US 20140363454A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0363454 A1 JacksOn et al. (43) Pub. Date: Dec. 11, 2014

(54) ANTIBODY-DRUG CONJUGATES, Publication Classification COMPOSITIONS AND METHODS OF USE (51) Int. Cl. (71) Applicant: Igenica Biotherapeutics, Inc., A647/48 (2006.01) Burlingame, CA (US) A613 L/454 (2006.01) (52) U.S. Cl. (72) Inventors: David Y. Jackson, Belmont, CA (US); CPC ...... A61K 47/48384 (2013.01); A61 K3I/454 Edward Ha, San Francisco, CA (US); (2013.01) Gary D. Probst, San Francisco, CA USPC ... 424/181.1; 530/391.9; 546/256; 546/209; (US) 548/547 (73) Assignee: Igenica Biotherapeutics, Inc., Burlingame, CA (US) (57) ABSTRACT (21) Appl. No.: 14/298,798 (22) Filed: Jun. 6, 2014 Antibody-cytotoxin antibody-drug conjugates and related compounds, such as linker-cytotoxin conjugates and the link Related U.S. Application Data ers used to make them, tubulysin analogs, and intermediates (60) Provisional application No. 61/832,068, filed on Jun. in their synthesis; compositions; and methods, including 6, 2013. methods of treating cancers. Patent Application Publication Dec. 11, 2014 Sheet 1 of 7 US 2014/0363454 A1

Figure I Antibody Only RT = 7.12

8 Reisaff:EHA.J.C.S. 2313-48-39:38-42-8738:38:3) : s ; :

Figure 2 Antibody + dibromosuccinimide - RT - 7.24 388x8: 8:88-83.38:3:38 23:3:3-2: 33-38.83x33-8-1833;

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Figure 3 Antibody + dibromo-N-benzyl succinimide RT = 7.58 38:1. Sigs2288 Reissfish:3:K. OS33292-3-2-18-08-04081-3:03.3;

Patent Application Publication Dec. 11, 2014 Sheet 2 of 7 US 2014/0363454 A1

Figure 4 Conventional me-MMAF ADC

A: , Sig-220.83. Reixoff -:C:CEQ83:...C 23:2-3-23 38-3&-8839 & 3) :-}

Figure 5 "Stapled" or "Snapped" dts-ADC

Figure 6

Patent Application Publication Dec. 11, 2014 Sheet 3 of 7 US 2014/0363454 A1

Figure 7 18-2A-mc-MMAF (conventional ADC) "OAS Sigca2.3 resea;i -8&sh:ESC-3:20-2-3-2: 14-12-563-3381:8) Pix

Figure 8 18-2A-dts-MMAF ("stapled" or "snapped" ADC)

& & 88.

Figure 9 Potency of T2 and T4 in Tubulin Polymerization Assay. T2 & T4 Tubulin Assay (2/27|14)

as PBS a 32 (3 M) the 74 (3 uM)

Time (min) Patent Application Publication Dec. 11, 2014 Sheet 4 of 7 US 2014/0363454 A1

Figure 10 Potency of T2 ADCs in Tubulin Polymerization Assay. T2ADCs in Tubulin Assay

a PBS --- Herceptin-mPEG12-VAP-EDA-T2 4. 00 . . . GN523-PEG12-WAP-EDA-2

Figure 11 T2 and T4 Tubulin Polymerization ASSays O O als ro\ N O r 'it':H - C -o O HO HO

Tubulin Polymerization Assay-T2 tubulin Polyimerization Assay-T4

M - 30% DMSO -- 10% DSO 8000 x 6xyrr - T2 (0.1 uk) 8000 * - T4 (0.1 und) • T2 (0.5uM) 3 T4 (0.5uM) 60 & T2 (UM) 6000 -- T4 (1 uM) s & T2 (5uM) & 4 (5uM) 2 x - 2 (10 My e 4-T4 (10 uM) - 4000 400

200 2009 2C 4. 6 20 49 60 Time (min) Time (min)

Patent Application Publication Dec. 11, 2014 Sheet 6 of 7 US 2014/0363454 A1

Figure 13 T2 ADCs inhibit microtubule formation in vitro and are more potent to T4 ADCs: Whole AEC tubulin Polymerization inhibition (2:27:4} 28 50 ug of Tubulin

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Figure 14 T2 ADC Tubulin Assay T2ADCs in Tubulin Assay 1200 1000 800 - PBS 600 --- Herceptin-mPEG12-VAP-EDA-T2 400 ... IGN523-mREG2-WAP-EDA-2

200

200 60 Patent Application Publication Dec. 11, 2014 Sheet 7 of 7 US 2014/0363454 A1

Wicie. At futsalin Polyrnerization inhibition (2:27:14 &Sig: S0 tag of attain

S38: t}a} Six & : Sgs ise tia

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Figure 15 ADC Conjugation Protocol for "Stapled" or "Snapped" Linkers

xx xx Step 1: Step 2: disulfide reduction Conjugation -e- -b 5 ea. TCEP 5 eq, inker pH 7.4, 37°C 4C, O.N 2 r- - - - -

Antibody Reduced Antibody Stapled ADC US 2014/0363454 A1 Dec. 11, 2014

ANTIBODY-DRUG CONJUGATES, 52, 328-337; and Chari, “Targeted Cancer Therapy Confer COMPOSITIONS AND METHODS OF USE ring Specificity to Cytotoxic Drugs'. Acc. Chem. Res., 2008, 41(1), 98-107. These ADCs have three components: (1) a RELATED APPLICATION conjugated through a (2) linker to a (3) cytotoxin. The cytotoxins are attached to either lysine or 0001. This application claims the benefit of U.S. Provi cysteine sidechains on the antibody through linkers that react sional Application No. 61/832,068, filed Jun. 6, 2013, which selectively with primary amines on lysine or with sulfhydryl is incorporated herein by reference. groups on cysteine. The maximum number of linkers/drugs that can be conjugated depends on the number of reactive BACKGROUND OF THE INVENTION amino or sulfhydryl groups that are present on the antibody. A 0002 1. Field of the Invention typical antibody contains up to 90 lysines as potential conju 0003. This invention relates to antibody-drug conjugates gation sites; however, the optimal number of cytotoxins per (ADCs) and related compounds, such as linkers used to make antibody for most ADCs is typically between 2 and 4 due to them and intermediates in their synthesis; compositions; and aggregation of ADCs with higher numbers of cytotoxins. As methods, including methods of treating cancers. a result, conventional lysine linked ADCs currently in clinical 0004 2. Description of the Related Art development are heterogeneous mixtures that contain from 0 0005 Cancer is the second most prevalent cause of death to 10 cytotoxins per antibody conjugated to different amino in the U.S., yet there are few effective treatment options groups on the antibody. Key factors in the Success of an ADC beyond Surgical resection. Of the medical treatments for can include that the monoclonal antibody is cancer antigen spe cers, the use of monoclonal antibodies targeting antigens cific, non-immunogenic, low toxicity, and internalized by present on the cancer cells has become common. Anticancer cancer cells; the cytotoxin is highly potent and is suitable for antibodies approved for therapeutic use in the USA include linker attachment; while the linker may be specific for cys (CAMPATHR), a humanized anti-CD52 anti teine (S) or lysine (N) binding, is stable in circulation, may be body used in the treatment of chronic lymphocytic leukemia; protease cleavable and/or pH sensitive, and is suitable for (AVASTINR), a humanized anti-VEGF anti attachment to the cytotoxin. body used in ; (ERBITUXR), a 0008 Anticancer ADCs approved for therapeutic use in chimeric anti-epidermal growth factor antibody used in col the USA include (ADCETRIS(R), a chi orectal cancer, head and neck cancer, and squamous cell mericanti-CD30 antibody conjugated to monomethylaurista carcinoma; (YERVOYR), a human anti-CTLA-4 tin E used in anaplastic large cell lymphoma and Hodgkin antibody used in ; (ARZERRAR), a lymphoma; and (MYLOTARG(R), human anti-CD20 antibody used in chronic lymphocytic leu a humanized anti-CD33 antibody conjugated to calicheami kemia; (VECTIBIX(R), a human anti-epider ciny used in acute myelogeneous leukemia—though this was mal growth factor receptor antibody used in colorectal can withdrawn in 2010 for lack of efficacy. cer; (RITUXANR), a chimeric anti-CD20 0009. Although several ADCs have demonstrated recent antibody used in non-Hodgkin lymphoma; to situmomab clinical success, the utility of most ADCs currently in devel (BEXXARR), a murine anti-CD20 antibody used in non opment may be limited by cumbersome synthetic processes Hodgkin lymphoma; and (HERCEPTINR), a resulting in high cost of goods, insufficientanti-tumor activity humanized anti-HER2 antibody used in breast cancer. While associated with limited potency of the cytotoxic drug, and these antibodies have proven useful in the treatments of the questionable safety due to linker instability and ADC hetero cancers for which they are indicated, they are rarely curative geneity. See, for example, Ducry et al., “Antibody-Drug Con as single agents, and are generally used in combination with jugates: Linking Cytotoxic Payloads to Monoclonal Antibod standard for the cancer. ies’, Bioconjugate Chem. 2010, 21, 5-13; Chari, “Targeted 0006. As an example, trastuzumab is a recombinant DNA Cancer Therapy: Conferring Specificity to Cytotoxic Drugs'. derived humanized monoclonal antibody that selectively Acc. Chem. Res. 2008, 41, 98-107; and Senter, “Recent binds with high affinity to the extracellular domain of the advancements in the use of antibody drug conjugates for human epidermal growth factor receptor2 protein, HER2 cancer therapy'. Biotechnol. Pharma. Aspects, 2010, 11, (ErbB2) (Coussenset al., Science 1985,230, 1132-9; Salmon 309-322. et al., Science 1989, 244, 707-12), thereby inhibiting the 0010. As an example, trastuzumab has been conjugated to growth of HER2-positive cancerous cells. Although HER the maytansinoid drug mertansine to form the ADC trastu CEPTIN is useful in treating patients with HER2-overex Zumab emtansine, also called trastuzumab-DM1 or trastu pressing breast cancers that have received extensive prior Zumab-MC-DM1, abbreviated T-DM1 (LoRusso et al., anti-cancer therapy, some patients in this population fail to “Trastuzumab Emtansine: A Unique Antibody-Drug Conju respond or respond only poorly to HERCEPTIN treatment. gate in Development for Human Epidermal Growth Factor Therefore, there is a significant clinical need for developing Receptor 2-Positive Cancer, Clin. Cancer Res. 2011, 17. further HER2-directed cancer therapies for those patients 6437-6447; Burris et al., “Trastuzumab emtansine: a novel with HER2-overexpressing tumors or other diseases associ antibody-drug conjugate for HER2-positive breast cancer. ated with HER2 expression that do not respond, or respond Expert Opin. Biol. Ther: 2011, 11,807-819). It is now in Phase poorly to HERCEPTIN treatment. III studies in the US for that indication. The mertansine is 0007 Antibody drug conjugates (ADCs), a rapidly grow conjugated to the trastuzumab through a maleimidocaproyl ing class of targeted therapeutics, represent a promising new (MC) linker which bonds at the maleimide to the 4-thiovaleric approach toward improving both the selectivity and the cyto acid terminus of the mertansine side chain and forms an toxic activity of cancer drugs. See, for example, Trail et al., amide bond between the carboxyl group of the linker and a "Monoclonal antibody drug immunoconjugates for targeted lysine basic amine of the trastuzumab. Trastuzumab has 88 treatment of cancer, Cancer Immunol. Immunother: 2003, lysines (and 32 cysteines). As a result, trastuzumab emtansine US 2014/0363454 A1 Dec. 11, 2014 is highly heterogeneous, containing dozens of different mol natural products. This reactive functionality is labile in both ecules containing from 0 to 8 mertansine units per trastu acidic and basic reaction conditions, and therefore may play Zumab, with an average mertansine?trastuzumab ratio of 3.4. a key role in the function of the tubulysins. (Iley et al., Pharm. 0011 Antibody cysteines can also be used for conjugation Res. 1997, 14, 1634-1639). Recently, the total synthesis of to cytotoxins through linkers that contain maleimides or other tubulysin D was reported, which represents the first synthesis thiol specific functional groups. A typical antibody contains of any member of the tubulysin family that incorporates the 4, or sometimes 5, interchain disulfide bonds (2 between the O-acyl N.O-acetal functionality. (Peltier et al., J. Am. Chem. heavy chains and 2 between heavy and light chains) that Soc. 2006, 128, 16018-16019). Other tubulysins, including covalently bond the heavy and light chains together and con tubulysins U and V, have been synthesized by Donating et al., tribute to the stability of the antibodies in vivo. These inter “Total Synthesis of Tubulysins U and V, Angew. Chem. Int. chain disulfides can be selectively reduced with dithiothrei Ed. 2006, 45, 7235-7239; including the synthesis of tubul tol, tris(2-carboxyethyl)phosphine, or other mild reducing ysins via multi-component reactions; i.e. using the Ugi or agents to afford 8 reactive sulfhydryl groups for conjugation. Cysteine linked ADCs are less heterogeneous than lysine Passerinni methods. linked ADCs because there are fewer potential conjugation (0015 US Patent Application Publication No. US2011/ sites; however, they also tend to be less stable due to partial 0021568 A1 (Ellman et al.) discloses the synthesis and activi loss of the interchain disulfide bonds during conjugation, ties of a number of tubulysin analogs, including compounds since current cysteine linkers bond to only one Sulfur atom. (40) and (10), referred to here as T1 and T2, respectively: The optimal number of cytotoxins per antibody for cysteine linked ADCs is also 2 to 4. For example, ADCETRIS is a heterogeneous mixture that contains 0 to 8 monomethylau ristatin E residues per antibody conjugated through cysteines. 0012. The tubulysins, first isolated by the Hofle/Reichen bach group from myxobacterial cultures (Sasse et al., J. Anti biot. 2000, 53,879-885), are exceptionally potent cell-growth inhibitors that act by inhibiting tubulin polymerization and thereby induce apoptosis. (Khalil et al., Chem. Biochem. 2006, 7, 678-683; and Kaur et al., Biochem. J. 2006, 396, 235-242). The tubulysins, of which tubulysin D is the most potent, have activity that exceeds most other tubulin modifi ers including, the epothilones, vinblastine, and paclitaxel (TAXOL(R), by 10- to 1000-fold. (Steinmetz et al., Angew: Chem. 2004, 116,4996-5000; Steinmetz et al., Angew. Chem. Int. Ed. 2004, 43, 4888-4892; and Hale et al., Pure App. Chem. 2003, 75, 167-178). Paclitaxel and vinblastine are current treatments for a variety of cancers, and epothilone derivatives are under active evaluation in clinical trials. Syn thetic derivatives of tubulysin D would provide essential information about the mechanism of inhibition and key bind ing interactions, and could have Superior properties as anti cancer agents either as isolated entities or as chemical war heads on targeted antibodies or ligands. 0013 Tubulysin D is a complex pseudo-tetrapeptide that can be divided into four regions, Mep (D-N-methylpipe colinic acid), Ile (isoleucine), Tuv (tubuvaline), and Tup (tu 0016 Schumacher et al., “In Situ Maleimide Bridging of buphenylalanine), as shown in the formula: Disulfides and a New Approach to Protein PEGylation', Bio conjugate Chem. 2011, 22, 132-136, disclose the synthesis of 3,4-disubstituted maleimides such as 3,4-bis(2-hydroxyeth Tuv ylsulfanyl)pyrrole-2,5-dione referred to by Schumacher et Mep Ile O al. as “dimercaptoethanolmaleimide' and 3,4-bis(phenyl Tup sulfanyl)pyrrole-2,5-dione “dithiophenolmaleimide', and a their N-PEGylated derivatives as PEGylating agents for N -N ? somatostatin, where the substituted maleimide bonds to the two Sulfur atoms of an opened cysteine-cysteine disulfide bond. --> w 0017. It would be desirable to develop potent, homoge OH neous ADCs, compositions containing them and methods for O their use in treating cancers, and methods and intermediates in their preparation. 0014 Most of the more potent derivatives of tubulysin, 0018. The disclosures of the documents referred to in this including tubulysin D, also incorporate the interesting O-acyl application are incorporated into this application by refer N.O-acetal functionality, which has rarely been observed in CCC. US 2014/0363454 A1 Dec. 11, 2014

