Proc. Natl. Acad. Sci., 96:4256-4261 (1999: Kim et al., subramanian, R.; et al. J. Med. Chem., 2009, 52, 238-240. Bull. Korean Chem. Soc., 20, 1389-1390 (1999); Miller, et al. Wipf, P.; etal. Org. Lett., 2004, 6,4057-4060. Pando, O.; etal. J. Med. Chem., 47, 4802-4805(2004); U.S. Pat. Nos. 5,475, J. Am. Chem. Soc., 2011, 133,7692-7695. Reddy, J. A.; etal. 011 5,728,849, 5,811,452; 6,340,701; 6,372,738; 6,391,913, Mol. Pharmaceutics, 2009, 6, 1518-1525. Raghavan, B.; etal. 6,436,931; 6,589,979; 6,596,757; 6,706,708; 7,008,942: J. Med. Chem., 2008, 51, 1530-1533. Patterson, A. W.; et al. 7,186,851; 7,217,819; 7,276,499; 7,598,290; 7,667,054. J. Org. Chem., 2008, 73, 4362-4369. Pando, O... et al. Org. 0.134 CC-1065 analogues and doucarmycin analogs are Lett., 2009, 11 (24), pp 5567-5569. Wipf, P; et al. Org. Lett., also preferred to be used for a conjugate with the hydrophilic 2007, 9(8), 1605-1607. Friestad, G. K.; Org. Lett., 2004, 6, pp linkers of the present patent. The examples of the CC-1065 3249-3252. Hillary M. Peltier, H. M.; etal.J. Am. Chem. Soc., analogues and doucarmycin analogs as well as their synthesis 2006, 128, 16018-16019. Chandrasekhar, S.; et al. J. Org. are described in: e.g. Warpehoski et al., J. Med. Chem. 31:590 Chem., 2009, 74,9531-9534. Liu, Y.; etal. Mol. Pharmaceu 603 (1988), D. Boger et al., J. Org. Chem, 66; 6654–6661, tics, 2012, 9, 168-175. Friestad, G. K.; etal. Org. Lett., 2009, 2001; U.S. Pat. Nos: 4,169,888, 4.3919,04, 4,671,958, 4,816, 11, 1095-1098. Kubicek, K., et al., Angew Chem Int Ed Engl, 567, 4,912,227, 4,923,990, 4,952,394, 4,975,278, 4,978,757, 2010. 49: p. 4809-12. Chai, Y.; et al., Chem Biol, 2010, 17: 4,994,578, 5,037,993, 5,070,092, 5,084,468, 5,101,038, 296-309. Ullrich, A.; et al., Angew Chem Int Ed Engl, 2009, 5,117,006, 5,137,877, 5,138,059, 5,147,786, 5,187,186, 48. 4422-5. Sani, M.; et al. Angew Chem Int Ed Engl, 2007, 5,223,409, 5,225,539, 5,288,514, 5,324.483, 5,332,740, 46, 3526-9. Domling, A.; et al., Angew Chem Int Ed Engl, 5,332,837, 5,334,528, 5,403,484, 5,427,908, 5,475,092, 2006. 45, 7235-9. Patent applications: Zanda, M.; etal, Can. 5,495,009, 5,530,101, 5,545,806, 5,547,667, 5,569,825, Pat. Appl. CA2710693 (2011). Chai, Y.; et al. Eur. Pat. Appl. 5,571,698, 5,573,922, 5,580,717, 5,585,089, 5,585,499, 2174947 (2010), PCT WO 2010.034724. Leamon, C.; etal, 5,587,161, 5,595,499, 5,606,017, 5,622,929, 5,625,126, PCT WO 2010033733, WO 2009002993. Ellman, J.; etal, 5,629,430, 5,633,425, 5,641,780, 5,660,829, 5,661,016, PCT WO 2009134279; PCT WO 2009012958, US appl. 5,686,237, 5,693,762, 5,703,080, 5,712,374, 5,714,586, 20110263650, 2011 0021568, Matschiner, G.; etal, PCTWO 5,739,116, 5,739,350, 5,770,429, 5,773,001, 5,773,435, 2009095447. Vlahov, I., et al., PCT WO 2009055562, WO 5,786,377 5,786,486, 5,789,650, 5,814,318, 5,846,545, 2008112873. Low, P; etal, PCT WO 2009026177. Richter, 5,874,299, 5,877,296, 5,877,397, 5,885,793, 5,939,598, W., PCT WO 2008138561. Kjems, J.; et al., PCT WO 5,962,216, 5,969,108, 5,985,908, 6,060,608, 6,066,742, 20081251 16. Davis, M.; etal, PCT WO 2008076333. Diener, 6,075,181, 6,103,236, 6,114,598, 6,130,237, 6,132,722, J.; et al, U.S. Pat. Appl. 20070041901, WO 2006096754. 6,143,901, 6,150,584, 6,162,963, 6,172,197, 6,180,370, Matschiner, G.; etal, PCT WO 2006056464. Vaghefi, F.; etal, 6,194.612, 6,214,345, 6,262,271, 6,281,354, 6,310,209, 5 PCT WO 2006033913. Doemling, A., Ger. Offen. DE 6,329,497, 6,342,480, 6,486,326, 6,512,101, 6,521,404, 102004030227; PCT WO 2004005327; WO 2004005326; 6,534,660, 6,544,731, 6,548,530, 6,555,313, 6,555,693, WO2004.005269. Stanton, M.: et al., U.S. Pat. Appl. Publ. 6,566,336, 6,586,618, 6,593,081, 6,630,579, 6,756,397, 20040249130. Hoefle, G.; etal, Ger. Offen. DE 10254439; 6,759,509, 6,762,179, 6,884,869, 6,897,034, 6,946,455, DE 10241152: DE 10008089. Leung, D.; et al., PCT WO 7,049,316, 7,087,600, 7,091,186, 7,115,573, 7,129,261, 2002077036. Reichenbach, H. et al, Ger. Offen. DE 7,214,663, 7,223,837, 7,304,032, 7,329,507, 7,329,760, 19638870; Wolfgang, R.; US 20120129779, Chen, H., US 7,388,026, 7,655,660, 7,655,661, 7,906,545, 8,012,978. appl. 20110027274. The preferred structure of tubulysins for 0.135 Daunorubicin/Doxorubicin Analogues are also pre conjugation of cell binding molecules are described in the ferred for conjugation via the hydrophilic linkers of the patent application of PCT/IB2012/053554 present patent. The preferred structures and their synthesis 0131 Calicheamicins and their related enediyne antibiot are exampled in: Hurwitz, E., et al., Cancer Res. 35, 1175 ics that are preferred for cell-binding molecule-drug conju 1181 (1975). Yang, H. M., and Reisfeld, R. A., Proc. Natl. gates of this patent are described in: Nicolaou, K. C. et al. Acad. Sci. 85, 1189-1193 (1988); Pietersz, C.A., E., et al., E., Science 1992, 256, 1172-1178; Proc. Natl. Acad. Sci USA. et al.” Cancer Res. 48,926-9311 (1988); Trouet, et al., 79, 1993, 90,5881-5888), U.S. Pat. Nos. 4,970, 198; 5,053,394; 626-629 (1982); Z. Brich et al., J. Controlled Release, 19, 5,108,912; 5,264,586; 5,384,412: 5,606,040; 5,712,374; 245-258 (1992); Chen et al., Syn. Comm, 33, 2377-2390, 5,714,586; 5,739,116; 5,770,701; 5,770,710; 5,773,001; 2003; King et al., Bioconj. Chem., 10,279-288, 1999: King et 5,877,296; 6,015,562: 6,124.310; 8,153,768. al., J. Med. Chem., 45, 4336-4343, 2002: Kratz et al., J Med 0132 Maytansinoids that are preferred to be used in the Chem. 45,5523-33. 2002: Kratz et al., Biol Pharm Bull. Jan. present invention including maytansinol and maytansinol 21, 56-61, 1998: Lau et al., Bioorg. Med. Chem. 3, 1305 analogues are described in U.S. Pat. Nos. 4.256,746, 4.361, 1312, 1995; Scott et al., Bioorg. Medl Chem. Lett. 6, 1491 650 and 4,307,016, 4,294,757, 4,294,757, 4,371,533, 4,424, 1496; 1996; Watanabe et al., Tokai J. Experimental Clin. 219, 4,331,598, 4.450,254, 4,364,866, 4,313,946, 4,315,929 Med. 15,327-334, 1990; Zhou et al., J. Am. Chem. Soc. 126, 4,362,663, 4,322,348, 4,371,533, 4,424,219, 5,208,020, 15656-7, 2004: WO 01/38318; U.S. Pat. Nos. 5,106,951; 5,416,064, 5,208,020; 5,416,064; 6,333.410; 6,441,163: 5,122,368; 5,146,064; 5,177,016; 5,208,323; 5,824.805; 6,716,821, 7,276,497, 7,301,019, 7,303,749, 7,368,565, 6,146,658; 6,214,345; 7,569,358; 7.803,903; 8,084,586: 7,411,063, 7,851,432, 8,163,888. 8,053,205. 0.133 Taxanes, which includes Paclitaxel (Taxol), a cyto 0.136 Auristatins and dolastatins are preferred in conjuga toxic natural product, and docetaxel (Taxotere), a semi-syn tion via the hydrophilic linkers of this patent. The auristatins thetic derivative, and their analogs which are preferred for (e.g. auristain E (AE) auristatin EB (AEB), auristatin EFP conjugation via the hydrophilic linkers of the present patent (AEFP), monomethyl auristatin E (MMAE), Monomethylau US 2015/0250896 A1 Sep. 10, 2015