SUMMARY OF THE INVENTION 0035. In another embodiment, there is provided a linker cytotoxin conjugate of formula A, B or C: 0019. In one embodiment, the present application dis closes antibody-cytotoxin antibody-drug conjugates (ADCs) of the formula: A X R1 S Z-L-CTX R Ns 0020 wherein: X 0021 A is an antibody; B 0022 PD is a pyrrole-2,5-dione or derivative thereof, a X pyrrolidine-2,5-dione or derivative thereof; R1 S 0023 CTX is a cytotoxin: Z-L-CTX 0024 each L', Land L is independently a linker selected R Ns from the group consisting of —O—, —C(O)— —S , X S(O)-, -S(O), , -NH-, -NCH, -, -(CH2) , C —NH(CH)NH , —OC(O)—, —CO , X -NHCHCHC(O) , C(O)NHCHCH-NH , S —NHCHC(O) , – NHC(O) , C(O)NH-, - NCHC R1 (O) , C(O)NCH, , -(CH2CH2O), , -(CH2CH2O) Z-L-CTX CHCH , —CH2CH2 (CH2CH2O). , OCH (CH2O—) , cyclopentanyl, cyclohexanyl, unsubstituted X phenylenyl, phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF , CFO , 0036 where each RandR' is independently selected from CHO , —C(O)CH, —C(O)OC-alkyl, —C(O)CH, the group consisting of Calkyl optionally substituted with —CN, NH, —OH, -NHCH, N(CH), Calkyl and halo or hydroxyl; phenyl optionally substituted with halo, -(AA),-: hydroxyl, carboxyl, Calkoxycarbonyl, or C-alkyl, naph 0025 a, b and c are each independently 0, 1, 2 or 3, pro thyl optionally substituted with halo, hydroxyl, carboxyl, vided that at least one of a, b or c is 1: Calkoxycarbonyl, or C-alkyl, 2-pyridyl optionally Sub 0026 each p is independently an integer of 1 to 14; stituted with halo, hydroxyl, carboxyl, Calkoxycarbonyl or 0027 each q is independently an integer from 1 to 12; Calkyl, Calkylsulfonyloxy, Caocycloalkylsulfony 0028 each AA is independently an amino acid; loxy, Carylsulfonyloxy; Calkyl-S , Caryl-S— and Coheteroaryl-S—; 0029) each r is 1 to 12; and 0037 X is O, S or NR' where R' is H or Calkyl: 0030 m is an integer of 1 to 4; and n is an integer of 1 to 4: 0038 X is O, S or NR where R is H or Calkyl: 0031 with the proviso that when -(L)-(L)-(L)- 0039 Z is selected from the group consisting of N—, together is —(CH2) - or —(CH2CH2O)2CH2CH2— CH CR - and CR CRR where R, R and Rare then L', Land L are not bonded to CTX by an amide bond. each independently H or C-alkyl. 0032. In one aspect of the linkers of the present applica 0040 L is a linker defined by L-L-L, wherein each L', tion, the cyclopentanyl, cyclohexanyl, and phenylenyl may be Land L is independently a linker selected from the group divalent linkers or trivalent linkers that may be attached to consisting of —O—, —C(O)— —S , —S(O)— —S(O) one, two or more CTX residues. In another aspect of the ADC , -NH-, -NCH, ,-(CH2). , -NH(CH2)NH-, of the present application, the linker is attached to the CTX by –OC(O) , —CO. , -NHCHCHC(O) C(O) a group selected from the group consisting of NHC(O)—, NHCHCH-NH NHCHC(O) , NHC(O) , NHC(O)C , —N(Calkyl)C(O)O—, NH , - C(O)NH-, - NCHC(O) , —C(O)NCH , —N(Calkyl)-, - N(Calkyl)C(O)NH and —N(C. -(CH2CH2O), , -(CH2CH2O). CHCH. , 3alkyl)C(O)N(Calkyl)-. —CHCH-(CH2CH2O), , —OCH(CHO ) , cyclo 0033. Because of the bidentate binding of the PD to the pentanyl, cyclohexanyl, unsubstituted phenylenyl, phenyle two Sulfur atoms of an opened cysteine-cysteine disulfide nyl substituted by 1 or 2 substituents selected from the group bond in the antibodies, these ADCs are homogeneous and consisting of halo, CF , CFO CHO , —C(O)OH, have enhanced stability over ADCs with monodentate linkers. —C(O)CC-alkyl, —C(O)CH —CN, NH, —OH, They will therefore have increased half-lives in vivo, reduc —NHCH, -N(CH), —C-alkyl and -(AA)-; ing the amount of cytotoxin released systemically, and be 0041 a, b and care each independently 0, 1, 2 or 3, pro safer than ADCs with monodentate linkers linking one anti vided that at least one of a, b or c is 1: body amino acid to one linkage point which may attach one or 0042 each p is independently an integer of 1 to 14; more drug entities. 0043 each q is independently an integer from 1 to 12: 0034. In another embodiment, there is provided pharma 0044 each AA is independently an amino acid; each ris 1 ceutical compositions containing ADCs as disclosed herein, to 12; and and methods of treatment of cancers targeted by the relevant 0045 CTX is a cytotoxin bonded to L by an amide bond; antibodies by administering ADCs of the present application with the proviso that when L or -(L)-(L)-(L)-together is or pharmaceutical compositions thereof. (CH2)-2-or-(CH2CH2O)2CH2CH2 then L', Land US 2014/0363454 A1 Dec. 11, 2014

L are not bonded to CTX by an amide bond. In one aspect of 0049. In another embodiment, there is provided a linker of the above, L is (CH), or (CHCHO), CHCH. formula AA, BB or CC: 0046. In another aspect, the C-alkyl-S—, Coaryl-S- and Coheteroaryl-S—is selected from the group consisting of: AA X R1 S Z-L-D R-S-O S- O R Ns O O X Sulfonates tosylate BB O O X HC-S-O FC-S-O R1 S Z-L-D O O R meSylate triflate Ns X S S CC N X S A-4 R1 R" Z-L-D, Thiophenyls & Substituted thiophenyls X

S S 0050 where each RandR is independently selected from the group consisting of Calkyl optionally substituted with halo or hydroxyl; phenyl optionally substituted with halo, hydroxyl, carboxyl, Calkoxycarbonyl or C-alkyl, naph thyl optionally substituted with halo, hydroxyl, carboxyl, R" Calkoxycarbonyl or C-alkyl, or 2-pyridyl optionally Sub Thionaphthyls & Substituted stituted with halo, hydroxyl, carboxyl, Calkoxycarbonyl or thionaphthyls Calkyl, Calkylsulfonyloxy, Cocycloalkylsulfonyloxy and Coarylsulfonyloxy; N S N S I0051 L is a linker defined by -(L)-(L)-(L)-, wherein C n n each L', Land L is independently a linker selected from the group consisting of —O—, —C(O)— —S— —S(O)—, R" S(O). , NH , NCH, ,-(CH.) , NH(CH.) A-4 NH , OC(O) , CO. , – NHCHCHC(O) , R" C(O)NHCHCH-NH , NHCHC(O) NHC Thiopyridyll & Substituted (O) , —C(O)NH-, - NCHC(O) , —C(O)NCH. , thiopyridyls -(CH2CH2O), , -(CH2CH2O). CHCH. , —CH2CH2 -(CH2CH2O), , —OCH(CHO ) , cyclo pentanyl, cyclohexanyl, unsubstituted phenylenyl, phenyle wherein R is Calkyl, Caryl, Coheteroaryl, each of nyl substituted by 1 or 2 substituents selected from the group which is optionally substituted by R" that is selected from the consisting of halo, CF , CFO CHO , —C(O)OH, group consisting of halo, CF , CFO , CHO , —C(O) OH, -C(O)CC alkyl, -C(O)CH, CN, -NH-OH, —C(O)CC alkyl, —C(O)CH, —CN, NH, -OH, —NHCH, N(CH) and Calkyl. —NHCH, -N(CH), —C-alkyl and -(AA)-; 0047. These bidentate linkers are also useful in preparing 0.052 a, b and care each independently 0, 1, 2 or 3, pro the linker-cytotoxin conjugates of the present application, vided that at least one of a, b or c is 1: and are useful in preparing the linkers as disclosed herein. 0053 each p is independently an integer of 1 to 14; 0048. In another embodiment, there is provided novel auristatins, derivatives of the auristatins, tubulysin and 0054 each q is independently an integer from 1 to 12: derivatives of the tubulysins, wherein the auristatins, tubul 0055 each AA is independently an amino acid; each ris 1 ysins and their derivatives represented as their respective residues are selected from the group consisting of CTX-I. to 12: CTX-II, CTX-III, CTX-IV, CTX-V, CTX-VI, CTX-VII and 0056 D is carboxyl, Calkoxycarbonyl or amino, and m CTX-VIII, wherein the squiggly line (-) on the bond of the is an integer of 1 to 12. In one aspect of the above, L is residue is attached to a hydrogen. —(CH2)— or —(CH2CH2O)CH2CH2—. US 2014/0363454 A1 Dec. 11, 2014

0057. In one embodiment, there is provided a linker of nesulfonyloxy and 4-toluenesulfonyloxy. In another aspect of formula AAA, BBB, CCC or DDD: the above, L is (CH), or (CHCHO). CHCH and m is an integer of 1 to 12. 0062 Preferred embodiments of this invention are charac AAA terized by the specification and by the features of the claims of X this application as filed, and of corresponding pharmaceutical R compositions, methods and uses of these compounds. Z-L-D DETAILED DESCRIPTION OF THE INVENTION R X BRIEF DESCRIPTION OF THE DRAWINGS BBB X 0063 FIG. 1: Antibody Only; RT=7.12 R 0064 FIG. 2: Antibody--dibromosuccinimide-RT-7.24 0065 FIG.3: Antibody--dibromo-N-benzyl succinimide Z-L-D RT=7.58 R 0066 FIG. 4: Conventional mc-MMAF ADC X 0067 FIG. 5: “Stapled” or “Snapped” dts-ADC CCC 0068 FIG. 6: 18-2A Antibody only X 0069 FIG. 7: 18-2A-mc-MMAF (conventional ADC) R (0070 FIG. 8:18-2A-dts-MMAF (“stapled” or “snapped Z-L-D ADC) (0071 FIG. 9: Potency of T2 and T4 ADCs in Tubulin Polymerization Assay. X (0072 FIG. 10: Potency of T2 ADCs in Tubulin Polymer DDD ization Assay. R X (0073 FIG. 11: T2 and T4 Tubulin Polymerization Assays. (0074 FIG. 12: T2 and T4 Assays. M Z-L-D 0075 FIG. 13: T2 ADCs Inhibit microtubule formation in vitro and are more potent to T4 ADCs. (0076 FIG. 14: T2 ADC Tubulin Assay. 0058 where each RandR' is independently selected from (0077 FIG. 15: ADC Conjugation Protocol for “Stapled” the group consisting of chloro, bromo, iodo, Calkylsulfo nyloxy, Cocycloalkylsulfonyloxy, Coarylsulfonyloxy; or “Snapped Linkers. 0059 L is a linker defined by -(L)-(L)-(L)-, wherein DEFINITIONS each L', Land L is independently a linker selected from the group consisting of —O—, —C(O)— —S— —S(O)—, 0078. An “antibody', also known as an immunoglobulin, S(O). , NH , NCH, , -(CH2). , NH(CH.) is a large Y-shaped protein used by the immune system to NH , —OC(O)— —CO. , NHCHCHC(O)—, identify and neutralize foreign objects such as bacteria and C(O)NHCHCH-NH NHCHC(O) NHC viruses. The antibody recognizes a unique part of the foreign (O) , —C(O)NH-, - NCHC(O) , —C(O)NCH , target, called an antigen, because each tip of the “Y” of the -(CH2CH2O), , -(CH2CH2O). CHCH. , antibody contains a site that is specific to a site on an antigen, —CH2CH2-(CH2CH2O), , —OCH(CHO ) , cyclo allowing these two structures to bind with precision. An anti pentanyl, cyclohexanyl, unsubstituted phenylenyl, phenyle body consists of four polypeptide chains, two identical heavy nyl substituted by 1 or 2 substituents selected from the group chains and two identical light chains connected by cysteine consisting of halo, CF, , CFO , CHO , —C(O)OH, disulfide bonds. A "monoclonal antibody' is a monospecific —C(O)CC-alkyl, —C(O)CH —CN, NH, —OH, antibody where all the antibody molecules are identical —NHCH. —N(CH), C-alkyl and -(AA),-: a, b and c are because they are made by identical immune cells that are all each independently 0, 1, 2 or 3, provided that at least one of a, clones of a unique parent cell. Initially, monoclonal antibod b or c is 1; each p is independently an integer of 1 to 14; each ies are typically prepared by fusing myeloma cells with the q is independently an integer from 1 to 12; each AA is inde spleen cells from a mouse (or B-cells from a rabbit) that has pendently an amino acid; each ris 1 to 12; and D is carboxyl, been immunized with the desired antigen, then purifying the Calkoxycarbonyl or amino. resulting hybridomas by Such techniques as affinity purifica tion. Recombinant monoclonal antibodies are prepared in 0060. In one aspect of the above, each R and R' is inde viruses or yeast cells rather than in mice, through technolo pendently selected from the group consisting of H, Cl, Brand gies referred to as repertoire cloning or phage display/yeast I and iodo; and L is selected from the group consisting of display, the cloning of immunoglobulin gene segments to —(CH),C(O)-Val-Ala-NH-(p-CH)—CHOC(O)-(p- create libraries of antibodies with slightly different amino CH) NO —(CH2CH2O) (CH2CH2)C(O)-Val acid sequences from which antibodies with desired specifici Ala-NH-(p-CH)—CHOC(O)-(p-CH) NO —(CH) ties may be obtained. The resulting antibodies may be pre sC(O)-Val-Cit-NH-(p-CH)—CHOC(O)-(p-CH)— pared on a large scale by fermentation. “Chimeric' or NO. —(CH2CH2O). CHCH)C(O)-Val-Cit-NH-(p- "humanized' antibodies are antibodies containing a combi CH)—CHOC(O)-(p-CH)—NO. nation of the original (usually mouse) and human DNA 0061. In one aspect of the above, RandR' is selected from sequences used in the recombinant process, such as those in the group consisting of trifluoromethanesulfonyloxy, benze which mouse DNA encoding the binding portion of a mono US 2014/0363454 A1 Dec. 11, 2014 clonal antibody is merged with human antibody-producing comprising the population are identical except for possible DNA to yield a partially-mouse, partially-human monoclonal mutations, e.g., naturally occurring mutations, that may be antibody. Full-humanized antibodies are produced using present in minor amounts. The modifier term “monoclonal transgenic mice (engineered to produce human antibodies) or indicates the character of the antibody as not being a mixture phage display libraries. Antibodies (Abs) and “immunoglo of discrete antibodies. In certain aspects, such a monoclonal bulins’ (Igs) are glycoproteins having similar structural char antibody may include an antibody comprising a polypeptide acteristics. While antibodies exhibit binding specificity to a sequence that binds a target, wherein the target-binding specific antigen, immunoglobulins include both antibodies polypeptide sequence was obtained by a process that includes and other antibody-like molecules which generally lack anti the selection of a single target binding polypeptide sequence gen specificity. Polypeptides of antibody-like molecules are from a plurality of polypeptide sequences. For example, the produced at low levels by the lymph system and at increased selection process can be the selection of a unique clone from levels by myelomas. The terms “antibody' and “immunoglo a plurality of clones, such as a pool of hybridoma clones, bulin' are used interchangeably in the broadest sense and phage clones, or recombinant DNA clones. In addition to include monoclonal antibodies (e.g., full length or intact their specificity, monoclonal antibody preparations are monoclonal antibodies), polyclonal antibodies, monovalent advantageous in that they are typically uncontaminated by antibodies, multivalent antibodies, multispecific antibodies other immunoglobulins. The modifier “monoclonal indi (e.g., bispecific antibodies so long as they exhibit the desired cates the character of the antibody as being obtained from a biological activity). These antibodies may also include cer Substantially homogeneous population of antibodies, and is tain antibody fragments. An antibody can be chimeric, not to be construed as requiring production of the antibody by human, hunanized and/or affinity matured. Antibodies of par any particular method. (See Harlow et al., Antibodies: A ticular interest in this invention are those that are specific to Laboratory Manual, (Cold Spring Harbor Laboratory Press, cancer antigens, are non-immunogenic, have low toxicity, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and are readily internalized by cancer cells; and Suitable and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), antibodies include alemtuzumab, bevacizumab, brentuX recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816, imab, cetuximab, gemtuzumab, ipilimumab. ofatumumab, 567), and technologies for producing human or human-like panitumumab, rituximab, , inotuZumab, glem antibodies in animals that have parts or all of the human batumumab, lovortuzumab and trastuzumab. Antibodies also immunoglobulin loci or genes encoding human immunoglo include , afutuZumab, , beli bulin sequences (see, WO98/24893: WO96/34096; WO96/ mumab, bivatuZumab, cantuzumab, citatuZumab, cixutu 33735 and WO91/10741). The monoclonal antibodies herein mumab, , , , etaraci specifically include “chimericantibodies in which a portion Zumab, , , , of the heavy and/or light chain is identical with or homolo , , , , gous to corresponding sequences in antibodies derived from a , , , , particular species or belonging to a particular antibody class , , oportuZumab, , pritu or subclass, while the remainder of the chain(s) is identical mumab, ranibizumab, , , siltuX with or homologous to corresponding sequences in antibod imab, tacatuZumab, , tucotuZumab, , ies derived from another species or belonging to another Votumumab and . antibody class or Subclass, as well as fragments of Such anti 0079. The terms “full length antibody,” “intact antibody” bodies, so long as they exhibit the desired biological activity and “whole antibody' are used herein interchangeably to (U.S. Pat. No. 4,816,567). “Humanized forms of non-human refer to an antibody in its Substantially intact form, and are not (e.g., murine) antibodies are chimericantibodies that contain antibody fragments as defined below. The terms particularly minimal sequence derived from non-human immunoglobu refer to an antibody with heavy chains that contain the Fc lin. In one aspect, a is a human immu region. noglobulin (recipient antibody) in which residues from a 0080) “Antibody fragments' comprise only a portion of an hypervariable region of the recipient are replaced by residues intact antibody, wherein the portion retains at least one, two, from a hyperVariable region of a non-human species (donor three and as many as most or all of the functions normally antibody) Such as mouse, rat, rabbit, or nonhuman primate associated with that portion when present in an intact anti having the desired specificity, affinity, and/or capacity. In body. In one aspect, an antibody fragment comprises an anti another aspect, framework region (FR) residues of the human gen binding site of the intact antibody and thus retains the immunoglobulin are replaced by corresponding non-human ability to bind antigen. In another aspect, an antibody frag residues. In general, a humanized antibody will comprise ment, Such as an antibody fragment that comprises the Fc substantially all of at least one, and typically two, variable region, retains at least one of the biological functions nor domains, in which all or substantially all of the hypervariable mally associated with the Fc region when present in an intact loops correspond to those of a non-human immunoglobulin, antibody. Such functions may include FcRn binding, anti and all or substantially all the FRs are those of a human body half life modulation, ADCC function and complement immunoglobulin sequence. The humanized antibody may binding. In another aspect, an antibody fragment is a monova comprise at least a portion of an immunoglobulin constant lent antibody that has an in vivo half life substantially similar region (Fc), typically that of a human immunoglobulin. See to an intactantibody. For example, Such an antibody fragment Vaswani and Hamilton, Ann Allergy, Asthma & Immunol. may comprise on antigen binding arm linked to an Fc 1:105-115 (1998); Harris, Biochem. Soc. Transactions sequence capable of conferring in vivo stability to the frag 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. ment. 5:428-433 (1994). 0081. The term “monoclonal antibody” as used herein 0082 “Framework” or “FR residues are those variable refers to an antibody obtained from a population of Substan domain residues other than the hyperVariable region residues. tially homogeneous antibodies, i.e., the individual antibodies “Fc receptor or “FeR’ is a receptor that binds to the Fc region US 2014/0363454 A1 Dec. 11, 2014

of an antibody. In some embodiments, an FcR is a native double bond (as in maleimide) or a leaving group Such as a human FcR. In one aspect, an FcR is one which binds an IgG chloro, bromo or iodo or an R-sulfanyl group or Sulfonyl antibody (a gamma receptor) and includes receptors of the group, oranamine-reactive group Such as a carboxyl group or FcyRI. FcyRII and FcyRIII subclasses. (See Daeron, Annu. as defined herein; while the antibody conjugation reactive Rev. Immunol. 15:203-234 (1997)). terminus of the linker is typically a site that is capable of 0083. An "amino acid' (or AA) or amino acid residue conjugation to the cytotoxin through formation of an amide includebut are not limited to the 20 naturally occurring amino bond with a basic amine or carboxyl group on the cytotoxin, acids acids commonly designated by three letter symbols and and so is typically a carboxyl or basic amine group. In one also includes citrulline (Cit), 4-hydroxyproline, hydroxyl embodiment, when the term “linker' is used in describing the ysine, demosine, isodemosine, 3-methylhistidine, norvalin, linker in conjugated form, one or both of the reactive termini beta-alanine, gamma-aminobutyric acid, homocysteine, will be absent (such as the leaving group of the thiol-reactive homoserine, ornithine and methionine Sulfone. The amino group) or incomplete (such as the being only the carbonyl of acid residue of the present application also include the corre the carboxylic acid) because of the formation of the bonds sponding N-methylamino acids, such as —N(CH)CHC(O) between the linker and/or the cytotoxin. O-, -NHC(O)CHCH-CH(NHCH)C(O)O etc....The I0086. The term “leaving group, or “LG', as used herein, amino acids, dipeptides, tripeptides, oligomers and polypep refers to any group that leaves in the course of a chemical tides designated as -(AA), of the present application may reaction involving the group as described herein and includes include the corresponding non-N-alkylated amino acids and but is not limited to halogen, Sulfonates (brosylate, mesylate, peptides (such as non-N-methylated amino acids in the pep tosylate, triflate etc. . . . ), p-nitrobenzoate and phosphonate tides), as well as a mixture of the non-N-alkylated amino groups, for example. acids and the N-alkylated amino acids of the peptides. I0087 An “antibody-drug conjugate' (ADC) is an anti 0084. A “cytotoxin' (CTX) is a molecule that has a cyto body that is conjugated to one or more cytotoxins, through toxic effect on cells (e.g., when released within a cancer cell, one or more linkers. The antibody is typically a monoclonal is toxic to that cell). Cytotoxins of particular interest in this antibody specific to a therapeutic target Such as a cancer invention are the tubulysins (such as the tubulysins of the antigen. formulae T3 and T4, and CTX-I", CTX-II", CTX-III", CTX I0088 “Phenyl' means a CHs group as known in the art. IV, CTX-V, CTX-VI", CTX-VII' and CTX-VIII' disclosed “Phenylene' means a divalent phenyl group, wherein the herein), the auristatins (such as monomethylauristatin E and phenyl group is Substituted at two positions on the phenyl ring monomethylauristatin F), the maytansinoids (such as mer that may be ortho (o-CH) or para (p-CH). tansine), the calicheamicins (such as calicheamicin Y); those I0089. “Tubulysin' includes both the natural products cytotoxins that, like the tubulysins of the formulae T3 and T4. described as tubulysins, such as by Sasse et al. and other and those disclosed herein are capable of coordination authors mentioned in the Description of the related art, and through an amide bond to a linker, Such as by possessing a also the tubulysin analogs described in US Patent Application basic amine or a carboxyl group. Publication No. US 2011/0021568 A1. Tubulysins disclosed I0085. A “linker” (noted as L or L', Land L) is a molecule in the present application are noted herein and may include with two reactive termini, one for conjugation to an antibody the tubulysins of the formulae T3 and T4, and CTX-I", CTX or to another linker and the other for conjugation to a cyto II", CTX-III', CTX-IV, CTX-V, CTX-VI", CTX-VII' and toxin. The antibody conjugation reactive terminus of the CTX-VIII' and other tubulysins where the terminal N-meth linker is typically a site that is capable of conjugation to the ylpiperidine has been replaced by an unsubstituted piperidine antibody through a cysteine thiol or lysine amine group on the (the des-methyl analogs), allowing amide bond formation antibody, and so is typically a thiol-reactive group Such as a with a linker.

H N\' Na,'.