ristatin (MMAF), Auristatin F phenylene diamine (AFP) and under nitrogen. All other reagents and solvents were pur a phenylalanine variant of MMAE) which are synthetic ana chased as the highest grade available and used without further logs of dolastatins, are described in Int. J. Oncol. 15:367-72 purification. NMR spectra were recorded on Varian Mercury (1999); Molecular Cancer Therapeutics, vol. 3, No. 8, pp. 300 MHz Instrument. Chemical shifts (delta.) are reported in 921-932 (2004); U.S. application Ser. Nos. 11/134,826, parts per million (ppm) referenced to tetramethylsilane at 2006007.4008, 2006022925. U.S. Pat. Nos. 4,414,205, 4,753, 0.00 and coupling constants (J) are reported in HZ. Low 894, 4,764,368, 4,816,444, 4,879,278, 4,943,628, 4,978,744, resolution mass spectral data were acquired on a Waters 5,122,368, 5,165,923, 5,169,774, 5,286,637, 5,410,024, Micromass ZMD mass spec with Waters 2795 HPLC sepa 5,521,284, 5,530,097, 5,554,725, 5,585,089, 5,599,902, rations module and the 2996 photodiode array detector. 5,629, 197, 5,635,483, 5,654,399, 5,663,149, 5,665,860, 5,708,146, 5,714,586, 5,741,892, 5,767,236, 5,767,237, 5,780,588, 5,821,337, 5,840,699, 5,965,537, 6,004,934, Example 1 6,033,876, 6,034,065, 6,048,720, 6,054,297, 6,054,561, 6,124,431, 6,143,721, 6,162,930, 6,214,345, 6,239,104, (2-Bromoethyl)(3-ethoxy-3-oxopropyl)phosphinic 6,323,315, 6,342,219, 6,342,221, 6,407,213, 6,569,834, acid (or ethyl 3-2-Bromoethyl(hydroxy)phosphinyl 6,620,911, 6,639,055, 6,884,869, 6,913,748, 7,090,843, propanoate) (4) 7,091,186, 7,097,840, 7,098,305, 7,098,308, 7,498,298, 7,375,078, 7.462,352, 7,553,816, 7,659,241, 7,662,387, 7,745,394, 7,754,681, 7,829,531, 7,837,980, 7,837,995, 0141 7,902,338, 7,964,566, 7,964,567, 7,851,437, 7,994,135. 0.137 The benzodiazepine dimers (e.g. dimmers of pyr O rolobenzodiazepine (PBD) or (tomayimycin), indolinobenzo diazepines, imidazobenzothiadiazepines, or oxazolidinoben Br Zodiazepines) which are preferred cytotoxic agents according n- lausOEt to the present invention are exampled in the art: U.S. Pat. Nos. OH 8,163,736; 8,153,627; 8,034,808; 7,834,005; 7,741,319; 7,704,924; 7.691,848; 7,678,787; 7,612,062; 7,608,615; 7,557,099: 7,528,128; 7,528,126; 7,511,032; 7,429,658; 0142. A mixture of ammonium hypophosphite (8.00 g,96 7,407,951; 7,326,700; 7,312,210; 7,265,105; 7,202,239; mmol) and hexamethydisilazane (20.0 mL, 96 mmol) was 7,189,710; 7,173,026; 7,109,193; 7,067,511; 7,064,120; heated at 120° C. for 1 h under argon. After the mixture was 7,056,913; 7,049,311; 7,022,699; 7,015,215; 6,979,684; cooled to 0° C., ethyl acrylate (10.4 mL, 96 mmol) was 6,951,853; 6,884,799; 6,800,622: 6,747,144; 6,660,856; carefully added dropwise, and the resulting mixture was 6,608, 192: 6,562,806; 6,977,254; 6,951.853; 6,909,006; stirred at 50° C. for 2 h. Then the mixture was cooled to room 6,344,451; 5,880,122; 4,935,362; 4,764,616; 4,761412: 4,723,007; 4,723,003: 4,683,230; 4,663,453; 4,508,647: temperature, dibromoethane (40.0 mL) was added, and the 4,464,467: 4,427,587; 4,000,304; US patent appl. mixture was stirred for 5 hat 120° C. The formed trimethyl 20100203007, 20100316656, 20030195196. bromosilane and excess dibromoethane were removed under 0138 Analogues and derivatives of the cytotoxic drugs/ vacuum. Then 100 mL of aqueous ethanol (1:1) were added agents described in the present patent can be conjugated via a dropwise to the residue and refluxed for 0.5 h. Then the hydrophilic linker of the present patent. One skilled in the art solvent was removed under vacuum and extracted with ethyl of drugs/cytotoxic agents will readily understand that each of acetate. The organic layer was dried over magnesium Sulfate the drugs/cytotoxic agents described herein can be modified and the solvent was removed under vacuum to give the title in Such a manner that the resulting compound still retains the compound 4 (10.85 g, 41% yield). H NMR (300 MHz, specificity and/or activity of the starting compound. The CDOD): 81.26 (t, J=7.1 Hz,3H), 2.07 (m, 2H), 2.42 (m. 2H), skilled artisan will also understand that many of these com 2.62 (m, 2H), 3.59 (m, 2H), 4.15 (q, J=7.1 Hz, 2H). PNMR pounds can be used in place of the drugs/cytotoxic agents (100 MHz, CDOD): 8 49.5; ESI MS m/z- CH13BrOP described herein. Thus, the drugs/cytotoxic agents of the (M-H), cacla. 271.98, found 271.97. present invention include analogues and derivatives of the compounds described herein. Example 2 0139 All references cited herein and in the examples that follow are expressly incorporated by reference in their entire ties. Ethyl 3-2-Bromoethyl(ethoxy)phosphinylpro panoate (5) and Ethyl 3-Ethoxy(vinyl)phosphinyl EXAMPLES propanoate (6) 0140. The invention is further described in the following examples, which are not intended to limit the scope of the 0143 invention. Cell lines described in the following examples were maintained in culture according to the conditions speci fied by the American Type Culture Collection (ATCC) or Deutsche Sammlung Von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany (DMSZ), unless otherwise specified. Cell culture reagents were obtained from Invitro gen Corp., unless otherwise specified. All anhydrous solvents were commercially obtained and stored in Sure-seal bottles US 2015/0250896 A1 Sep. 10, 2015 20

-continued 18.75 mmol). After being stirred under Arfor 3 h, the mixture was acidified with 3 MHPO to pH 3.0. The mixture was concentrated, and purified on a SiO, column eluted with water/MeCN/HOAc (1:10:0.01), pooled the fraction, added DMF (-5 ml), evaporated to dryness to afford the title com pound 9 (5.38 g, 83% yield). ESIMS, m/z-433.10 (M-H). Example 5 0144. An amount of 10.84 g of 4 (20 mmol) was treated with 100.0 mL of triethyl orthoformate, and the mixture was 3-(Hydroxy(2-(hydroxy(2-(pyridin-2-yldisulfanyl) refluxed with a Dean-Stark trap to remove ethanol and ethyl ethyl)phosphoryl)ethyl)phosphoryl)propanoic acid formate. Excess triethyl orthoformate was removed under (10) vacuum to give 5 and 6 (39.2:60.8 'P NMR ratio), 11.83 g). 6:'HNMR (300 MHz, CDOD) & 1.27(m, 6H), 2.19 (m. 2H), 0149 2.57 (m, 2H), 4.11 (m, 4H), 6.36 (m, 3H). 'P NMR (100 MHz, CDOD): & 44.9; 5: P NMR (100 MHz, CDOD) & 53.3: ESIMS m/z+, 5:323.01 (M+Na), 6: 243.09 (M+Na). Example 3 Clusa-ul-us -N/1u| 1uls OH (2-bromoethyl)(2-(ethoxy(3-ethoxy-3-oxopropyl) OH OH phosphoryl)ethyl)phosphinic acid (8) (O150 Compound 9 (5.00 g, 11.51 mmol) in 100 ml of (0145 methanol was added 50 ml of 3 MNaOH. After being stirred under Arfor 3 h, the mixture was neutralized with 3 MHPO O to pH 7.2 under Ar. The mixture was added dropwise to the O solution of 1.2-bis(pyridin-2-yl)disulfane (10.0 g, 45.45 mmol) in 200 ml of methanol. After being stirred for 4 hunder B1M Is/> Pi-us OEt Ar, the mixture was concentrated, diluted with EtOAc/Hex OEt be ane (1:1), separated, and the organic layer was washed with pure water (3x25 ml) while the generated each of aqueous layer was washed with EtOAc/Hexane (1:1, 35 ml). The 0146 A mixture of ammonium hypophosphite (8.00 g,96 aqueous layers were combined, acidified with HCl/HOAc to mmol) and hexamethy-disilaZane (20.0 mL. 96 mmol) was pH -2, concentrated to ~10 ml, diluted with MeCN (60 ml), heated at 120° C. for 1 h under argon. After the mixture was sonicated (or quickly stirred) for 1 h, filtered, washed the cooled to 0°C., ethyl acrylate (10.4 mL. 96 mmol) was pellet with water/MeCN (1:10). The solution was then con carefully added dropwise, and the resulting mixture was centrated and purified on a SiO, column eluted with water/ stirred at 50° C. for 2 h. Then the mixture of compound 5 and MeCN/HOAc (1:8:0.01), pooled the fraction, added DMF 6 (10.0 g, 384 mmol estimated from above ratio) was added (-5 ml), evaporated to dryness to afford the title compound 10 and heated at 120° C. for 2 h under argon, followed by (3.62g, 79% yield). ESIMS, m/z-398.02 (M-H). addition of 1,2-dibromoethane (40 ml) and the mixture was stirred for 5 hat 120° C. After the solvent was removed under Example 6 vacuum, 100 mL of aqueous ethanol (1:1) were added drop wise to the residue and refluxed for 0.5 h. The mixture was (3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)(2- concentrated and purified by SiO, chromatographic column (hydroxy(2-(pyridin-2-yldisulfanyl)ethyl)phospho (1:20 to 1:10 MeOH/CHC1,) to afford the title compound 8 ryl)ethyl)phosphinic acid (11) (6.48g, 43%% yield). ESIMS m/z-391.2 (M-H). 0151 Example 4