CH O w

Nw H O

Desmethyl US 2014/0363454 A1 Dec. 11, 2014

-continued

H N\' N, X CH O w w

OH

O

N\ H O

Desmethyl OH

O

Des-methyl tubulysin compounds correspond to the terminal non-N-methylated piperidine analogs

0090. The terms “cell proliferative disorder” and “prolif 0094. As used herein, the term “pharmaceutically accept erative disorder” refer to disorders that are associated with able salt” refers to those salts of the ADCs formed by the Some degree of abnormal cell proliferation. In one aspect, the process of the present application which are Suitable for use in cell-proliferative disorder is cancer. contact with the tissues of humans and lower animals without 0091 “Tumor refers to all neoplastic cell growth and undue toxicity, irritation, allergic response and the like. Phar proliferation, whether malignant or benign, and all pre-can maceutically acceptable salts are well known in the art. For cerous and cancerous cells and tissues. The terms "cancer.” example, S. M. Berge, et al. describes pharmaceutically “cancerous.” “cell proliferative disorder.”99 “proliferative&g dis acceptable salts in detail in J. Pharmaceutical Sciences, 66: order and “tumor are not mutually exclusive. The terms 1-19 (1977). The salts can be prepared in situ during the final “cancer and “cancerous” refer to the physiological condition isolation and purification of the ADC compounds, or sepa in mammals that is typically characterized by unregulated rately by reacting the free base function or group of a com cell growth. Examples of cancer include, but are not limited pound with a Suitable organic acid. Examples of pharmaceu to, carcinoma, lymphoma, blastoma, sarcoma and leukemia tically acceptable salts include, but are not limited to, or lymphoid malignancies. nontoxic acid addition salts, or salts of an amino group 0092 A“basic amine', such as the amine forming a part of formed with inorganic acids such as hydrochloric acid, the terminal piperidine group of the tubulysins. Such as that of hydrobromic acid, phosphoric acid etc. . . . or with organic the formulae T3 and T4, CTX-I", CTX-II", CTX-III", CTX acids such as acetic acid, maleic acid, tartaric acid, citric acid, IV, CTX-V, CTX-VI", CTX-VII' and CTX-VIII', is a pri Succinic acid or malonic acid. Other pharmaceutically mary or secondary amine that is not part of an amide acceptable salts include, but are not limited to, adipate, algi 0093. A “therapeutically effective amount’ means that nate, ascorbate, benzenesulfonate, benzoate, bisulfate, cit amount of an ADC of the first aspect of this invention or rate, digluconate, dodecylsulfate, ethanesulfonate, formate, composition of the second aspect of this invention which, fumarate, gluconate, 2-hydroxy-ethanesulfonate, lactate, lau when administered to a human Suffering from a cancer, is rate, malate, maleate, malonate, methanesulfonate, oleate, sufficient to effect treatment for the cancer. “Treating or oxalate, palmitate, phosphate, propionate, Stearate. Succinate, “treatment of the cancer includes one or more of: Sulfate, tartrate, p-toluenesulfonate, Valerate salts, and the (1) limiting/inhibiting growth of the cancer, i.e. limiting its like. Representative alkalioralkaline earth metal salts include development; Sodium, lithium, potassium, calcium, or magnesium salts, (2) reducing/preventing spread of the cancer, i.e. reducing/ and the like. Further pharmaceutically acceptable salts preventing metastases; include, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, (3) relieving the cancer, i.e. causing regression of the cancer, hydroxide, carboxylate, Sulfate, phosphate, nitrate, alkyl (4) reducing/preventing recurrence of the cancer; and groups having from 1 to 6 carbon atoms (i.e., Calkyl). (5) palliating symptoms of the cancer. Sulfonate and aryl Sulfonate. US 2014/0363454 A1 Dec. 11, 2014

0.095 Cancers of interest for treatment include, but are not -continued limited to, carcinoma, lymphoma, blastoma, Sarcoma, and leukemia or lymphoid malignancies. More particular examples of Such cancers include squamous cell cancer (e.g. conjugation epithelial squamous cell cancer), lung cancer including N-L-CTX --> Small-cell lung cancer, non-Small cell lung cancer, adenocar cinoma of the lung and squamous carcinoma of the lung, O cancer of the peritoneum, hepatocellular cancer, gastric or conventional maleimide stomach cancer including gastrointestinal cancer, pancreatic linker-drug cancer, glioblastoma, cervical cancer, ovarian cancer, oral conjugate cancer, liver cancer, bladder cancer, cancer of the urinary O tract, hepatoma, breast cancer including, for example, HER2 positive breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland car N-L-CTX cinoma, kidney or renal cancer, prostate cancer, Vulval can cer, thyroid cancer, hepatic carcinoma, anal carcinoma, Antibody/Ns O penile carcinoma, melanoma, acute myeloid leukemia SH

(AML), chronic lymphocytic leukemia (CML), multiple -- myeloma and B-cell lymphoma, brain cancer, head and neck O cancers and associated metastases.

Abbreviations/Acronyms N-L-CTX

0096 ADC: antibody-drug conjugate; DEA: diethy Antibody /Ns O lamine; DCC: 1,3-dicyclohexylcarbodiimide: DIAD: diiso S O propyl azodicarboxylate; DIPC: 1,3-diisopropylcarbodiim ide; DIPEA. diisopropylethylamine; DMF: N,N- dimethylformamide: DPBS: Dulbecco's phosphate-buffered N-L-CTX. saline; DTPA; diethylenetriaminepentaacetic acid; DTT: dithiothreitol: EDC: ethyl 3-(3-dimethylaminopropyl)carbo O diimide; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tet ramethyluronium hexafluorophosphate: HOBT: N-hydroxy benzotriazole; NHS: N-hydroxysuccinimide: NMM: However, the bifunctional pyrrole-2,5-dione- and pyrroli N-methylmorpholine: MMAE: monomethylauristatin E: dine-2,5-dione-based linkers of this invention contain two MMAF: monomethylauristatin F, monomethylauristatin phe reactive functional groups (X in the scheme below) that react nylalanine; MC: maleimidocaproyl, 6-(2,5-dioxopyrrolyl) with the two Sulfur atoms of an opened cysteine-cysteine hexanoyl; PBS: phosphate-buffered saline; PEG: poly(ethyl disulfide bond. Reaction of the bifunctional linker with the eneglycol); TBTU: 2-(1H-bentotriatol-1-yl)-1,1,3,3- two cysteines gives a “stapled' or “snapped dithiosuccinim tetrasethyluronium tetrafluoroborate TCEP: tris(2- ide or dithiomaleimide antibody conjugate with one linker carboxyethyl)phosphine: TGI: tumor growth inhibition. per disulfide connected through two thioether bonds, as shown in the scheme below (double bond absent from the ring: Succinimide linkers of formulae AA and AAA; double The ADCs of the Invention bond present in the ring: maleimide linkers of formulae BB 0097. As mentioned in the Description of the related art, and BBB). ADCs of the prior art that coordinate to cysteine thiols of the antibody have employed monofunctional linkers, of which disulfide the MC linker is an example. Reduction and opening of the /N S reduction /N SH cysteine-cysteine disulfide bonds to give free thiols for con Antibody -- Antibody -- jugation decreases the stability of the antibody, and the for S Nu-SH mation of the ADC by reaction of the reduced thiols does not re-form a bond, as illustrated in the general scheme below: conjugation N-L-CTX -->

disulfide /N i reduction /N SH -- Antibody -> Antibody O bifunctional linker-drug conjugate US 2014/0363454 A1 Dec. 11, 2014 10

-continued R O O Br S Antibody /Ns NH R-SH NH HO-L-Z S base Mitsunobu Br S reaction O PPh3, DIAD R “Activated for 0098. Unlike conventional methods for cysteine conjuga disulfide exchange tion, the reaction re-forms a covalently bonded structure R between the 2 cysteine sulfur atoms and therefore does not O compromise the overall stability of the antibody. The method S also enables conjugation of an optimal 4 drugs per antibody to N-L-Z. afford a homogeneous ADC since the reactive cysteines are used. The overall result is replacement of a relatively labile S O disulfide with a stable “staple' or "snapp' between the cys R teines. The monosubstituted maleimide linkers (formulae CC and CCC) are also effectively bifunctional in conjugation 0103 2.3-Dibromomaleimide, 1 equivalent, and a base with the antibody because the double bond of the maleimide Such as Sodium bicarbonate, about 5 equivalents, are dis is capable of conjugation to one of the cysteine Sulfur atoms Solved in methanol, and a solution of 2-pyridinethiol, slightly and the X group with the other. more than 1 equivalent, in methanol, is added. The reaction is stirred for 15 min at ambient temperature. The solvent is Preparation of the Compounds of the Invention removed under vacuum and the residue is purified. Such as by flash chromatography on silica gel (petroleum ether:ethyl 0099. The compounds of the invention, such as ADCs, acetate, gradient elution from 9:1 to 7:3, to give 3,4-bis(2- linker-cytotoxin conjugates, linkers, and tubulysins, are pre pyridylsulfanyl)pyrrole-2,5-dione. pared by conventional methods of organic and bio-organic 0104. The coupling of the 3,4-bis(2-pyridylsulfanyl)pyr chemistry. See, for example, Larock, “Comprehensive role-2,5-dione with the sidechain is performed under strictly Organic Transformations”, Wiley-VCH, New York, N.Y., dry conditions. To the 3,4-bis(2-pyridylsulfanyl)pyrrole-2,5- U.S.A. Suitable protective groups and their methods of addi dione, 1 equivalent, and triphenylphosphine, 1 equivalent, in a mixture of tetrahydrofuran and dichloromethane, is added tion and removal, where appropriate, are described in Greene dropwise DIAD, 1 equivalent, at -78° C. The reaction is et al., “Protective Groups in Organic Synthesis”, 2" ed., stirred for 5 min and the sidechain, 0.5 equivalent, in dichlo 1991, John Wiley and Sons, New York, N.Y., US. Reference romethane is added dropwise. After stirring for 5 min, neo may also be made to the documents referred to elsewhere in pentyl alcohol. 1 equivalent, in tetrahydrofuran and dichlo the application, such as to the Schumacher et al. article romethane is added, and stirred for a further 5 min, then the referred to earlier for the synthesis of linkers, US Patent 3,4-bis(2-pyridylsulfanyl)pyrrole-2,5-dione, 1 equivalent, is Application Publication No. US 2011/0021568 A1 for the added and stirred for another 5 min. The reaction is allowed to preparation of tubulysins, etc. warm to ambient temperature with stirring for 20 hr, then the solvents are removed under vacuum. The residue is purified, Preparation of the Tubulysins Such as by flash chromatography on silica gel (methanol: dichloromethane, gradient elution from 0-10% methanol), to 0100 Tubulysins T3 and T4, CTX-I", CTX-II", CTX-III', give the linker. The sidechain may be used in protected form, CTX-IV, CTX-V, CTX-VI", CTX-VII' and CTX-VIII', are and deprotected following the Mitsunobu reaction, if appro prepared by methods analogous to those of Peltier et al. and priate. US Patent Application Publication No. US 2011/0021568 A1, 0105. Alternatively, the sidechain, optionally protected if by substituting D-pipecolinic acid for the D-N-methylpipe appropriate, may be coupled to a 3,4-dibromomaleimide by colinic acid, protecting and deprotecting if appropriate. Tubu Mitsunobu coupling; and the resulting compound activated lysin analogues may be prepared using conventional Syn for disulfide exchange by reaction with an R-thiol in the thetic procedures known in the art, such as those described by presence of base; in the reverse of the synthesis described in Larock, above. the two previous paragraphs. 0106 A similar method may be used for linkers containing the pyrrolidine-2,5-dione moiety rather than the pyrrole-2,5- Preparation of the Linkers dione moiety shown above, by starting with 2,3-dibromosuc cinimide; but more usually these linkers are prepared by 0101 The comparator MC linker is prepared by methods preparing the linker with an unsubstituted maleimide and known to the art for its preparation. brominating the linker to give the dibromosuccinimide moi 0102 Linkers of this invention are prepared by methods ety after coupling with the sidechain, and then “activating analogous to those of Schumacher et al., as follows (in this the linker with the R-thiol as a last step. reaction Scheme, R. L. and Zhave the meanings given them in 0.107 Mono-substituted maleimide linkers are conve the discussion of the fifth and sixth aspects of the invention niently prepared by dehydrobromination of the dibromosuc above): cinimide linkers under basic conditions, and related methods. US 2014/0363454 A1 Dec. 11, 2014

Preparation of the Linker-Cytotoxin Conjugates -continued 0108 Linker-cytotoxin conjugates may be prepared by n X methods analogous to those of Doronina et al., Bioconjugate c3Nshi -- N-L-CTX Chem. 2006, 17, 114-124, and similar documents. The linker, 1 equivalent, and HATU, 1 equivalent, are dissolved in anhy c1Nshi X drous DMF, followed by the addition of DIPEA, 2 equiva O lents. The resulting solution is added to the cytotoxin, 0.5 linker-cytotoxin conjugate equivalents, dissolved in DMF, and the reaction stirred at (X represents a leaving group, ambient temperature for 3 hr. The linker-cytotoxin conjugate Such as 2-pyridinesulfanyl) is purified by reverse phase HPLC on a C-18 column Preparation of ADCs 0109 Antibodies, typically monoclonal antibodies are raised against a specific cancer target (antigen), and purified and characterized. Therapeutic ADCs containing that anti n O body are prepared by standard methods for cysteine conjuga tion, such as by methods analogous to those of Hamblett et al., c1\s “Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate’. Clin. Cancer Res. N-L-CTX

2004, 10, 7063-7070; Doronina et al., “Development of c1\s pi potent and highly efficacious monoclonal antibody auristatin conjugates for cancer therapy'. Nat. Biotechnol., 2003, 21 (7). O 778-784; and Francisco et al., “cAC10-vcMMAE, an anti CD30-monomethylauristatin E conjugate with potent and selective antitumor activity, Blood, 2003, 102, 1458-1465. 0111 Typically, n will be 4, where all of the reactive Antibody-drug conjugates with four drugs per antibody are cysteine disulfide bonds are replaced by linker-drug conju prepared by partial reduction of the antibody with an excess gates. of a reducing reagent such as DTT or TCEP at 37° C. for 30 min, then the buffer exchanged by elution through SEPHA The Antibody-Drug Conjugates (ADC) of the Present DEXOR) G-25 resin with 1 mM DTPA in DPBS. The eluent is Application: diluted with further DPBS, and the thiol concentration of the antibody may be measured using 5,5'-dithiobis(2-nitroben O112 In one embodiment, there is provided an ADC of the Zoic acid) Ellman's reagent. An excess, for example 5-fold, formula: of the linker-cytotoxin conjugate is added at 4°C. for 1 hr., and the conjugation reaction may be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The wherein A is an antibody, the double bond (—) represents resulting ADC mixture may be purified on SEPHADEXG-25 bonds from the 3- and 4-positions of the PD wherein PD is a equilibrated in PBS to remove unreacted linker-cytotoxin pyrrole-2,5-dione or derivative thereof, a pyrrolidine-2,5-di conjugate, desalted if desired, and purified by size-exclusion one or derivative thereof; L is a linker as defined herein, and chromatography. The resulting ADC may then be then sterile CTX is a cytotoxin bonded to L. filtered, for example, through a 0.2 uM filter, and lyophilized if desired for storage. The Antibody (A): 0110. The formation of an ADC of this invention is illus trated by the reaction scheme below, where the “Y”-shaped 0113. In one embodiment, the antibody (A) is a mono structure denotes the antibody, only one disulfide bond is clonal antibody or a humanized antibody. In another embodi shown, and details of the linker-cytotoxin conjugate are omit ment, the antibody is specific to a cancer antigen. In another ted for simplicity in showing the concept of the ADC. embodiment, the antibody employed in the ADC of the present application is selected from the group consisting of alemtuzumab, bevacizumab, cetuximab, ipilimumab. ofatu mumab, anitumumab, rituximab, to situmomab, inotuZumab, n a DTT or TCEP glembatumumab, lovortuZumab, milatuZumab and trastu c1a S reduction Zumab. c1a S The PD Group: antibody with one disulfide bond 0114. In one embodiment, PD is a pyrrole-2,5-dione or shown derivative thereof, a pyrrolidine-2,5-dione or derivative thereof. In another embodiment, the PD group is selected from the group consisting of US 2014/0363454 A1 Dec. 11, 2014 12

CN, NH , NH, O—, OH, NHCH PD1 —N(CH), —C-alkyl and -(AA),-, I0123 a, b and care each independently 0, 1, 2 or 3, pro vided that at least one of a, b or c is 1: 0.124 each p is independently an integer of 1 to 14; 0.125 each q is independently an integer from 1 to 12: 0.126 each AA is independently an amino acid; X PD2 0127 each r is 1 to 12; and X I0128 m is an integer of 1 to 4; and n is an integer of 1 to 4: with the proviso that when -(L)-(L)-(L)- together is -(CH2)2 or -(CH2CH2O)2CH2CH2— then L', Li and L are not bonded to CTX by an amide bond. I0129. In another embodiment of the above ADC, each L', X Land L is independently selected from the group consisting PD3 of (CH) , NH(CH.).NH , OC(O), CO. ,

-(CH2CH2O)CH2CH2—, —CH2CH2 -(CH2CH2O), , —OCH (p-CH-) NH , —OCH(o-CH-) NH , —NH-(p-CH) CHO , —NH-(o-CH)—CHO , —OCH(CH2O—)—and -(AA)-; a, b and c are each inde where: pendently 0, 1 or 2; each p, q and r is independently 1, 2, 3 or 0115 X is O, S or NR' where R' is H or Calkyl: 4; m is 1; and n is an integer of 1 to 4. In another embodiment 0116 X" is O, S or NR where R is H or Calkyl; and of the ADC of the present application, the linker is attached to the CTX by a group selected from the group consisting of 0117 Z is selected from the group consisting of N , NHC(O)-, -NHC(O)O-, - N(Calkyl)C(O)O , CH , CR - and CR CRR where R, R and —NH , N(Calkyl)-, - N(Calkyl)C(O)NH and 0118 Rare each independently H or Calkyl. —N(Calkyl)C(O)N(Calkyl)-. 0119. In another embodiment of the PD group PD1, PD2 I0130. In another embodiment of the above ADC, each L', or PD3, X and X" are 0, and Z is N. In another embodiment of Land L is independently selected from the group consisting the PD group, X and X’ are S, and Z is N. In another embodi of (CH) , NH(CH2).NH , OC(O), OC, , ment of the PD group, X and X’ are NCH, and Z is N. In NHCHCHC(O) , C(O)NHCHCH-NH , another embodiment of the PD group, X and X’ are 0, and Z - NHCHC(O) , – NHC(O) C(O)NH-, - NCHC is CH-. In another embodiment of the PD group, X and X" (O)— —OCH(CHO )—and —C(O)NCH. : a, b and c are S, and Z is CH-. In another embodiment of the PD group, are eachindependently 0, 1 or 2; each p and q is independently X and X’ are NCH, and Z is CH-. 1 or 2; m is 1; and n is an integer of 1 to 4. 0120 In one aspect of the above ADC, when PD is a I0131. In another embodiment of the above ADC, each L', pyrrole-2,5-dione or a pyrrolidine-2,5-dione, L is —(CH) Land L is independently selected from the group consisting — or -(CH2CH2O)CH2CH2 and then L is not attached of NH(CH)NH-, -NHCHCHC(O)—, C(O) to CTX by an amide bond. NHCHCH-NH NHCHC(O) , NHC(O) , - C(O)NH-, - NCHC(O) , OCH(CHO ) and The Linker L: —C(O)NCH. : a, b and care each independently 0 or 1; m 0121. In one embodiment, there is provided an antibody is 1; and n is an integer of 1 to 4. drug conjugate (ADC) of the formula: (0132) In another embodiment of the above ADC, each L', Land L is independently selected from the group consisting of NHC(O) , C(O)NH (CH2CH2O), , wherein A is an antibody, PD is a pyrrole-2,5-dione orderiva -(CH2CH2O). CHCH , —CH2CH2 -(CH2CH2O), . tive thereof, a pyrrolidine-2,5-dione or derivative thereof; —OCH(CH2O—)—and -(AA)-; a, b and c are each inde CTX is a cytotoxin; pendently 0 or 1; each p and r is independently 1, 2 or 3; m is 0122 each L', Land L is independently a linker selected 1; and n is an integer of 1 to 4. from the group consisting of —O—, —C(O)— —S , 0133. In another embodiment of the above ADC, each AA S(O)-, -S(O), , -NH-, -NCH, , -(CH2) , is an amino acid selected from the group consisting of Ala, —NH(CH)NH , —OC(O)—, —CO , Arg, Asn. Asp, Cit, Cys, Glu, Gln, Gly. His, Ile, Lys, Met, Phe, —NHCHCHC(O) , C(O)NHCHCH-NH , Pro, Ser. Thr, Trp, Tyr and Val. In one variation of the above, -NHCHC(O)-, -NHC(O) , —C(O)NH-, -NCHC (AA), is a single amino acid selected from the group consist (O) , C(O)NCH, , -(CH2CH2O), , -(CH2CH2O) ing of Cit, Gly, Arg, Val, Ala, Cys, Gln, Leu, Ile, Lys and Ser CHCH. , —CHCH (CHCHO). , OCH or their N-methylated analogues. In another variation of the (CH2O—) , cyclopentanyl, cyclohexanyl, unsubstituted above, (AA), is selected from the group consisting of Ala-Val. phenylenyl, phenylenyl substituted by 1 or 2 substituents Val-Ala., Gly-Gly, Gly-Arg, Gly-Val, Gly-Ala., Gly-Cys, Gly selected from the group consisting of halo, CF , CFO , Gln, Gly-Ile, Lys-Leu, Gly-Lys, Val-Arg, Ala-Cit, Val-Cit and CHO , —C(O)OH, - C(O)OC-alkyl, —C(O)CH, Gly-Ser or their N-methylated analogues. US 2014/0363454 A1 Dec. 11, 2014

0134. In another variation of the above, (AA), is selected -continued from the group consisting of Gly-Gly-Gly, Gly-Arg-Gly, CTX-Ia

Gly-Val-Gly, Gly-Ala-Gly, Gly-Cys-Gly, Gly-Gln-Gly, Gly Ile-Gly, Lys-Leu-Gly, Gly-Lys-Gly and Gly-Ser-Gly or their R5 R7 N-methylated analogues. In another variation of the above, (AA), is selected from the group consisting of Ala-Ala, Ala Gly, Ala-Arg, Ala-Val. Ala-Ala, Ala-Cys, Ala-Gln, Ala-Ile, '-'.O$/ \ Ala-Leu, Ala-Lys, Ala-Cit and Ala-Ser or their N-methylated H analogues. In another variation of the above, (AA), is selected from the group consisting of Ala-Ala-Ala., Ala-Gly-ALa, Ala Arg-Ala., Ala-Val-Ala, Ala-Ala-Ala., Ala-Cys-Ala., Ala-Gln Ala, Ala-Ile-Ala, Ala-Leu-Ala, Ala-Lys-Ala and Ala-Ser-Ala O or their N-methylated analogues. wherein: The CTX Residue: 0.137 i is 0 or 1; I0138) R' is a C-alkyl; R is a Calkyl; R is Calkyl: 0135) In one embodiment, the CTX residue is a tubulysin I0139) R' is selected from the group consisting of residue of the formula T3 or T4: Calkyl, —OC-alkyl, —OC(O)Calkyl, OC(O)NHC 6alkyl and —OC(O)NHC-aryl; and I0140 R is selected from the group consisting of OH, —OC-alkyl, -COC-alkyl, -COC-aryl, -CH(C.

6alkyl)COR. —CH(Caryl)COR. - NH-CH(CSH) —NHC-alkyl, - NH(CH) COR. - NH(CHCH) Caryl, - NHCH(CHCaryl)CH-CH(CH)COR and NHCH(CHCOR)CH-p-CH NHC alkyl: where each R is independently H or C-alkyl; and R'' is selected from the group consisting of H. —CH and —C(O) CHs. 0141 In one embodiment, the CTX residue comprises the formula:

CTX-II

OH

0136 In one embodiment, the CTX residue comprises the formula:

O

CTX-I wherein:

0.142 i is 0 or 1: I0143 R' is a C-alkyl; R is a Calkyl: 10144) R' is selected from the group consisting of C1-alkyl, Co-oaryl; (0145 R7 is selected from the group consisting of Calkyl, —OC-alkyl, - NHC(O)Calkyl, —OC(O)Cl. 6alkyl, —OC(O)Caryl, —OC(O)NHC alkyl and —C(O)NHC-aryl; and

US 2014/0363454 A1 Dec. 11, 2014

0180 R is selected from the group consisting C-alkyl, wherein: -phenyl, 1-naphthyl and 2-mapthyl, wherein each -phenyl, (0193 each R is independently a C-alkyl or Coaryl; 1-naphthyl and 2-maphthyl group is unsubstituted or Substi (0194 R is a Calkyl or Coaryl; tuted by 1 or 2 substituents selected from the group consisting (0195 each R is independently selected from the group of halo, cyano, nitro, CF , CFO CHO , —C(O)CH, consisting of H. Calkyl, Caryl, —CH2OCOC-alkyl, —CH2COC-alkyl, —CH2CONHC alkyl, -COC. —NH —OH, -SH, -NHCH, N(CH), —SMe and 6alkyl, —CH(Calkyl)COH and —CH(Calkyl)COC Calkyl; and 6alkyl; 0181) R' is selected from the group consisting of (0196) each R" is independently selected from the group Calkyl, Calkenyl, -O-C-alkyl and —OC-aryl; consisting of —CN, —OC-alkyl, Calkyl, -NHC(O)Cl. R'' is H or C-alkyl: 6alkyl, —OC(O)Calkyl, —OC(O)Caryl, —OC(O) 0182 wherein R is selected from the group consisting of NHC alkyl and —OC(O)NHC-aryl; H. C. galkyl and Coaryl; and (0197) R'' is Hor Calkyl; 0183 wherein * designates an R chiral center, an Schiral (0198 each R' is independently selected from the group center or a mixture of R and S isomers. consisting of halo, cyano, nitro, CF , CFO , CHO—, 0184. In one aspect, there is provided the CTX residue of COH, -NH -OH, -SH, -NHCH, N(CH), the formula CTX-V or CTX-Va wherein: R is a Calkyl; R - SMe, Cisalkyl and Co-oaryl; is a C-alkyl, (0199 R' is H or is selected from the group consisting of 0185. R is selected from the group consisting of Calkyl, -CF, -C-alkyl-phenyl and Coaryl; Calkyl, —CH2OCOC-alkyl, —CH2COC-alkyl, (0200) R' is selected from the group consisting of H, —COC-alkyl and CH(Calkyl)COC-alkyl; —CH and —C(O)CH; and q is 0, 1 or 2. 0186 R is selected from the group consisting C-alkyl, 0201 In one aspect, there is provided the CTX residue of -phenyl, 1-naphthyl and 2-mapthyl, wherein each -phenyl, the formula CTX-VI or CTX-VIa: wherein: each R is inde 1-naphthyl and 2-naphthyl is unsubstituted or substituted by pendently a C-alkyl; R is a C-alkyl: 1 or 2 substituents selected from the group consisting of (0202) each R is independently selected from the group CF, , CHO , —C(O)CH, -NHCH, N(CH), and consisting of H. Calkyl, —CHOCOC alkyl, Cisalkyl, —CH2COC-alkyl, CH(Calkyl)COH and CH(C- 0187 R' is selected from the group consisting of alkyl)COC-alkyl; Calkyl, Calkenyl, -O-C-alkyland—O-phenyl; and 0203 each R7 is independently selected from the group R'' is selected from the group consisting of H, —CH, and consisting of —OC-alkyl, C-alkyl, -NHC(O)C-alkyl, —C(O)CH. —OC(O)Calkyl and —OC(O)Caryl; R" is H or 0188 In another aspect, there is provided the CTX residue C1-alkyl, of the formula CTX-V or CTX-Va: 0204 each R'' is independently selected from the group (0189 wherein: R is a Calkyl; R is a Calkyl; R is consisting of halo, CF , CFO CHO —NHCH Cisalkyl, —N(CH), and C-alkyl; (0190. R is selected from the group consisting Calkyl, 0205) R' is H or is selected from the group consisting of -phenyl, 1-naphthyl and 2-mapthyl; and Calkyl, -CF, -C-alkyl-phenyl. (0191) R' is selected from the group consisting of 0206. In another aspect, there is provided the CTX residue Cisalkyl and C2-galkenyl. of the formula CTX-VI or CTX-VIa wherein: each R is 0.192 In another embodiment, the CTX residue comprises independently a C-alkyl; R is a C-alkyl; the formula: 0207 each R is independently H or C-alkyl;