(2-(ethoxy(3-ethoxy-3-oxopropyl)phosphoryl)ethyl) O (2-((ethoxycarbonothioyl)thio)ethyl)phosphinic acid (9) 0147 s -s/-urus OH OH O S O 0152 Compound 10 (200 g, 5.01 mmol) in DMA (50 ml) 1No ! S 1\-- s/>at OEt was added 0.2 ml of HCl (conc) and the mixture was evapo OH OEt rated to dryness. Then the compound redissolved in dry DMA (60 ml) was added, NHS (0.80g, 6.95 mmol) and EDC (3.00 g, 15.62 mmol). The mixture was stirred under Ar overnight, 0148 Compound 8 (6.01 g, 15.30 mmol) in 150 ml of evaporated and purified on short C-18 chromatography eluted ethanol was added Potassium ethyl xanthogenate (3.00 g, with water/dioxane at 4°C. The fractions containing the US 2015/0250896 A1 Sep. 10, 2015 product were pooled, freezed at -78°C., lyophilized to afford Example 9 the title compound (1.26 g. 51% yield). MS m/z- 495.2 (M-H). Ethyl 3-((2-((2-(3, 6-endoxo-A-tetrahydrophthalido) ethyl)(ethoxy)phosphoryl)ethyl)(ethoxy)phosphoryl) Example 7 propanoate, or 3,6-endoxo--tetrahydrophthalhide tetrahydro-1H-4,7-epoxyisoindol-2(3H)-yl)ethyl) 0153 (ethoxy)phosphoryl)ethyl)(ethoxy)phosphoryl)pro panoate (24) O157 O O O O NH + W W Hs120° C. f NH O O OEt O O W N 1\- -\- N/ OEt OEt O 0154) Maleimide (10.0 g, 103.0 mmol) in ethylether (350 O ml) was added furan (11.0 ml, 151.2 mmol). The mixture was heated inside a 1 L of autoclave bomb at 100° C. for 8 h. The 0158 3.6-Endoxo-A-tetrahydrophthalhide (2.40 g, 14.55 bomb was cooled down to room temperature, and the inside mmol) in DMA (60 ml) was added KCO (4.2 g, 30.39 Solid was rinsed with methanol, concentrated and crystallized mmol) and KI (0.40 g, 3.45 mmol). After stiffing under Arfor in ethyl acetate/hexane to afford 16.9 g (99%) of the title 1 hr., the mixture of compound 13 and 14 (2.6 g., -6.80 mmol compound. "H NMR (DMF-d7, 300 MHz): 11.06 (s, 1H) estimated) in DMA (10 ml) was added. The mixture was (NH), 6.61 (m, 2H), 5.15 (m, 2H), 2.97 (m, 2H). ''C NMR stirred under Arovernight, evaporated, re-dissolved in EtOAc 178.86, 137.72, 82.05, 49.93. MS m/z+ 188.4 (M+Na). (100 ml), washed with water (2x50 ml) and 1.0 M NaH2PO (2x50 ml), dried over NaSO, filtered, evaporated and puri Example 8 fied by SiO, chromatography eluted with EtOAc/hexane (1:10-1:5) to afford the title compound (2.64g, 77% yield). Ethyl 3-((2-((2-bromoethyl)(ethoxy)phosphoryl) ESIMS m/z+ 528.60 (M+Na). ethyl)(ethoxy)phosphoryl)propanoate (13), Ethyl 3-(ethoxy(2-(ethoxy(vinyl)phosphoryl)ethyl)phos Example 10 phoryl)propanoate (14) 3-((2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) 0155 ethyl)(hydroxy)phosphoryl)ethyl)(hydroxy)phospho ryl)propanoic acid (25) 0159) 13 O O O B1M s/> P --- OEt O OEt be | it OH P P 14 O OH OH O O O | O N-s/h OEt OEt be 0160 Compound 24 (2.60 g, 5.14 mmol), in the mixture of DMA (20 ml), toluene (20 ml) and HCl (8N, 10 ml) was 0156 Compound 8 (4.51 g, 11.47 mmol) was treated with heated at 120-140°C. for 8 h. During the reaction time, 5x10 100.0 mL of triethyl orthoformate, and the mixture was ml of water was gradually added to keep the reaction volume refluxed with a Dean-Stark trap to remove ethanol and ethyl around 40 ml. The mixture was concentrated and purified by formate. Excess triethyl orthoformate was removed under C-18 chromatography eluted with (1:10:0.01 to 1:3:0.01) vacuum to give the mixture of 13 and 14. ESIMS m/z+, 13: water/CHCN/HOAc to afford the title compound (1.12 g, 443.10 (M+Na), 14:363.20 (M+Na). 62% yield). ESIMS m/z-352.10 (M-H). US 2015/0250896 A1 Sep. 10, 2015 22

Example 11 combined, dried over NaSO, filtered, concentrated and purified on SiO, column eluted with EtOAc/Hexane (1:10-1: (2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl) 5) to afford the title compound (4.05 g, 79% yield). ESIMS (hydroxy)phosphoryl)ethyl)(3-((2,5-dioxopyrrolidin m/z+ 281.02 (M+Na). 1-yl)oxy)-3-oxopropyl)phosphinic acid (26) Example 14 (0161 3-(Vinylsulfonyl)propanoic acid (54) (0167

O

-v-\-s/r , 4\ ti-us OH O

(0162 Compound 25 (1.10g, 3.11 mmol) in DMA (50 ml) (0168 The compound 53 (4.0 g, 15.50 mmol) in 50 ml of was added 0.1 ml of HCl (conc) and the mixture was evapo THF was added 1 MNaOH (50 ml). After stirred under Arfor rated to dryness. Then the compound redissolved in dry DMA 24h, the mixture was adjusted pH=-7 with 1M HCl at 4°C., (40 ml) was added NHS (0.41 g, 3.56 mmol) and EDC (2.00 concentrated, diluted with EtOAc (100 ml), separated. The g, 10.42 mmol). The mixture was stirred under Ar overnight, aqueous layer was extracted with EtOAc (4x80 ml). The evaporated and purified on SiO, chromatography eluted with organic layers were combined, dried over NaSO, filtered, 1:1:1% Acetone/DCM/HOAc, pooled the fractions, evapo evaporated, and SiO, chromatographic purification (1:15 to rated and Solidified with EtOHATol/Hexane to afford the title 1:10 MeOH/CH2Cl2) to afford the title compound 54 (1.80g, compound (712 mg, 51% yield). ESI MS m/z- 449.10 71% yield). ESIMS m/z- 163.10 (M-H). (M-H). Example 15 Example 12 2,5-dioxopyrrolidin-1-yl 3-(vinylsulfonyl)propanoate methyl 3-((2-bromoethyl)thio)propanoate (52) (55) (0163 (0169

O O 's-s-s-s- O laus N 0164. A mixture of methyl 3-mercaptopropanoate (5.20g, 1. o1 51.6 mmol) and 1,2-dibromoethane (30 ml, 348.1 mmol) in O O DIPEA (100 ml) was stirred at 45° C. for 8h. The mixture was concentrated under vacuum and purified on SiO, eluted with (0170 Compound 54 (1.70 g, 10.36 mmol) in DMA (50 EtOAc/Hexane (1:12 to 1:5) to give the title compound 52 ml) was added NHS (1.75 g, 15.21 mmol) and EDC (5.00 g, (9.09 g, 78% yield). ESIMS m/z-249.20 (M+Na). 26.04 mmol). The mixture was stirred under Ar overnight, evaporated and purified on SiO, chromatography eluted with Example 13 EtOAc/Hexane (1:10-1:4), pooled the fractions and evapo methyl 3-((2-bromoethyl)sulfonyl)propanoate (53) rated to afford the title compound (2.24 g, 83% yield). ESI MS m/z+284.10 (M+Na). (0165 Example 16 O methyl 3-((3-mercaptopropyl)thio)propanoate (64) Brn- ti-us o1 0171 O

0166 Methyl 3-((2-bromoethyl)thio)propanoate 52 (4.50 g, 19.91 mmol) in acetic acid (40ml), H.O. (30%, 20 ml) and KMnO (1.00 g. 6.33 mmol) was stirred overnight. The mix ture was concentrated, diluted with EtOAc (100 ml) and 1 M (0172 Compound 63 (10.010 g, 60.25 mmol) in DMA (40 NaH2PO (150 ml), separated, the aqueous layer was ml) was added propane-1,3-dithiol (40.0 g, 370.3 mmol) and extracted with EtOAc (2x100 ml). The organic layers were DIPEA (100 ml). The mixture was stirred at 45° C. under Ar US 2015/0250896 A1 Sep. 10, 2015

for 8 h, evaporated and purified on SiO, chromatography (0178 Compound 66 (6.60 g, 17.46 mmol) in 100 ml of eluted with 1:10:0.01% EtOAc/DCM/HOAc, pooled the frac THF was added thioacetic acid (3.0 ml, 41.97 mmol) and tions, and evaporated to afford the title compound (9.58 g. DIPEA (20 ml, 115.0 mmol). The mixture was stirred at 40° 82% yield). ESIMS m/z+ 217.2 (M+Na). C. overnight, evaporated, co-evaporated with acetic acid (5 ml)/toluene (200 ml), and purified with SiO, chromatography Example 17 eluted with EtOAc/Hexane (1:10 to 1:4) to afford the title compound (5.22 g, 80% yield). ESI MS m/z+ 397.10 Methyl 3-((3-((3-bromopropyl)thio)propyl)thio)pro (M+Na). panoate (65) Example 20 0173 3-((3-((3-(pyridin-2-yldisulfanyl)propyl)sulfonyl) O propyl)sulfonyl)propanoic acid (68) ~~~~ 0179 O (0174 Compound 64 (5.01 g, 25.76 mmol) in DMA (40 ml) was added 1,3-dibromopropane (30.0g, 150.0 mmol) and N laus DIPEA (100 ml). The mixture was stirred at 45° C. under Ar 4 /~~~ OH for 8 h, evaporated and purified on SiO, chromatography O O eluted with EtOAc/hexane (1:10 to 1:5), pooled the fractions, evaporated to afford the title compound 65 (6.87 g. 85% 0180 Compound 67 (5.20 g, 13.90 mmol) in 100 ml of yield). ESIMS m/z+ 337.2 (M+Na). methanol was added 50 ml of 3 MNaOH. After being stirred under Arfor 3 h, the mixture was neutralized with 3 MHPO Example 18 to pH 7.2 under Ar. The mixture was added dropwise to the methyl 3-((3-((3-bromopropyl)sulfonyl)propyl)sulfo solution of 1,2-bis(5-pyridin-2-yl)disulfane (15.0 g, 68.18 mmol) in 200 ml of methanol. After being stirred for 24h nyl)propanoate (66) under Ar, the mixture was concentrated, diluted with EtOAc/ 0175 Hexane (1:1), separated, and the organic layer was washed with pure water (3x25ml) while the generated each of aque ous layer was washed with EtOAc/Hexane (1:1, 35 ml). The O aqueous layers were combined, acidified with HCl/HOAc to pH 3-4, concentrated to ~10 ml, diluted with MeCN (60 ml), 1N1 aO 1-n ti-us o1 sonicated (or quickly stirred) for 1 h, filtered, washed the pellet with water/MeCN (1:10). The solution was then con O O centrated and purified on a SiO, column eluted with CH-OH/ CHCl/HOAc (1:10:0.01), pooled the fraction, evaporated to 0176 Compound 65 (6.70g, 21.33 mmol) was in acetic dryness to afford the title compound 68 (5.93 g, 82% yield). acid (40 ml) was added H2O (33%, 20 ml) and KMnO (1.01 ESIMS, m/z-426.10 (M-H). g, 6.33 mmol). The mixture was stirred at 40°C. overnight. The mixture was concentrated, diluted with EtOAc (100 ml) Example 21 and 1 M NaH2PO (100 ml), separated. The aqueous layer was extracted with EtOAc (2x80 ml). The organic layers were 2,5-dioxopyrrolidin-1-yl 3-((3-((3-(pyridin-2-yldisul combined, dried over NaSO, filtered, evaporated, purified fanyl)propyl)sulfonyl)propyl)sulfonyl)propanoate by SiO, chromatography eluted with EtOAc/hexane (1:10-1: (69) 4) to afford the title compound (6.69 g, 83% yield). ESIMS m/z+ 401.10 (M+Na). 0181

Example 19 O methyl 3-((3-((3-(acetylthio)propyl)sulfonyl)propyl) N O O O sulfonyl)propanoate (67) I I 0177) O O O

O 0182 Compound 68 (2.50 g. 6.08 mmol) in DMA (50 ml) was added NHS (0.80g, 6.96 mmol) and EDC (3.00 g, 15.62 laus mmol). The mixture was stirred under Ar overnight, evapo --~~~~ o1 rated and purified on SiO, chromatography eluted with O O EtOAC/DCM (1:10 to 1:5), pooled the fractions and evapo rated to afford the title compound (2.74 g, 86% yield). ESI MS m/z+547.10 (M+Na). US 2015/0250896 A1 Sep. 10, 2015 24

Example 22 Example 25 methyl 3-((3-mercaptopropyl)thio)propanoate (71) methyl 3-((2-((3-(acetylthio)butyl)sulfonyl)ethyl) 0183) Sulfonyl)propanoate (84) (0189 O HS N-1 ns --- o1 0184 Methyl 3-bromopropanoate 51 (10.010 g, 60.24 mmol) in DMA (80 ml) was added ethane-1,2-dithiol (40.0g, O 425.4 mmol) and DIPEA (150 ml). The mixture was stirred at O 1. 45° C. under Ar for 8 h, evaporated and purified on SiO, --> ti-usS O chromatography eluted with 1:10:0.01% EtOAc/DCM/ AcS ~ O HOAc, pooled the fractions, and evaporated to afford the title O compound 71 (8.56 g. 79% yield). ESI MS m/z+ 203.10 (M+Na). (0190. Compound 83 (6.0 g, 15.87 mmol) in 100 ml of Example 23 THF was added thioacetic acid (3.0 ml, 41.97 mmol) and DIPEA (20 ml, 115.0 mmol). The mixture was stirred at 50° methyl 3-((2-((3-bromobutyl)thio)ethyl)thio)pro C. overnight, evaporated, co-evaporated with acetic acid (5 panoate (82) ml)/toluene (200 ml), and purified with SiO, chromatography eluted with EtOAc/Hexane (1:10 to 1:4) to afford the title 0185 compound 84 (4.27 g., 72% yield). ESI MS m/z+ 397.20 (M+Na). --> O Example 26 Br N----- 3-((2-((3-(pyridin-2-yldisulfanyl)butyl)sulfonyl) ethyl)sulfonyl)propanoic acid (85) 0186 Compound 71 (8.51 g, 47.26 mmol) in DMA (40 ml) was added 1,3-dibromobutane (30.0g, 140.25 mmol) and (0191) DIPEA (100 ml). The mixture was stirred at 45° C. under Ar for 8 h, evaporated and purified on SiO, chromatography eluted with EtOAc/hexane (1:10 to 1:5), pooled the fractions, evaporated to afford the title compound 82 (12.16 g. 82% yield). ESIMS m/z+ 337.2 (M+Na). O Example 24 N2 g-1--1a,laus OH methyl 3-((2-((3-bromobutyl)sulfonyl)ethyl)sulfo N S-S nyl)propanoate (83) O O 0187. (0192 Compound 84 (2.10 g, 5.61 mmol) in 100 ml of O methanol was added 50 ml of 2 MNaOH. After being stirred O O under Arfor 1 h, the mixture was neutralized with 3 MHPO to pH 7.2 under Ar. The mixture was added dropwise to the solution of 1,2-bis(5-pyridin-2-yl)glisulfane (6.0 g, 27.27 mmol) in 100 ml of methanol. After being stirred for 15 h --~~~~O O under Ar, the mixture was concentrated, diluted with EtOAc/ Hexane (1:1), separated, and the organic layer was washed 0188 Compound 82 (6.00 g, 19.10 mmol) was in acetic with pure water (3x25ml) while the generated each of aque acid (40 ml) was added H2O (33%, 20 ml) and KMnO (1.01 ous layer was washed with EtOAc/Hexane (1:1, 35 ml). The g, 6.33 mmol). The mixture was stirred at 40°C. overnight. aqueous layers were combined, acidified with HCl/HOAc to The mixture was concentrated, diluted with EtOAc (100 ml) pH 3-4, concentrated to ~10 ml, diluted with MeCN (60 ml), and 1 M NaH2PO (100 ml), separated. The aqueous layer sonicated for 1 h, filtered, washed the pellet with water/ was extracted with EtOAc (2x80 ml). The organic layers were MeCN (1:10). The solution was then concentrated and puri combined, dried over NaSO, filtered, evaporated, purified fied on a SiO, column eluted with CH-OH/CHC1/HOAc by SiO, chromatography eluted with EtOAc/hexane (1:10-1: (1:10:0.01), pooled the fraction, evaporated to dryness to 4) to afford the title compound 83 (6.01 g, 83% yield). ESI afford the title compound 85 (1.98 g, 82% yield). ESIMS, MS m/z+ 401.02 (M+Na). m/Z-426.10 (M-H). US 2015/0250896 A1 Sep. 10, 2015 25