CTX-VI R4 O R5 O-R or NHus N R4 7 \ R6 O 4 R6 R7 O R7 SX (R'), N W R11 CTX-VIa

R4 O R5 RSn N 1. Nulls N R6 O 4 R6 R7 O US 2014/0363454 A1 Dec. 11, 2014

(0208 each R" is independently selected from the group consisting of OC-alkyl, —OC(O)C-alkyl, - OC(O)Cl. 1oaryl, —OC(O)NHC alkyl and —OC(O)NHC-aryl; R'' is H or Calkyl: (0209 each R" is independently selected from the group consisting of CFO CHO and Calkyl; and R' is Hor is selected from the group consisting of Calkyl, -CF, —C-alkyl-phenyl. 0210. In another embodiment, the CTX residue comprises the structure of the formula:

CTX-VII A,

CTX-VIIa

R R N H N u N 1N | 1

wherein: 0214 In one aspect, there is provided the CTX residue of the formula CTX-VII: wherein: R'' is H: R' is selected from 0211) R'' is H or Calkyl: the group consisting of CF , CFO CH-O-, -CO2H. 0212 each R" is independently selected from the group —NHCH, -N(CH), —C-alkyl and phenyl: consisting of halo, cyano, nitro, CF , CFO , CHO—, 0215I R' is H or is selected from the group consisting of COH, -NH –OH, -SH, -NHCH, -N(CH), Cisalkyl, -C-2alkyl-phenyl and phenyl; R' is selected —SMe, Calkyl and Coaryl; from the group consisting of H. —CH and —C(O)CH, and q is 1. In one aspect, there is provided the CTX residue of the 0213 R' is H or is selected from the group consisting of formula CTX-VII: wherein: R'' is Hand R' is H, Calkyl Calkyl, —CF – Calkyl-phenyl and Coaryl; and q is or—C-alkyl-phenyl; and q is 0. In another embodiment, the 0, 1 or 2. CTX residue comprises the formula:

CTX-VIII O R5

N N 4 R R 14 4 R6 R7 O E A.1.us R7 N 1N-R" O O US 2014/0363454 A1 Dec. 11, 2014 18

-continued

CTX-VIIIa R4 O 5 RS NHus s R6 O R4 R6 R7 O

wherein: 0222 each R is independently selected from the group 0216) each Risindependently a Calkylor Caryl; R consisting of H. Calkyl, —CH2OCOC alkyl, —CH-COC alkyl, CH(Calkyl)COH and CH(C. is a C-alkyl or Co-oaryl; alkyl)COC alkyl, 0217 each R is independently selected from the group 0223) each R7 is independently selected from the group consisting of H, Calkyl, C-aryl, —CHOCOC-alkyl, consisting of —OC-alkyl, C-alkyl, -NHC(O)C-alkyl, —CH2COC-alkyl, —CH2CONHC alkyl, -COC. —OC(O)Calkyl, —OC(O)Caryl, —OC(O)NHC 6alkyl, CH(Calkyl)COH and CH(Calkyl)COC. 6alkyl and —OC(O)NHC-aryl; 6alkyl; 0224) R'' is Hor Calkyl; R'' is Calkyl; R is Horis 0218 each R" is independently selected from the group selected from the group consisting of OH, NH, -NHCH consisting of CN, —OC-alkyl, C-alkyl, -NHC(O)Cl. and —OC-alkyl; and R' is Coaryl. 6alkyl, —OC(O)Calkyl, —OC(O)Caryl, —OC(O) 0225. In another aspect, there is provided the CTX residue of the formula CTX-VIII or CTX-VIIIa wherein: each R is NHC alkyl and —OC(O)NHC-aryl; independently a C-alkyl; R is a C-salkyl: 0219 R'' is Hor Calkyl; R'' is selected from the group 0226 each R is independently H or Calkyl: consisting of C-alkyl and Co-oaryl; 10227 each R" is independently selected from the group 0220) R' is H or is selected from the group consisting of consisting of OC-alkyl, —OC(O)Calkyl, —OC(O)Cl. —OH, NH, —NHCH, C-alkyl, OC-alkyland—OC 1oaryl, - OC(O)NHCalkyl and OC(O)NHCaryl; 10aryl; R' is selected from the group consisting of Calkyl, 0228) R' is H or Calkyl: R'' is Calkyl; R' is Coaryland heteroaryl; and R' is selected from the group selected from the group consisting of —OH, NH and consisting of H. —CH and —C(O)CH. NHCH; and R' is Coaryl. 0221. In one aspect, there is provided the CTX residue of 0229. In one embodiment, there is provided the non-con the formula CTX-VIII or CTX-VIIIa, wherein: each R is jugated cytotoxins CTX-I", CTX-II", CTX-III', CTX-IV, independently a C-alkyl; R is a C-alkyl; CTX-V, CTX-VI", CTX-VII' and CTX-VIII' of the formulae:

CTX-IV US 2014/0363454 A1 Dec. 11, 2014

-continued CTX-VI R4 O R5 NHus N O-R18 HN N Z \ E R4 R6 O R4 R6 R7 O S7-(R12) R7 g N O \ R11 CTX-VII

O O-R18 NH N

O 1N -O O O S/(R'),ca. 12 / N O V R11 CTX-VIII R4 O R5

HN NHus N N R4 E R 14 R6 O R4 R6 R7 O R7 R 16 N 1n O wherein the variables i, q R. R. R. R7, R. R. R. R'', 0234. In one embodiment of the above ADC, A is selected R'', R', R'', R'' and Rare as defined herein in the corre from the group consisting of alemtuzumab, bevacizumab, sponding cytotoxin conjugated residues CTX-I, CTX-II, cetuximab, ipilimumab. ofatumumab, anitumumab, ritux CTX-III, CTX-IV, CTX-V, CTX-VI, CTX-VII and CTX imab, to situmomab, inotuZumab, glembatumumab, lovortu VIII, respectively. In one embodiment of the above non Zumab and trastuzumab, conjugated cytotoxins, the cytotoxin is not T3 or T4. 0230. In one aspect of the above variables R' to R', any 0235 PD is a pyrrole-2,5-dione, a pyrrolidine-2,5-dione: designated aryl group. Such as a Coaryl, may be a phenyl 0236 each L', Land L is independently a linker selected group, a 1-naphthyl or 2-naphthyl group, and the aryl group is from the group consisting of (CH2). , -NH(CH2)NH-, unsubstituted or substituted with 1 or 2 substituents selected - NH(CHCH)C(O) , C(O)NH(CHCH)NH , from the group consisting of halo, cyano, nitro, CFs— NHCHC(O)-, -NHC(O) , —C(O)NH-, -NCHC CFO CHO , —COH, - C(O)CH, -NH2, —OH, (O)— —C(O)NCH , cyclopentanyl, cyclohexanyl. —SH, -NHCH, N(CH), SCH and —Calkyl. unsubstituted phenylenyl, phenylenyl substituted by 1 or 2 0231. In one embodiment of the above ADC, A is selected Substituents selected from the group consisting of halo, from the group consisting of alemtuzumab, bevacizumab, CHO , —C(O)OC-alkyl, —C(O)CH, -NHCH cetuximab, ipilimumab. ofatumumab, anitumumab, ritux —N(CH), —C-alkyl; and -(AA)-; where the AA is imab, to situmomab, milatuZumab and trastuzumab; selected from the group consisting of Gly, Arg, Val, Ala, Cys, 0232 PD is a pyrrole-2,5-dione, a pyrrolidine-2,5-dione: Gln, Leu, Ile, Lys and Seror their N-methylated analogues; a, 0233 each L', Land L is independently selected from band c are each independently 0 or 1; the group consisting of NHC(O)— —C(O)NH . -(CH2CH2O), , -(CH2CH2O). CHCH. , 0237 each p and r is independently 1 or 2: m is 1; and n is —CH2CH2 (CH2CH2O), and -(AA)- where the AA is 1, 2, 3 or 4; and CTX is a tubulysin residue or derivative selected from the group consisting of Gly, Arg, Val, Ala, Cys, thereof, oran auristatin residue or a derivative thereof. In one Gln, Leu, Ile, Lys and Seror their N-methylated analogues; a, aspect, when -(L)-(L)-(L)-togetheris-(CH2)2 or b and c are each independently 0 or 1; each p and r is inde —(CH2CH2O)2CHCH then L', L and L are not pendently 1 or 2: m is 1; and n is 1, 2, 3 or 4; and CTX is a bonded to CTX by an amide bond. tubulysin residue orderivative thereof, oran auristatin residue 0238. In one embodiment of the above ADC, A is selected or a derivative thereof; with the proviso that when -(L)-(L) from the group consisting of alemtuzumab, bevacizumab, -(L)- together is (CH2)2 - or -(CH2CH2O). cetuximab, ipilimumab. ofatumumab, anitumumab, ritux 12CHCH then L. L and L are not bonded to CTX by an imab, to situmomab, inotuZumab, glembatumumab, lovortu amide bond. Zumab, milatuZumab and trastuzumab,

US 2014/0363454 A1 Dec. 11, 2014 29

TABLE 1-continued Antibody-Drug Conjugates

US 2014/0363454 A1 Dec. 11, 2014 47

TABLE 1-continued Antibody-Drug Coniugates

En- i R4 R R6 R7 R8 try A PD Ll L2 L3 CTX II 225 BTZ O —(CH2)CO— —NHCH2CH2— —NCHC(O)– 1 —CH —CH —OC(O)CH —NH (CH3)2 (CH2)2- p-MeO N phenyl w O

226 BTZ O —(CH2)CO— —NHCH2CH2— —NCHC(O)– 1 —CH —CH —OC(O)CH —NH (CH3)2 (CH2)2- p-MeO N phenyl w O

228 GTZ. O (CH2)2 OC(O) 1 —CH —CH —OC(O)CH —OCH (CH3)2

w O

229 TTZ O (CH2)2 OC(O) 1 —CH —CH —OC(O)CH —OCH (CH3)2

O w O

230 GBZ O (CH2)2 OC(O) 1 —CH —CH —OC(O)CH —OCH (CH3)2

w O

231 LVT O (CH2)2 OC(O) 1 —CH —CH —OC(O)CH —OCH (CH3)2

O w O

A (Antibodies): TZ (trastuzumab), BTX (brentuximab), GTZ (gemtuzumab), ITZ (inotuzumab), GBT (glembatumumab) and LVT (lovortuzumab).