Example 27 pH 6.5). The extent of drug conjugation to antibody was assessed by measuring A2s and A2so of the conjugate. 2,5-dioxopyrrolidin-1-yl 3-((2-((3-(pyridin-2-yldisul fanyl)butyl)sulfonyl)-ethyl)sulfonyl)propanoate (86) Example 30 0193 In Vitro Cytotoxicity Evaluation of a Tubulysin (ZT041) Conjugates of Antibodies with a Disulfide Linkers Containing Sulfonyl Groups O O (0197) The targeted cells (e.g. Ramos cells, 20,000 cells) were cultured in the presence of various concentrations of Ol2 J- 1-1ag O1 N unconjugated antibody or the antibody conjugate for 96 hours N S-S I I after which cell viability was measured by propidium iodide O O O exclusion and analyzed by flow cytometry using a Becton Dickinson FACSort (Becton Dickinson, Franklin Lakes, (0194 Compound 85 (1.00g, 2.34 mmol) in DMA (50 ml) N.J.). Red fluorescent intensity (emission at 617 nm in the was added NHS (0.41 g, 3.56 mmol) and EDC (2.00 g, 10.42 FL2 channel) of the cells excited at 488 nm was measured. mmol). The mixture was stirred under Ar overnight, evapo The regions for viable cells were also set using both the rated and purified on SiO, chromatography eluted with forward light scatter and right-angle light scatterproperties of EtOAC/DCM (1:10 to 1:5), pooled the fractions and evapo the cells. The loss of viability was determined by the loss of rated to afford the title compound 86 (993 mg, 81% yield). cells from within the gated region defining viable cells. The ESI MS m/z+547.10 (M+Na). average number of viable cells per 6 replicate cultures was calculated. The Survival fraction was plotted versus conjugate Example 28 concentration to determine the ICs value (50% cell killing concentration) of the conjugate. Modification of Antibody with Phosphinate Linker REFERENCE 0.195 The antiHer2 antibody is modified with phosphinate linker at 8 mg/mL antibody, a 10 fold molar excess of phos (0198 U.S. Pat. Nos. 4,680,338, 5,122,368, 5,141,648, phinate linker (-30mM stock solution in DMA). The reaction 5,208,020, 5,416,064; 5,475,092, 5,543,390, 5,563,250 is carried out in 100 mMNaHPO, pH 7.4 buffer with DMA 5,585,499, 5,880,270, 6,214,345, 6,436,931, 6,372,738, (5% v/v) for 15, 30, 60, 120, and 240 minutes at 25°C. The 6,340,701, 6,989,452, 7,129,261, 7,375,078, 7,498.302, modified antiHer2 was purified by G25 column with 50 mM 7,507,420, 7,691.962, 7,910,594, 7,968,586, 7,989.434, NaH2PO 50 mM. NaCl, and 2 mM EDTA, pH 6.5 to remove 7,994,135, 7,999,083, 8,153,768, 8.236,319. the excess phosphinate linker. (0199 US Patent Application Nos. 20120201809, 20120082617, 2012.0034295, 20110293513, 20110076722, Example 29 20110064753, 20110064752, 20110.064666, 20100189727, 2010013.6652, 20100104589, 20100074840, 20100062008, Conjugate Synthesis 20090176253, 20090088390 0200 Albrecht, H.; et al. J Immunol Methods 2006, 310, 0196. A hydrophilic linker containing thiopyridine (SPP) 100. linker was dissolved in DMA at a concentration of approxi 0201 Alley, S. C.; et al. Curr Opin Chem Biol 2010, 14, mately 10 mM. An antibody was dialyzed into buffer A (50 529. mM NaH2PO 50 mM NaCl, 2 mM EDTA, pH 6.5). For the 0202 Anderson, D.C.; etal. Bioconjug Chem 1993, 4, 10. linker reaction, the antibody was at 8 mg/ml, and 4-6 equiva (0203 Antczak, C.; etal. Bioconjug Chem 2006, 17, 1551. lents of linker were added while stirring in the presence of 5% (0204 Aoki, S.; et al. Bioorg Med Chem 2009, 17, 3405. (v/v) DMA. The reaction was allowed to proceed at ambient 0205 Austin, C. D.; et al. Proc Natl AcadSci USA 2005, temperature for 90 minutes. Unreacted linker was removed from the antibody by Sephadex G25 gel filtration using a 102, 17987. Sephadex G25 column equilibrated with Buffer Aat pH 6.5 or 0206 Barbour, N. P. et al. Pharm Res 1995, 12, 215. 150 mM potassium phosphate buffer containing 100 mM 0207 Beeson, C.; etal. Bioconjug Chem 2003, 14,927. NaCl, pH 7.4 as indicated. For the SPP containing linker, the (0208 Bickel, U.: et al. Bioconjug Chem 1995, 6, 211. extent of modification was assessed by release of pyridine-2- (0209 Chen, J.; etal. Expert Opin Drug Deliv 2005, 2,873. thione using 50 mM DTT and measuring the absorbance at 0210 DiJoseph, J. F.; et al. Blood 2004, 103, 1807. 343 nm as described below (e343–8080 M' cm' for free 0211 Doronina, S.O., et al. Bioconjugate Chem., 2006, pyridine-2-thione). For the conjugation reaction, thiol-con 17, 114. taining drug (such tubulysin TZ041) was dissolved in DMA 0212 Doronina, S.O., et al. Bioconjug Chem 2008, 19, (N,N-dimethylacetamide) at a concentration of approxi 1960. mately 10 mM. The drug (1-1.5-fold molar excess relative to 0213 Ebner, A.; et al. Bioconjug Chem 2007, 18, 1176. the number of linker molecules per antibody as indicated) was 0214 Erickson, H. K.; etal. Bioconjug Chem 2010, 21, 84. slowly added with stirring to the antibody which was at a 0215 Garsky, V. M.: et al. J. Med. Chem., 2001, 4216. concentration of 2.5 mg/ml in buffer A (pH 6.5 or pH 7.4) in 0216 Greenwald, R. B., et al. J. Med. Chem., 1999, 42, a final concentration of 3% (v/v) DMA. The reaction was 3657. allowed to proceed at ambient temperature for the indicated 0217. Haenseler, E.; etal. Biochemistry, 1992, 31, 891. times. Drug-conjugated antibody was purified using a Sepha 0218 Hamann, P. R.; etal. Bioconjugate Chem., 2002, 13, dex G25 column equilibrated with buffer B (PBS (NaH2PO), 40. US 2015/0250896 A1 Sep. 10, 2015 26

0219 Hamann, P. R.; etal. Bioconjugate Chem., 2005, 16, What is claimed is: 354. 1. A hydrophilic linker of the formula (I) 0220 Jeffrey, S.C.; et al. J. Med. Chem., 2005, 48, 1344 0221 Johnson, D.A.; et al. Cancer Res 1991, 51, 5774. 0222 Jones, D. S.; etal. Bioconjug Chem 2001, 12, 1012. (I) 0223 Jones, D. S.; et al. Bioconjug Chem 1999, 10, 480. Y-R-Q-R- T-R4-7 0224 Jones, D. S.; et al. Bioconjug Chem 1994, 5,390. 0225 Kashef, N.; et al. J Med Microbiol 2006, 55, 1441. 0226 Kellogg, B.A.; etal. Bioconjug Chem 2011, 22,717. Wherein: 0227 Kelly, R. K. et al. Eurj Cancer 2011, 47, 1736. Y represents a functional group that enables reaction with 0228 King, H. D.; et al. Bioconjug Chem 1999, 10,279. a cell-binding agent; 0229 King, H. D.; etal, J. Med. Chem., 2002, 45,4336 Q and T are either —P(=O)(OM)-, or —S(O)—, or —S(O)—: 0230 Klussman, K.; etal. Bioconjug Chem 2004, 15,765. m and n are integer from 0 to 5, but not 0 at the same time; 0231 Kovaráf. M.: et al. Bioconjugate Chem., 2002, 13, In addition, when m=1, n=0, Q is not—P(=O)(OM)-; 2O6. or when n=1, m=0, T is not —P(=O)(OM)-; 0232 Kratz, F., et al. J. Med. Chem., 2002, 45, 5523. Z represents a functional group that enables linkage of a 0233 Kumaresan, P. R. et al. Bioconjug Chem 2008, 19, cytotoxic drug via a disulfide, thioether, thioester, pep 1313. tide, hydraZone, ether, ester, carbamate, carbonate, 0234 Kumaresan, P. R. et al. Bioconjug Chem 2007, 18, amine (secondary, tertiary, or quartary), imine, cyclohet 175. eroalkyane, heteroaromatic, alkoxime or amide bond; 0235 Lee, L. S.; et al. Bioconjug Chem 1999, 10,973. R. R. R. R. Rs and Re, are the same or different and are 0236. Li, L.; et al. Bioconjug Chem 2002, 13,985. H. linear alkyl having from 1-6 carbon atoms, branched 0237 Lipinski, T.: et al. GlycoconjJ 2011, 28, 149. or cyclic alkyl having from 3 to 6 carbon atoms, linear, 0238 Meyer-Losic, F.; et al. J. Med. Chem., 2006, 49, branched or cyclic alkenyl or alkynyl, or 1-6 carbon 6908 atoms of esters, ether, amide, or polyethyleneoxy unit of 0239 Mikolajczyk, S. D.; et al. Bioconjug Chem 1994, 5, formula (OCHCH.), wherein p is an integer from 0 to 636. about 1000, or combination thereof. Additionally R. R. R. and Ra are respectively a chain of 0240 Miller, M. L.; et al. J. Med. Chem., 2004, 47, 4802 atoms selected from C, N, O, S, Si, and P that covalently 0241 Mitchell, J. S.; etal. Bioconjug Chem 2007, 18, 268. connects the cell-surface binding ligand, the phosphi 0242 Moon, S.-J., et al. J. Med. Chem., 2008, 51,6916 nate or Sulfonyl group, the conjugated drug and among 0243 Ojima, I., et al. J. Med. Chem., 2002, 45,5620 themselves (R,R,R, and Ra). The atoms used inform 0244 Ruppert, C.; et al. Bioconjug Chem 2002, 13,804. ing the hydrophilic linker may be combined in all chemi 0245 Safavy, A.; et al. Bioconjug Chem 2003, 14, 302. cally relevant ways, such as forming alkylene, alk 0246 Safavy, A.; et al. Bioconjug Chem 2004, 15, 1264. enylene, and alkynylene, ethers, polyoxyalkylene, 0247. Senter, P. et al. Photochem. Photobio., 1985, 42, esters, amines, imines, polyamines, hydrazines, hydra 231. Zones, amides, ureas, semicarbazides, carbazides, 0248 Scott, C. F., Jr.; et al. J Natl Cancer Inst 1987, 79, alkoxyamines, alkoxylamines, urethanes, amino acids, 1163. peptides, acyloxylamines, hydroxamic acids, or combi 0249 Sharkey, R. M.; et al. Mol Cancer Ther 2012, 11, nation thereof. 224. M is H, or Na, or K, or NRRR or a pharmaceutical salt. 0250 Siiman, O... et al. Bioconjugate Chem., 2000, 11, R. R. and R are described above. 549. 2. A cell-binding agent-drug conjugate of formula (II) 0251 Skwarczynski, M.: et al. J. Med. Chem., 2006, 49, 7253 0252 Srinivasachar, K. Neville, D. M., Jr. Biochemistry (II) Cb-HR--Q-R2- -T-R-R-Drug 1989, 28, 2501. / V 0253 Studer, M.: et al. Bioconjug Chem 1992, 3,424. Rs R6 0254 Sun, X.; et al. Bioconjug Chem 2011, 22,728. g 0255 Suzawa, T.; etal. Bioorg Med Chem 2000, 8, 2175. 0256 Tadayoni, B. M.; et al. Bioconjug Chem 1993, 4, wherein: 139. Cb represents a cell-binding agent/molecule; 0257 ten Hoeve, W.; et al. Bioconjug Chem 1997, 8, 257. Drug represents the drug linked to the cell-binding agent via the hydrophilic linker by a disulfide, thioether, 0258 Tsai, N. M.: et al. Biotechniques 2001, 30, 396. thioester, peptide, hydrazone, ether, ester, carbamate, 0259 Walker, M. A.; rt al. Bioorg Med Chem Lett 2004, carbonate, cycloheteroalkyane, heteroaromatic, 14,4323. alkoxime or amide bond; 0260 Wilbur, D. S.; etal. Bioconjug Chem 2011, 22, 1089. Q and T are either - P(=O)(OM)-, or —S(O)—, or 0261 Widdison, W. C. et al. J. Med. Chem., 2006, 49, —S(O)—: 4392. m and n are integer from 0 to 5, but not 0 at the same time; 0262 Zhao, R.Y.; et al. J. Med. Chem., 2011, 54, 3606 In addition, when m=1, n=0, Q is not—P(=O)(OM)-; 0263. Zhao, R.Y.; et al. J. Med. Chem., 2012, 55, 766 or when n=1, m=0, T is not —P(=O)(OM)-; US 2015/0250896 A1 Sep. 10, 2015 27