Assays in determining Suitable doses to test in humans as anticancer 0247 The ADCs of the present application may be agents, and, from the results of those tests, in determining assayed for binding affinity to and specificity for the desired Suitable doses to use to treat cancers in humans. antigen by any of the methods conventionally used for the assay of antibodies; and they may be assayed for efficacy as Formulation and Administration anticancer agents by any of the methods conventionally used 0248. The ADCs of the first aspect of this invention will for the assay of cytostatic/cytotoxic agents, such as assays for typically be formulated as solutions for intravenous adminis potency against cell cultures, Xenograft assays, and the like. A tration, or as lyophilized concentrates for reconstitution to person of ordinary skill in the art will have no difficulty, prepare intravenous Solutions (to be reconstituted, e.g., with considering that skill and the literature available, in determin normal saline, 5% dextrose, or similar isotonic solutions). ing Suitable assay techniques; from the results of those assays, They will typically be administered by intravenous injection US 2014/0363454 A1 Dec. 11, 2014 48 or infusion. A person of ordinary skill in the art of pharma -continued ceutical formulation, especially the formulation of anticancer antibodies, will have no difficulty, considering that skill and rs the literature available, in developing suitable formulations. N 2 O EXAMPLES S NH. Synthesis of Linkers S 0249. The following procedures may be employed for the O preparation of the compounds of the present invention, Such N21 as the compounds described in Table 1. The starting materials and reagents used in preparing these compounds are either N available from commercial suppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well known to a person of ordinary skill in the art, following procedures described in such references as Fieser and Fies er's Reagents for Organic Synthesis, vols. 1-17, John Wiley 0253 3,4-Dibromopyrrole-2,5-dione 2,3-dibromomale and Sons, New York, N.Y., 1991, Rodd's Chemistry of Car imide, 1 g, was added to a clean 100 mL round bottom flask bon Compounds, vols. 1-5 and supps. Elsevier Science Pub with a rubber stopper and bubbler, and dissolved in 50 mL lishers, 1989, Organic Reactions, vols. 1-40, John Wiley and HPLC grade methanol. 2-Pyridinethiol, 2 equivalents, was Sons, New York, N.Y., 1991; March J.: Advanced Organic added to a 20 mL scintillation vial, and dissolved in 10 mL Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and methanol. Under nitrogen and with stirring, the 2-pyridi Larock: Comprehensive Organic Transformations, VCH nethiol/methanol solution was added dropwise to the 3,4- Publishers, New York, 1989. dibromopyrrole-2,5-dione via a 20 mL syringe with a 16 0250 In some cases, protective groups may be introduced gauge needle, and the reaction mixture was stirred for an and finally removed. Suitable protective groups for amino, additional 3-4 hours. The methanol was evaporated and the hydroxy and carboxy groups are described in Greene et al., crude product was dissolved in ethyl acetate and loaded onto Protective Groups in Organic Synthesis, Second Edition, about 2 g silica gel. The silica gel-loaded crude product was John Wiley and Sons, New York, 1991. Standard organic eluted through a 12 g silica gel cartridge with a hexane:ethyl chemical reactions can be achieved by using a number of acetate gradient from 9:1 to 0:1 over 25 column volumes. The different reagents, for examples, as described in Larock: enriched fractions were identified, pooled and lyophilized to Comprehensive Organic Transformations, VCH Publishers, dryness. The final product was recrystallized from ethyl New York, 1989. acetate and diethyl ether to provide yellow needle crystals which were collected by filtration. 0251 While a number of exemplary embodiments, aspects and variations have been provided herein, those of 0254. Similar syntheses may be performed using the skill in the art will recognize certain modifications, permuta methods of Schumacher et al. for other 3,4-di(R-sulfanyl) tions, additions and combinations and certain Sub-combina pyrrole-2,5-diones (see the Supplementary Materials at pages tions of the embodiments, aspects and variations. It is S17-S18). Similar syntheses may also be performed starting intended that the following claims are interpreted to include with (3,4-dibromo-2,5-dioxopyrrolyl)-terminated linkers all such modifications, permutations, additions and combina i.e. compounds where a sidechain has already been added to tions and certain Sub-combinations of the embodiments, the pyrrole nitrogen to give the corresponding (2,5-dioxo-3, aspects and variations are within their scope. 4-di(R-sulfanyl)pyrrolyl)-terminated linkers; and/or with other thiols (such as the benzenethiol and 2-hydroxy Example 1 ethanethiol of Schumacher et al.) to give the corresponding linkers; and/or with other pyrrolediones or pyrrolidinediones, such as 3,4-dichloropyrrole-2,5-dione or 3,4-dibromopyrro Synthesis of lidine-2,5-dione, or based on them, to give the corresponding 3,4-bis(2-pyridylsulfanyl)pyrrole-2,5-dione 3,4-di(R-sulfanyl) pyrrole-2,5-diones or 3,4-di(R-sulfanyl) 0252) pyrrolidine-2,5-diones or linkers based on them. 0255 General procedures for the synthesis of the linkers (L. L. L and L) may be performed using standard synthetic O procedures as described in Larock, above, or Modern Syn Br N thetic Reactions, Second Edition, H. O. House, The Ben BH + Hs jamin/Cummings Publishing Company, Menlo Park, Calif., 2 1972; the chemistry of amino acids and peptide synthesis Br N SH described in The Chemistry of the Amino Acids, J. P. Green O stein, M. Winitz, Robert E. Krieger Publishing Company, Malabar, Fla., 1986, Volumes 1, 2 and 3. US 2014/0363454 A1 Dec. 11, 2014 49

Example 2 dioxo-3,4-bis(2-pyridylsulfanyl)pyrrolyl)-3,6,9,12,15, 18, 21,24,27.30,33,36-dodecaoxanonatriacontanoic acid, or Synthesis of 39-(3,4-dibromo-2,5-dioxopyrrolyl)-3, starting with other 3,4-di(R-sulfanyl)pyrrole-2,5-diones to 6,9,12,15, 18.21,24,27.30,33,36-dodecaoxanonatria give the corresponding linkers; and/or starting with other contanoic acid hydroxyl-terminated sidechains, e.g. using tert-butyl 6-hy droxyhexanoate to give 6-(3,4-dibromo-2,5-dioxopyrrolyl) 0256 hexanoic acid, etc. Similar syntheses starting with maleimide rather than 2,3-dibromomaleimide give comparator linkers of O the prior art, such as 6-(2,5-dioxopyrrolyl)hexanoic acid, the Br MC linker. NH + HO(CH2CH2O)2CH2CH2C(O)OtBu Example 3 Br O Synthesis of 39-(3,4-dibromo-2,5-dioxopyrrolidi nyl)-3,6,9,12,15, 18.21,24,27.30,33,36-dodecaox phyDIAD anonatriacontanoic acid the dBrPEG linker O Br 0259 N-(CH2CH2O)2CH2CH3C(O)OtBu O Br O FADCM + HO(CH2CH2O)2CH2CH2C(O)OtBu O O Br N-(CH2CH2O)2CHCHC(O)OH. phyDIAD O Br O - (CH2CH2O)2CH2CH2C(O)OtBu 0257. A 100 mL two-necked round bottom flask was flame dried and cooled under nitrogen. The cooled flask was O charged with 200 mg (0.296 mmol) of tert-butyl 39-hydroxy 3,6,9,12,15, 18.21,24,27.30,33,36-dodecaoxanonatriacon taxy tanoate. Triphenylphosphine, 106 mg, was dissolved in about O 5 mL anhydrous tetrahydrofuran in a vial, and the solution was added to the 100 mL flask via cannula under nitrogen. The 100 mL flask was cooled in an ice-water bath for 15 minutes. To the cooled solution was added 55 mg (0.217 - (CH2CH2O)2CH2CH2C(O)OH mmol) 3,4-dibromopyrrole-2,5-dione with stirring until a clear solution was observed. DIAD, 58.3 ul, was added to the O cooled reaction mixture, which was stirred in the ice bath for an additional 10 minutes. The reaction mixture was stirred pictic and allowed to reach room temperature over about 20 hours, O then concentrated on a rotary evaporator until dry, giving a Br yellow viscous oil, which was absorbed onto about 1 g silica gel and dry-loaded onto a Reveleris normal phase chroma tography unit. The oil was eluted over a 12 g silica gel car Br tridge with a methanol:dichloromethane gradient from 1:0 to D 9:1 over 28 column volumes. The fractions containing the desired product were pooled and concentrated to dryness. The purified product was suspended in 50:50 acetonitrile: water 0260 39-(2,5-dioxopyrrolyl)-3,6,9,12,15, 18.21,24,27, and lyophilized overnight to provide a clear light yellow 30.33.36-dodecaoxanonatriacontanoic acid was prepared in viscous oil. By LC-MS analysis, the tert-butyl-protected car the same manner as the 39-(3,4-dibromo-2,5-dioxopyrrolyl)- boxylic acid product had been partially deprotected during 3,6,9,12,15, 18.21,24,27.30,33,36-dodecaoxanonatriacon the work-up. To fully deprotect the material to the free acid, tanoic acid of Example 2, but starting with maleimide rather the lyophilized material was treated with 5% trifluoroacetic than 2,3-dibromomaleimide. The acid was treated with 0.5 acid in dichloromethane, concentrated to dryness and lyo equivalents of bromine in chloroform followed by refluxing philized in acetonitrile:water (50:50) overnight. overnight to give 39-(3,4-dibromo-2,5-dioxopyrrolidinyl)-3, 0258 Similar syntheses may be performed starting with 6,9,12,15, 18.21,24,27.30,33,36-dodecaoxanonatriacon 3,4-bis(2-pyridylsulfanyl)pyrrole-2,5-dione to give 39-(2,5- tanoic acid after flash purification on silica gel. US 2014/0363454 A1 Dec. 11, 2014 50

0261 Similar syntheses may be performed using other hydroxyl-terminated sidechains, e.g. using tert-butyl 6-hy droxyhexanoate to give 6-(3,4-dibromo-2,5-dioxopyrrolidi nyl)hexanoic acid, etc. The dibrominated linkers that are products of this synthesis may be dehydrobrominated with base in an additional step to give (3-bromo-2,5-dioxopyrro lyl)-terminated linkers, such as 6-(3-bromo-2,5-dioxopyrro lyl)hexanoic acid. Synthesis of Linker-Cytotoxin Conjugates: Synthesis of T2 0262 Different methods for the preparation of T2 are shown in the Schemes.