R. R. R. R. Rs and Re, are the same or different and are M is H, or Na, or K, or NRRR or a pharmaceutical salt. H. linear alkyl having from 1-6 carbon atoms, branched R. R. and R are described above. or cyclic alkyl having from 3 to 6 carbon atoms, linear, 4. A compound of formula (IV): branched or cyclic alkenyl or alkynyl, or 1-6 carbon atoms of esters, ether, amide, or polyethyleneoxy unit of formula (OCHCH.), wherein p is an integer from 0 to (IV) about 1000, or combination thereof. Y-R--Q-R-it- T-R spng Additionally R. R. R. and R are respectively a chain of atoms selected from C, N, O, S, Si, and P that covalently connects the cell-surface binding ligand, the phosphi nate or Sulfonyl group, the conjugated drug and among Wherein: themselves (R. R. R. and Ra). The atoms used inform Y represents a functional group that enables reaction with ing the hydrophilic linker may be combined in all chemi a cell-binding agent; cally relevant ways, such as forming alkylene, alk Q and T are either —P(=O)(OM)-, or S(O)—, or enylene, and alkynylene, ethers, polyoxyalkylene, —S(O)—; esters, amines, imines, polyamines, hydrazines, hydra m and n are integer from 0 to 5, but not 0 at the same time; Zones, amides, ureas, semicarbazides, carbazides, In addition, when m=1, n=0, Q is not—P(=O)(OM)-; alkoxyamines, alkoxylamines, urethanes, amino acids, or when n=1, m=0, T is not —P(=O)(OM)-; peptides, acyloxylamines, hydroxamic acids, or combi “Drug represents the drug linked to the cell-binding agent nation thereof. via the hydrophilic linker by a disulfide, thioether, M is H, or Na, or K, or NRRR or a pharmaceutical salt. thioester, peptide, hydrazone, ether, ester, carbamate, R. R. and R are described above. carbonate, cycloheteroalkyane, heteroaromatic, 3. A compound of formula (III): alkoxime or amide bond; R. R. R. R. Rs and Re, are the same or different and are H. linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, (III) branched or cyclic alkenyl or alkynyl, or 1-6 carbon ck-to-R,+,+r-Rih V -z atoms of esters, ether, amide, or polyethyleneoxy unit of R5 R6 formula (OCH2CH2), wherein p is an integer from 0 to g about 1000, or combination thereof. Additionally R. R. R. and Ra are respectively a chain of wherein: atoms selected from C, N, O, S, Si, and P that covalently Cb represents a cell-binding agent/molecule; connects the cell-surface binding ligand, the phosphi Z represents a functional group that enables reaction with a nate or Sulfonyl group, the conjugated drug and among cytotoxic drug; themselves (R,R,R, and Ra). The atoms used inform Q and T are either —P(=O)(OM)-, or —S(O)—, or ing the hydrophilic linker may be combined in all chemi —S(O)—; cally relevant ways, such as forming alkylene, alk m and n are integer from 0 to 5, but not 0 at the same time: enylene, and alkynylene, ethers, polyoxyalkylene, In addition, when m=1, n=0, Q is not—P(=O)(OM)-; esters, amines, imines, polyamines, hydrazines, hydra or when n=1, m=0, T is not —P(=O)(OM)-; Zones, amides, ureas, semicarbazides, carbazides, Z represents a functional group that enables linkage of a alkoxyamines, alkoxylamines, urethanes, amino acids, cytotoxic drug via a disulfide, thioether, thioester, pep peptides, acyloxylamines, hydroxamic acids, or combi tide, hydraZone, ether, ester, carbamate, carbonate, nation thereof. amine (secondary, tertiary, or quartary), imine, cyclohet M is H, or Na, or K, or NRRR or a pharmaceutical salt. eroalkyane, heteroaromatic, alkoxime or amide bond; R. R. and R are described above. R. R. R. R. Rs and Re, are the same or different and are 5. The formula (II) and (IV) of claims 2 and 4, wherein the H. linear alkyl having from 1-6 carbon atoms, branched Drug is selected from: or cyclic alkyl having from 3 to 6 carbon atoms, linear, 1). Chemotherapeutic agents: a). Alkylating agents: Such branched or cyclic alkenyl or alkynyl, or 1-6 carbon as Nitrogen mustards: chlorambucil, chlornaphazine, atoms of esters, ether, amide, or polyethyleneoxy unit of cyclophosphamide, dacarbazine, estramustine, ifosfa formula (OCH2CH2), wherein p is an integer from 0 to mide, mechlorethamine, mechlorethamine oxide hydro about 1000, or combination thereof. chloride, mannomustine, mitobronitol, melphalan, Additionally R. R. R. and R are respectively a chain of mitolactol, pipobroman, novembichin, phenesterine, atoms selected from C, N, O, S, Si, and P that covalently prednimustine, thiotepa, trofosfamide, uracil mustard; connects the cell-surface binding ligand, the phosphi CC-1065 (including its adozelesin, carzelesin and bize nate or Sulfonyl group, the conjugated drug and among lesin synthetic analogues); duocarmycin (including the themselves (R, RRs and Ra). The atoms used in form synthetic analogues, KW-2189 and CBI-TMI); benzo ing the hydrophilic linker may be combined in all chemi diazepine dimers (e.g., dimmers of pyrrolobenzodiaz cally relevant ways, such as forming alkylene, alk epine (PBD) or tomayimycin, indolinobenzodiazepines, enylene, and alkynylene, ethers, polyoxyalkylene, imidazobenzothiadiazepines, or oxazolidinobenzodiaz esters, amines, imines, polyamines, hydrazines, hydra epines); Nitrosoureas: (carmustine, lomustine, chloro Zones, amides, ureas, semicarbazides, carbazides, Zotocin, fotemustine, nimustine, ranimustine); Alkylsul alkoxyamines, alkoxylamines, urethanes, amino acids, phonates: (buSulfan, treosulfan, improSulfan and peptides, acyloxylamines, hydroxamic acids, or combi piposulfan); TriaZenes: (dacarbazine); Platinum con nation thereof. taining compounds: (carboplatin, cisplatin, oxaliplatin); US 2015/0250896 A1 Sep. 10, 2015 28

aziridines, such as benzodopa, carboquone, meture carzinophilin; chromomycins, dactinomycin, daunoru dopa, and uredopa; ethylenimines and methy bicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxo lamelamines including altretamine, triethylen rubicin, morpholino-doxorubicin, cyanomorpholino emelamine, trietylenephosphoramide, doxorubicin, 2-pyrrolino-doxorubicin and triethylenethiophosphaoramide and trimethylolom deoxydoxorubicin, epirubicin, esorubicin, idarubicin, elaminel; b). Plant Alkaloids: such as Vinca alkaloids: marcellomycin, nitomycins, mycophenolic acid, (Vincristine, vinblastine, Vindesine, Vinorelbine, navel nogalamycin, olivomycins, peplomycin, potfiromycin, bin); Taxoids: (paclitaxel, docetaxol) and their analogs, puromycin, quelamycin, rodorubicin, Streptonigrin, Maytansinoids (DM1, DM2, DM3, DM4, DMS, DM6, streptozocin, tubercidin, ubenimex, Zinostatin, Zorubi DM7, maytansine and ansamitocins) and their analogs, cin: f). Others: Such as Polyketides (acetogenins), espe cryptophycins (particularly cryptophycin 1 and crypto cially bullatacin and bullatacinone; gemcitabine, epoxo micins (e. g. carfilzomib), bortezomib, thalidomide, phycin 8); epothilones, eleutherobin, discodermolide, lenalidomide, pomalidomide, tosedostat, Zybrestat, bryostatins, dolostatins, auristatins, tubulysins, cepha PLX4032, STA-9090, Stimuvax, allovectin-7, Xegeva, lostatins; pancratistatin; a sarcodictyin; spongistatin; c). Provenge, Yervoy, Isoprenylation inhibitors (such as DNA Topoisomerase Inhibitors: such as Epipodophyl Lovastatin), Dopaminergic neurotoxins (such as 1-me lins: (9-aminocamptothecin, camptothecin, crisinatol, thyl-4-phenylpyridinium ion), Cell cycle inhibitors daunomycin, etoposide, etoposide phosphate, irinote (such as staurosporine), Actinomycins (such as Actino can, mitoxantrone, novantrone, retinoic acids (retinols), mycin D, dactinomycin), Bleomycins (such as bleomy teniposide, topotecan, 9-nitrocamptothecin (RFS cin A2, bleomycin B2, peplomycin), Anthracyclines 2000)); mitomycins: (mitomycin C); d). Anti-metabo (such as daunorubicin, doxorubicin (adriamycin), ida lites: such as {|Anti-folate: DHFR inhibitors: (methotr rubicin, epirubicin, pirarubicin, Zorubicin, mitoxantrone, exate, trimetrexate, denopterin, pteropterin, aminop MDR inhibitors (such as Verapamil), Cat ATPase terin (4-aminopteroic acid) or the other folic acid inhibitors (such as thapsigargin). Histone deacetylase analogues); IMP dehydrogenase Inhibitors: (mycophe inhibitors (Vorinostat, Romidepsin, Panobinostat, Valp nolic acid, tiazofurin, ribavirin, EICAR); Ribonucle roic acid, Mocetinostat (MGCD0103), Belinostat, PCI otide reductase Inhibitors: (hydroxyurea, deferoxam 24781, Entinostat, SB939, Resminostat, Givinostat, ine); Pyrimidine analogs: Uracil analogs: (ancitabine, AR-42, CUDC-101, sulforaphane, Trichostatin A); aZacitidine, 6-aZauridine, capecitabine (Xeloda), car Thapsigargin, Celecoxib, glitaZones, epigallocatechin mofur, cytarabine, dideoxyuridine, doxifluridine, enoc gallate, Disulfiram, Salinosporamide A.; Anti-adrenals, itabine, 5-Fluorouracil, floxuridine, ratitrexed(Tomu Such as aminoglutethimide, mitotane, triloStane; acegla dex)); Cytosine analogs: (cytarabine, cytosine tone; aldophosphamide glycoside; aminolevulinic acid; arabinoside, fludarabine); Purine analogs: (azathio amsacrine, arabinoside, bestrabucil; bisantrene; prine, fludarabine, mercaptopurine, thiamiprine, edatraxate; defofamine; demecolcine; diaziquone; eflo thioguanine); folic acid replenisher, Such as frolinic rnithine (DFMO), elfomithine; elliptinium acetate, eto acid; e). Hormonal therapies: Such as Receptor glucid: gallium nitrate, gacytosine, hydroxyurea; iban antagonists: Anti-estrogen: (megestrol, raloxifene, dronate, lentinan; lonidamine; mitoguaZone; tamoxifen); LHRH agonists: (goScrclin, leuprolide mitoxantrone; mopidamol; nitracrine; pentostatin: phe acetate); Anti-androgens: (bicalutamide, flutamide, namet, pirarubicin; podophyllinic acid; 2-ethylhy calusterone, dromostanolone propionate, epitioStanol, drazide; procarbazine; PSKR); razoxane: rhizoxin; sizo goserelin, leuprolide, mepitioStane, nilutamide, testo firan; Spirogermanium; tenuaZonic acid; triaziquone; lactone, triloStane and other androgens inhibitors); Ret 2.2.2"-trichlorotriethylamine; trichothecenes (espe inoids/Deltoids: Vitamin D3 analogs: (CB 1093, EB 1089 KH 1060, cholecalciferol, ergocalciferol); Photo cially T-2 toxin, Verrucarin A, roridin A and anguidine); dynamic therapies: (verteporfin, phthalocyanine, photo urethane, siRNA, antisense drugs; sensitizer PC4, demethoxy-hypocrellin A); Cytokines: 2). Anti-autoimmune disease agents: cyclosporine, (Interferon-alpha, Interferon-gamma, tumor necrosis cyclosporine A, aminocaproic acid, azathioprine, bro factor (TNFs), human proteins containing a TNF mocriptine, chlorambucil, chloroquine, cyclophospha domain)}: f). Kinase inhibitors, such as BIBW 2992 mide, corticosteroids (e.g. amcinonide, betamethasone, (anti-EGFR/Erb2), imatinib, gefitinib, pegaptainib, sor budesonide, hydrocortisone, flunisolide, fluticasone afenib, dasatinib, Sunitinib, erlotinib, nilotinib, lapa propionate, fluocortolone danazol, dexamethasone, Tri tinib, axitinib, paZopanib. Vandetanib, E7080 (anti amcinolone acetonide, beclometasone dipropionate), VEGFR2), mubritinib, ponatinib (AP24534), bafetinib DHEA, enanercept, hydroxychloroquine, infliximab, (INNO-406), bosutinib (SKI-606), cabozantinib, vis meloxicam, methotrexate, mofetil, mycophenylate, modegib, iniparib, ruxolitinib, CYT387, axitinib, tivo prednisone, Sirolimus, tacrolimus. Zanib, Sorafenib, bevacizumab, cetuximab, Trastu 3). Anti-infectious disease agents, a). Aminoglycosides: Zumab, RanibiZumab, Panitumumab, ispinesib; g). amikacin, astromicin, gentamicin (netilmicin, Sisomi antibiotics, such as the enediyne antibiotics (e.g. cali cin, isepamicin), hygromycin B, kanamycin (amikacin, cheamicins, especially calicheamicin.Y1, Ö1, C.1 and B1; arbekacin, bekanamycin, dibekacin, tobramycin), neo dynemicin, including dynemicin A and deoxydynemi mycin (framycetin, paromomycin, ribostamycin), cin; esperamicin, kedarcidin, C-1027, maduropeptin, as netilmicin, spectinomycin, streptomycin, tobramycin, well as neocarzinostatin chromophore and related chro Verdamicin; b). Amphenicols: azidamfenicol, chloram moprotein enediyne antiobiotic chromomophores), phenicol, florfenicol, thiamphenicol; c). Ansamycins: aclacinomysins, actinomycin, authramycin, azaserine, geldanamycin, herbimycin; d). Carbapenems: biap bleomycins, cactinomycin, carabicin, carminomycin, enem, doripenem, ertapenem, imipenem/cilastatin, US 2015/0250896 A1 Sep. 10, 2015 29

meropenem, panipenem; e). Cephems: carbacephem ronidazole, mupirocin, mycolactone, NAM synthesis (loracarbef), cefacetrile, cefaclor, cefradine, cefadroxil, inhibitors (e.g. fosfomycin), nitrofurantoin, paclitaxel, cefalonium, cefaloridine, cefalotin or cefalothin, cefal platensimycin, pyrazinamide, quinupristin?dalfopristin, exin, cefaloglycin, cefamandole, cefapirin, cefatrizine, rifampicin (rifampin), taZobactam tinidazole, uvaricin; cefazaflur, cefazedone, cefazolin, cefbuperaZone, cef 4). Anti-viral drugs: a). Entry/fusion inhibitors: aplaviroc. capene, cefdaloXime, cefepime, cefiminox, cefoxitin, maraviroc, Vicriviroc. gp41 (enfuvirtide), PRO 140, cefprozil, cefroxadine, cefiezole, cefuroxime, cefixime, CD4 (ibalizumab); b). Integrase inhibitors: raltegravir, cefdinir, cefditoren, cefepime, cefetamet, cefimenoxime, elvitegravir, globoidnan A; c). Maturation inhibitors: cefodizime, cefonicid, cefoperaZone, ceforanide, cefo , Vivecon; d). Neuraminidase inhibitors: osel taxime, cefotiam, cefoZopran, cephalexin, cefpimizole, tamivir, Zanamivir, peramivir; e). Nucleosides & nucle cefpiramide, cefpirome, cefpodoxime, cefprozil, otides: abacavir, aciclovir, adefovir, amdoxovir, apricit abine, brivudine, cidofovir, clevudine, dexelvucitabine, cefauinome, cefsulodin, ceftazidime, cefteram, ceftib didanosine (ddI), , emitricitabine (FTC), uten, ceftiolene, ceftizoxime, ceftobiprole, ceftriaxone, entecavir, famciclovir, fluorouracil (5-FU), 3'-fluoro cefuroxime, cefuZonam, cephamycin (cefoxitin, Substituted 2',3'-dideoxynucleoside analogues (e.g. cefotetan, cefimetazole), oxacephem (flomoxef, lata 3'-fluoro-2',3'-dideoxythymidine (FLT) and 3'-fluoro-2', moxe?): f). Glycopeptides: bleomycin, Vancomycin (ori 3'-dideoxyguanosine (FLG), fomivirsen, ganciclovir, tavancin, telavancin), teicoplanin (dalbavancin), idoxuridine, lamivudine (3TC), 1-nucleosides (e.g. B-1- ramoplanin; g). Glycylcyclines: e. g. tigecycline; g). thymidine and B-1-2'-deoxycytidine), penciclovir, B-Lactamase inhibitors: penam (Sulbactam, taZobac tam), clavam (clavulanic acid): i). Lincosamides: clin racivir, ribavirin, stampidine, stavudine (d4T), taribavi damycin, lincomycin; ). Lipopeptides: daptomycin, rin (viramidine), telbivudine, tenofovir, trifluridine val A54145, calcium-dependent antibiotics (CDA); k). aciclovir, Valganciclovir, Zalcitabine (ddC). Zidovudine Macrollides: azithromycin, cethromycin, clarithromy (AZT): f). Non-nucleosides: amantadine, ateviridine, cin, dirithromycin, erythromycin, flurithromycin, josa capravirine, diarylpyrimidines (etravirine, rilpivirine), mycin, ketolide (telithromycin, cethromycin), mideca delavirdine, docosanol, emivirine, efavirenz, foScarnet mycin, miocamycin, oleandomycin, rifamycins (phosphonoformic acid), imiquimod, interferon alfa, (rifampicin, rifampin, rifabutin, rifapentine), rokitamy loviride, lodenosine, methisazone, nevirapine, NOV cin, roXithromycin, spectinomycin, spiramycin, tacroli 205, peginterferon alfa, podophyllotoxin, rifampicin, mus (FK506), troleandomycin, tellithromycin; 1). rimantadine, residuimod (R-848), tromantadine; g). Monobactams: aztreonam, tigemonam, m). Oxazolidi Protease inhibitors: amprenavir, atazanavir, boceprevir, nones: lineZolid; n). Penicillins: amoxicillin, amplicillin darunavir, fosamprenavir, indinavir, lopinavir, nelfi (pivampicillin, hetacillin, bacampicillin, metampicillin, navir, pleconaril, ritonavir, saquinavir, telaprevir (VX talampicillin), azidocillin, azlocillin, benzylpenicillin, 950), tipranavir; h). Other types of anti-virus drugs: benzathine benzylpenicillin, benzathine phenoxymeth abzyme, arbidol, , ceragenin, cyanovirin-n, ylpenicillin, clometocillin, procaine benzylpenicillin, diarylpyrimidines, (EGCG), carbenicillin (carindacillin), cloxacillin, dicloxacillin, foscarnet, griffithsin, taribavirin (viramidine), hydrox epicillin, flucloxacillin, mecillinam (pivmecillinam), yurea, KP-1461, miltefosine, pleconaril, portmanteau meZlocillin, meticillin, nafcillin, oxacillin, penamecil inhibitors, ribavirin, seliciclib. lin, penicillin, pheneticillin, phenoxymethylpenicillin, 5). The radioisotopes selected from (radionuclides) H. piperacillin, propicillin, Sulbenicillin, temocillin, ticar IC, 14C, 18F 32P 35S, (Cu, Ga, 86Y. 99Tc, 11 In, 123, cillin; o). Polypeptides: bacitracin, colistin, polymyxin 1241, 125I, 13 II, 133Xc, 77Lu, 21 At or 'Bi. B; p). Quinolones: alatrofloxacin, balofloxacin, ciprof 6). The pharmaceutically acceptable salts, acids or deriva loxacin, clinafloxacin, danofloxacin, difloxacin, enoxa tives of any of the above drugs. cin, enrofloxacin, floxin, garenoxacin, gatifloxacin, 6. The conjugate of claim 2 or 4, wherein “Drug is pre gemifloxacin, grepafloxacin, kano trovafloxacin, levof ferred from tubulysins, calicheamicins, auristatins, maytansi loxacin, lomefloxacin, marbofloxacin, moxifloxacin, noids, CC-1065 analogs, morpholinos doxorubicins, taxanes, nadifloxacin, norfloxacin, orbifloxacin, ofloxacin, cryptophycins, epothilones, and benzodiazepine dimers (e.g., pefloxacin, trovafloxacin, grepafloxacin, Sitafloxacin, dimmers of pyrrolobenzodiazepine (PBD) or tomayimycin), sparfloxacin, temafloxacin, to Sufloxacin, trovafloxacin; indolinobenzodiazepines, imidazobenzothiadiazepines, or q). Streptogramins: pristinamycin, quinupristin?dalfo oxazolidinobenzodiazepines), siRNA or a combination pristin); r). Sulfonamides: mafenide, prontosil, Sulfac thereof, and pharmaceutically acceptable salts, acids or etamide, Sulfamethizole, Sulfanilimide, SulfaSalazine, derivatives of any of the above. Sulfisoxazole, trimethoprim, trimethoprim-sul 7. The conjugate of claims 2 and 3 wherein the cell binding famethoxazole (co-trimoxazole); s). Steroid antibacteri agent/molecule is selected from antibodies, proteins, Vita als: e.g. fusidic acid; t). Tetracyclines: doxycycline, mins (e.g. folates), peptides, polymeric micelles, liposomes, chlortetracycline, clomocycline, demeclocycline, lyme lipoprotein-based drug carriers, nano-particle drug carriers, cycline, meclocycline, metacycline, minocycline, dendrimers, and combination thereof. oxytetracycline, penimepicycline, rollitetracycline, tet 8. The conjugate of claims 2 and 3, wherein the cell racycline, glycylcyclines (e.g. tigecycline); u). Other binding agent binds to target cells which are selected from types of antibiotics: annonacin, arsphenamine, bacto tumor cells, virus infected cells, microorganism infected prenol inhibitors (Bacitracin), DADAL/AR inhibitors cells, parasite infected cells, autoimmune cells, activated (cycloserine), dictyostatin, discodermolide, eleuther cells, myeloid cells, activated T-cells, B cells, or melanocytes; obin, epothilone, ethambutol, etoposide, faropenem, cells expressing the CD19, CD20, CD22, CD30, CD33, fusidic acid, furazolidone, isoniazid, laulimalide, met CD37, CD38, CD40, CD51, CD52, CD56, CD66, CD70, US 2015/0250896 A1 Sep. 10, 2015 30

CD74, CD79, CD80, CD98, CD125, CD221, CD227, 12. The immunoconjugate of claims 2 and 4, wherein the CD262, CD309, CD326, CEACAM3, CEACAM5 (carcino drug/cytotoxic agent are selected from a toxin, a chemothera embryonic antigen), DLL4 (A-like-4), EGFR, CTLA4, peutic agent, a drug moiety, an antibiotic, a radioactive iso CXCR4 (CD184), Endoglin (CD105), EPCAM (epithelial tope, and a nucleolytic enzyme. cell adhesion molecule), ERBB2 (Epidermal Growth Factor 13. The immunoconjugate of claim 2, the immunoconju Receptor 2), FCGR1, FOLR (folate receptor.), GD2 ganglio gate having the formula Cb-(-L-Drug), wherein Cb is a cell side, G-28 (a cell surface antigen gly volipid), GD3 idiotype, binding agent, L is the hydrophilic linker, Drug is a drug Heat shock proteins, HER1, HER2, HLA-DR10, HLA-DRB, molecule, and n is an integer from 1 to 20. human chorionic gonadotropin, IGF1R (insulin-like growth factor 1 receptor), IL-2 receptor ( receptor), 14. The immunoconjugate of claim 13, wherein the hydro IL-6R (interleukin 6 receptor), Integrins (CVB3, C5B1, C.6.f34, philic linker L. may be composed of one or more linker com Clf3, C.5 B5, CVB5), MAGE-1, MAGE-2, MAGE-3, MAGE ponents of 6-maleimidocaproyl (MC), maleimidopropanoyl 4, anti-transferrin receptor, p97, MS4A1 (membrane-span (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala ning 4-domains subfamily A member 1), MUC1 or MUC1 phe), p-aminobenzyloxycarbonyl (PAB), 4-thio-pentanoate KLH, MUC16 (CA125), CEA, gp100, MART1, MPG, (SPP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate MS4A1 (membrane-spanning 4-domains subfamily A), (MCC), 4-thio-butyrate (SPDB), maleimidoethyl containing Nucleolin, Neu oncogene product, P21, Paratope of anti-(N- (ME), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB) glycolylneuraminic acid), PLAP-like testicular alkaline and (4-acetyl)aminobenzoate (SIAB). phosphatase, PSMA, PSA, ROBO4, TAG 72 (tumor associ 15. The immunoconjugate of claims 2 and 13, wherein ated glycoprotein 72), T cell transmembrane protein, Tie Drug is preferably selected from tubulysins, maytansinoids, (CD202b), TNFRSF10B (tumor necrosis factor receptor taxanoids (taxanes), CC-1065 analogs, daunorubicin and superfamily member 10B), TNFRSF13B (tumor necrosis doxorubicin compounds, benzodiazepine dimers (e.g., factor receptor superfamily member 13B), TPBG (tropho dimers of pyrrolobenzodiazepine (PBD), tomayimycin, blast glycoprotein), TRAIL-R1 (Tumor necrosis apoprosis anthramycin, indolinobenzodiazepines, imidazobenzothiadi Inducing ligand Receptor 1), VCAM-1 (CD106), VEGF, azepines, or oxazolidinobenzodiazepines), calicheamicins VEGF-A, VEGF-2 (CD309), CanAg, CALLA, and cells and the enediyne antibiotics, actinomycin, azaserines, bleo expressing insulin growth factor receptor, or epidermal mycins, epirubicin, tamoxifen, idarubicin, dolastatins/aurist growth factor receptor. atins (e.g. monomethylauristatin E, MMAE, MMAF, aurista 9. The conjugate of claims 2 and 3, wherein the cell tin PYE. auristatin TP. Auristatins 2-AQ, 6-AQ, EB (AEB), binding agent is preferred an antibody, a single chain anti and EFP (AEFP)), duocarmycins, thiotepa, Vincristine, hemi body, an antibody fragment that binds to the target cell, a asterlins, esperamicins, and their analogues and derivatives monoclonal antibody, a single chain monoclonal antibody, or thereof. a monoclonal antibody fragment that binds the target cell, a 16. The immunoconjugate of claims 2 and 13, having in chimericantibody, a chimericantibody fragment that binds to vitro, in vivo or ex vivo cell killing activity. the target cell, a domain antibody, a domain antibody frag ment that binds to the target cell, a resurfaced antibody, a 17. The immunoconjugate of claim 13, wherein the linker resurfaced single chain antibody, or a resurfaced antibody comprises either a peptides of 1-20 units of natural or unnatu fragment that binds to the target cell, a or ralamino acids, or ap-aminobenzyl unit, or a 6-maleimidoca a resurfaced antibody, a humanized single chain antibody, or proyl unit, or a disulfide unit, or a thioether unit, or a hydro a humanized antibody fragment that binds to the target cell, a Zone unit, or an alkoxime unit. lymphokine, a hormone, a vitamin, a growth factor, a colony 18. The immunoconjugate of claim 13, wherein the linker stimulating factor, or a nutrient-transport molecule. can be cleavable by a protease. 10. The conjugate of claim 8, wherein the tumor cells are 19. A pharmaceutical composition comprising the immu selected from lymphoma cells, myeloma cells, renal cells, noconjugate of claim 2 or 13, and a pharmaceutically accept breast cancer cells, prostate cancer cells, ovarian cancer cells, able carrier. colorectal cancer cells, gastric cancer cells, squamous cancer cells, Small-cell lung cancer cells, testicular cancer cells, or 20. A pharmaceutical composition comprising a therapeu any cells that grow and divide at an unregulated, quickened tically effective amount of the immunoconjugate of claim 2 or pace to cause cancers. 13, administered concurrently with the other therapeutic 11. A pharmaceutical composition comprising a therapeu agents such as the chemotherapeutic agent, the radiation tically effective amount of the immunoconjugate of claims 2, therapy, agents, autoimmune disorder and a pharmaceutically acceptable salt, carrier, diluent, or agents, anti-infectious agents or the other immunoconjugates excipient therefore, or a combination therefore, for the treat for synergistically effective treatment or prevention of a can ment or prevention of a cancer, or an autoimmune disease, or cer, or an autoimmune disease, or an infectious disease. an infectious disease. k k k k k