Scheme 1: Synthesis of T2

Boc NN N s'X N. HCI H dioxane e O O O

240

OH N N N W US 2014/0363454 A1 Dec. 11, 2014 51

-continued o, ? O

O N N N w U

LiOH O O O MeOHIHO 25% over 3 steps

OH

O Ac2O - - N pyridine 91%

O OH

O Nl N Pentafluorophenol N N Nas New Ns -e-PS-DCC, DIEA H -Ns O 244

HCI

HN

PFP

O N OH

N N He )S O DIEA N 47% over 2 steps

245 US 2014/0363454 A1 Dec. 11, 2014 52

-continued

O N NH2. DC.s w N s s yO r US 2014/0363454 A1 Dec. 11, 2014 53

H

CD

N

. . . 14 OIOH. Ye

HO

US 2014/0363454 A1 Dec. 11, 2014 54

NIH

CD

O ponunuoo O IOH.

~~~~

US 2014/0363454 A1 Dec. 11, 2014 55

Scheme 3: Alternative Synthesis of T-2:

4N HC in dioxane -e- ethanol

COEt 246

OH

N O HN e IX ( TTDCC, HOAt, DIPEA, CHCI HN 251 HCI

COEt 247

MeCN, 1N NaOH aC.

O OH H O N N O r N e 4N hydrogen chloride

O s' S / -e-in 1,4-dioxane w HN

249 US 2014/0363454 A1 Dec. 11, 2014 56

-continued HCI O OH

INulls N e N O

w IX (HN

CO2H 250 INT-2

Example 4 The solution was brought to a pH-2 with 1 Naqueous hydro gen chloride, then frozen and lyophilized. The residue was 0263 Ethyl (2S,4R)-4-(2-((1R,3R)-1-acetoxy-3-((tert- flash chromatographed on silica gel (12 g) with methylene butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-4- chloride:ethyl acetate as the eluent 100:0 to 0:100 over 20 carboxamido)-2-methyl-5-phenylpentanoate (246, 323 mg, minutes to afford 30 mg (18% yield) ethyl (2S,4R)-4-(2-((1R, 523 umol) in 4 NHCl in 1,4-dioxane (6.0 ml) was stirred for 3R)-3-((2S,3S)-2-((tert-butoxycarbonyl)amino)-N,3-dim 30 min Ethanol (1.0 ml) was added and stirring for was ethylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4- continued for an additional 24 h. The solution was blown dry carboxamido)-2-methyl-5-phenylpentanoate (251), 96 mg with a stream of air then diluted with 1:1 acetonitrile: water, (61% yield) of (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2-((tert frozen and lyophilized to afford ethyl (2S,4R)-4-(2-((1R,3R)- butoxycarbonyl)amino)-N,3-dimethylpentanamido)-1-hy 1-hydroxy-4-methyl-3-(methylamino)pentyl)thiazole-4-car droxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl boxamido)-2-methyl-5-phenylpentanoate hydrochloride 5-phenylpentanoic acid (249), and 45 mg (21% recovery) of (247) as a light yellow solid. ethyl (2S,4R)-4-(2-((6S.9R,11R,14S)-6, 14-di(S)-sec-bu tyl)-9-isopropyl-2.2.8, 18, 18-pentamethyl-4,7,13,16-tet 0264. Ethyl (2S,4R)-4-(2-((1R,3R)-1-hydroxy-4-methyl raoxo-3,12,17-trioxa-5,8,15-triazanonadecan-11-yl)thiaz 3-(methylamino)pentyl)thiazole-4-carboxamido)-2-methyl ole-4-carboxamido)-2-methyl-5-phenylpentanoate (248) as 5-phenylpentanoate hydrochloride (247, material from GDP white solids after lyophilization. 131-66, ca. 523 umol), dicyclohexylmethanediimine (2265 mg, 11.0 mmol), (tert-butoxycarbonyl)-L-isoleucine (251. 2654 mg, 11.5 mmol), 3H-1.2.3 triazolo 4,5-b]pyridin-3-ol 0266 (R)-1-methylpiperidine-2-carboxylic acid (92 mg, (44 mg. 323 umol), and diisopropylethylamine (0.20 ml, 1.15 643 umol), 2,3,4,5,6-pentafluorophenol (122 mg, 662 umol), mmol) in methylene chloride (20 ml) was stirred for 18 h. The and dicyclohexylmethanediimine (198 mg, 960 umol) in heterogeneous mixture was filtered and the filtrate was con ethyl acetate (1.0 ml) was stirred for 24 h. The heterogeneous centrated under reduced pressure. The residue was dissolved mixture was filtered and the solid was washed with ethyl into methylene chloride and the solid was removed by filtra acetate. The perfluorophenyl (R)-1-methylpiperidine-2-car tion two additional times. The residue was flash chromato boxylate contained in the filtrate was used crude in the sub graphed on silica (80 g) with methylene chloride:ethyl acetate sequent reaction. 100:0 to 50:50 as the eluent over 10 minto afford 214 mg (45% yield over two steps) of ethyl (2S,4R)-4-(2-((6S.9R, 0267 (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2-((tert-butoxy 11R,14S)-6,14-di(S)-sec-butyl)-9-isopropyl-2.2.8, 18, 18 carbonyl)amino)-N.3-dimethylpentanamido)-1-hydroxy-4- pentamethyl-4,7,13,16-tetraoxo-3,12,17-trioxa-5,8,15-tria Zanonadecan-11-yl)thiazole-4-carboxamido)-2-methyl-5- methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenyl phenylpentanoate (248) as a white solid. pentanoic acid (249, 96 mg, 145 umol) in 4 N hydrogen chloride in 1,4-dioxane (2 ml) was stirring for 1 h. The solu 0265 Ethyl (2S,4R)-4-(2-((6S,9R,11R,14S)-6, 14-di(S)- tion was concentrated under a stream of air then diluted with sec-butyl)-9-isopropyl-2.2.8, 18, 18-pentamethyl-4,7,13,16 1:1 acetonitrile:water and lyophilized to yield 87 mg (100% tetraoxo-3,12,17-trioxa-5,8,15-triazanonadecan-11-yl)thiaz yield) of (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2-amino-N.3- ole-4-carboxamido)-2-methyl-5-phenylpentanoate (248,214 dimethylpentanamido)-1-hydroxy-4-methylpentyl)thiazole mg, 237 umol) and 1 Naqueous sodium hydroxide (0.35 ml, 4-carboxamido)-2-methyl-5-phenylpentanoic acid hydro 350 umol) in 1:1 acetonitrile:water (2 ml) was stirred for 4 h. chloride (250, INT-2) as a white solid. US 2014/0363454 A1 Dec. 11, 2014 57

Scheme 4: Alternative Synthesis of T2 OH

F F

OH | 1.DCC EtOAtuae N r F F N | || F

HCI O OH

N O e DIPEA, CHCl2, EtOAc IX {HN T - Y -

CO2H 250 INT-2 uu, C

CO2H 252

Cl

CO2H T2

Example 5 steps) of (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)- N.3-dimethyl 2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-1- 0268 Perfluorophenyl (R)-1-methylpiperidine-2-car hydroxy-4-methylpentyl)thiazole-4-carboxamido)-2-me boxylate (crude material from GDP-131-071, ca. 643 umol) thyl-5-phenylpentanoic acid (252) as a white solid. 30 mg of in ethyl acetate (2.0 ml), (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)- 2-amino-N,3-dimethylpentanamido)-1-hydroxy-4-methyl impure product was also recovered. pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpen 0269. Acetic anhydride (2.0 ml) was added to a solution of tanoic acid hydrochloride (250, material from GDP-131-070, (2S,4R)-4-(2-((1R,3R)-3-((2S,3S) N.3-dimethyl-2-((R)-1- ca. 145 umol), and diisopropylethylamine (0.05 ml, 287 methylpiperidine-2-carboxamido)pentanamido)-1-hydroxy umol) in methylene chloride (2.0 ml) was stirred for 24h. The 4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phe Solution was concentrated under a stream of air and the resi nylpentanoic acid (252, material from GDP-01-079) in due was flash chromatographed on silica (12 g) with methyl pyridine (2.0 ml). After stirring for 16 h, the solution was ene chloride:methanol as the eluent with a 100:0 to 80:20 concentrated under reduced pressure and the residue was gradient over 20 minto furnish 36 mg (36% yield over two purified by HPLC to yield 1.1 mg of (2S,4R)-4-(2-((1R,3R)- US 2014/0363454 A1 Dec. 11, 2014 58

1-acetoxy-34(2S,3S)- N,3-dimethyl-2-((R)-1-methylpip- -continued eridine-2-carboxamido)pentanamido)-4-methylpentyl)thia- 289 Zole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (T2). Analogs of T2 Prepared: 0270

287

COEt

290

OH H O 288 N1''' 1 Null N O

HO --~~ NH

CO2H

Scheme 6: Preparation of T4

4NHC dioxane Her EtOH 100%

COEt 246 US 2014/0363454 A1 Dec. 11, 2014 59

-continued

OH

N N 2N. O

/ HerDIPEA, DMF, 90° C. HN NO O

Boc1 O COEt s 247 -> 284

O OH

Boc1 N 2N. O E S 7 4NHC dioxane s' Hs w HN EtOH 100%

COEt 251

O OH

HN sus N e N O / DIPEA, CH2Cl2, EtOAc S w HN F

"... --O F

CO2Et Bocr O 255 F F F

OH

N sr N 2N. O

S X-(HN 1NaqEtOH NaOH.

COEt 256 US 2014/0363454 A1 Dec. 11, 2014 60

-continued O OH

N '', r Null N 2N. O Boc O is i/ Ac2O,2U, pypyridine w HN

CO2H 253

O OH H

“1 e N O Boc 5 is / 4NHC dioxane w HN EtOH 100%

CO2H 254

Overal Yield - 30%

Example 6 0271 (R)-1-(Tert-butoxycarbonyl)piperidine-2-carboxy lic acid (48 mg, 209 umol), 2,3,4,5,6-pentafluorophenol (38 mg, 206 umol), and dicyclohexylmethanediimine (60 mg. 291 umol) in ethyl acetate (1 ml) was stirred for 48 h. The heterogeneous mixture was filtered and the Solid was washed with ethyl acetate. This material was used crude in the sub sequent reaction.

Scheme 7: Synthesis of T4 HCI

O OH H '', Null N O N e MeOH, S / 1N NaOH ad. HN

COEt 256 US 2014/0363454 A1 Dec. 11, 2014 61

-continued

O OH H '', N N O N N e Ac2O, / pyridine Boc O ''' S HN -e-

CO2H

4N hydrogen chloride in 1,4-dioxane -e- Boc

CO2H 254

H N “... -N e O

O HCI HN

O HO T-4 HCI Scheme 8: Alternative Synthesis of T4 HCI

F

O F HCI O OH INull N 2N O >JC / F HN DIPEA CHCl2, EtOAc

CO2H

250 INT-2 US 2014/0363454 A1 Dec. 11, 2014 62

-continued OH

pyridine -e-

CO2H

4N HCI in 1,4-dioxane Her

254

HO

Example 7 min. The combined fractions were concentrated under reduced pressure and the residue was dissolved into 1:1 aceto 0272 (R)-1-(Tert-butoxycarbonyl)piperidine-2-carboxy nitrile: water and lyophilized to afford 30 mg (86% yield over lic acid (48 mg, 209 umol), 2,3,4,5,6-pentafluorophenol (38 two steps) of tert-butyl (R)-2-(((2S,3S)-1-(((1R,3R)-1-(4- mg, 206 umol), and dicyclohexylmethanediimine (60 mg. (((2R,4S)-5-ethoxy-4-methyl-5-oxo-1-phenylpentan-2-yl) 291 umol) in ethyl acetate (1 ml) was stirred for 48 h. The carbamoyl)thiazol-2-yl)-1-hydroxy-4-methylpentan-3-yl) heterogeneous mixture was filtered and the Solid was washed (methyl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl) with ethyl acetate. This material was used crude in the sub piperidine-1-carboxylate, 256, as an off-white solid. sequent reaction. 0274 Acetic anhydride (0.10 ml, 106 umol) tert-butyl (R)- 0273 1-(Tert-butyl) 2-(perfluorophenyl) (R)-piperidine 2-(((2S,3S)-1-(((1R,3R)-1-(4-(((2R,4S)-5-ethoxy-4-methyl 1,2-dicarboxylate in ethyl acetate (2 ml) from GDP-131-077 5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-1-hy was added to a solution of ethyl (2S,4R)-4-(2-((1R,3R)-3- droxy-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1- ((2S,3S)-2-amino-N,3-dimethylpentanamido)-1-hydroxy-4- oXopentan-2-yl)carbamoyl)piperidine-1-carboxylate (256. methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenyl 30 mg, 37.5umol)in pyridine (0.50 ml). After stirring for 6 h, pentanoate hydrochloride (255, 43.5 umol from GDP-131 the Solution was concentrated under a stream of air and the 078) and diisopropylethylamine (0.05 ml, 287 umol) in residue was purified by flash chromatography (12 g silica) methylene chloride (2 ml). After stirring for 18 h, the solution with methylene chloride:ethyl acetate as the eluent 100:0 to was concentrated under reduced pressure and the residue was 50:50 over 25 min. The combined fractions were concen purified by flash chromatography (12g silica) with methylene trated under reduced pressure and the residue was dissolved chloride:ethyl acetate as the eluent 100:0 to 50:50 over 25 into 1:1 acetonitrile: water and lyophilized to afford 30 mg

US 2014/0363454 A1 Dec. 11, 2014 66

OH

OH

OH

US 2014/0363454 A1 Dec. 11, 2014 69

-continued O OH HCI

ful N e N O

w HN

COEt

255 INT-1

Example 12 tanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylic acid, 267, as a white solid that was used crude in the subse 0282 Ethyl 2-((1R,3R)-1-hydroxy-4-methyl-3-(methy- quent Step. lamino)pentyl)thiazole-4-carboxylate hydrochloride (265, Example 14 638 mg, 1.98 mmol), (tert-butoxycarbonyl)-L-isoleucine 0284. 2-((1R,3R)-3-((2S,3S)-2-((tert-butoxycarbon (264, 4.8 g. 20.8 mmol), 3H-1.2.3 triazolo 4,5-b]pyridin-3- yearmino)-N.3-dimethylpentanamido)-1-hydroxy-4-methyl ol (0.8 g. 5.88 mmol), dicyclohexylmethanediimine (5.1 g, pentyl)thiazole-4-carboxylic acid (267, 134 mg, 284 umol), 24.7 mmol), diisopropylethylamine (0.5 ml, 2.87 mmol) in ethyl (2S,4R)-4-amino-2-methyl-5-phenylpentanoate hydro methylene chloride (100 ml) was stirred for 18 h. The hetero chloride (268, 84 mg. 309 umol), 3-(((ethylimino)methylene) geneous mixture was filtered and the filtrate was concentrated amino)-N,N-dimethylpropan-1-amine hydrochloride (104 under reduced pressure. Methylene chloride was added to the mg, 543 umol), 3H-1.2.3 triazolo 4,5-b]pyridin-3-ol (22 residue and the solid was removed by filtration. The filtrate mg, 162 umol), and diisopropylethylamine (0.10 ml, 574 was flash chromatographed on silica gel (80 g) with methyl umol) in methylene chloride (2 ml) was stirred for 18 h. The ene chloride:ethylacetate as the eluent 100:0 to 50:50 over 25 solution was directly flash chromatographed on silica gel (40 minto afford 1686 mg (120% yield likely impure with dicy g) with methylene chloride:ethyl acetate as the eluent 100:0 to clohexylurea) of ethyl 2-((6S,9R,11R,14S)-6, 14-di(S)-sec 50:50 over 25 minutes to afford 173 mg (88% yield) of (2R, butyl)-9-isopropyl-2.2.8, 18, 18-pentamethyl-4,7,13,16-tet 4S)-5-ethoxy-4-methyl-5-oxo-1-phenylpentan-2-yl 2-((1R, raoxo-3,12,17-trioxa-5,8,15-triazanonadecan-11-yl) 3R)-3-((2S,3S)-2-((tert-butoxycarbonyl)amino)-N,3-dim thiazole-4-carboxylate, 266, as a viscous yellow oil. ethylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4- carboxylate, 251, as a white solid after lyophilization. Example 13 Example 15 0285 4 N Hydrogen chloride in 1,4-dioxane (2 ml) was (0283 Ethyl 2-((6S,9R,11R,14S)-6,14-di(S)-sec-butyl)- added to (2R,4S)-5-ethoxy-4-methyl-5-oxo-1-phenylpentan 9-isopropyl-2.2.8, 18, 18-pentamethyl-4,7,13,16-tetraoxo-3, 2-yl 2-((1R,3R)-3-((2S,3S)-2-((tert-butoxycarbonyeamino)- 12, 17-trioxa-5,8,15-triazanonadecan-11-yl)thiazole-4-car N.3-dimethylpentanamido)-1-hydroxy-4-methylpentyl)thia boxylate (266, 96 mg, 135 umol) and 1 Naqueous sodium Zole-4-carboxylate (251,42 mg, 60.9 umol). After stirring for hydroxide (0.50 ml, 500 umol) in 1:1:1 methanol:acetonitrile: 2 h, the Solution was evaporated under a stream of air, diluted water (3 ml) was stirred for 18 h. The solution was brought to with 1:1 acetonitrile:water, and lyophilized to afford 38 mg an acidic pH with 1 Naqueous hydrogen chloride, frozen, and (100% yield) of ethyl (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2- lyophilized. The residue was diluted with methylene chloride amino-N.3-dimethylpentanamido)-1-hydroxy-4-methylpen and filtered. The solid was collected to afford 2-((1R,3R)-3- tyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate ((2S,3S)-2-((tert-butoxycarbonyl)amino)-N,3-dimethylpen hydrochloride, 255 as a white solid.

O

C us o1 N O H ethyl acetate, O C. 1N1N1N1 OH Dess-Martin N -- 1N1N1N1 OH periodinane, NaHCO3, HN N CHCl2 O NaHCO3, H2O, O C. 269 US 2014/0363454 A1 Dec. 11, 2014 70

-continued O NaBH(OAc)3. 1,2-dichloroethane N 1N1-1-4' -- N r" -es N H O O 270 271

HATU, DIPEA, DMF OH

O e

272 w 5 S X-(HN

COEt 255 INT-1

O NNull O O 1 N / OJN N Of 2N. O ls ls O N--- w 5 IX {HN O N

COEt

273

O O / N---N1 ". O

COEt 274

Example 16 over 20 min to afford 0.5 g (25% yield) of 1-(6-hydroxy hexyl)-1H-pyrrole-2,5-dione, 269, as a clear oil. 0286 Methyl chloroformate (1.0 ml, 13.0 mmol) was slowly added dropwise to a solution of H-pyrrole-2,5-dione Example 17 (1.0 g, 10.3 mmol) and N-methylmorpholine (1.5 ml, 13.6 mmol) in ethyl acetate (10 ml) at 0°C. After stirring for 30 0287 1-(6-Hydroxyhexyl)-1H-pyrrole-2,5-dione (269, min, 6-aminohexan-1-ol (1.4g, 11.9 mmol) was added fol 0.5 g, 2.54 mmol). DessMartin periodinane (2.2 g, 5.19 lowed by the addition of saturated aqueous sodium bicarbon mmol), and sodium bicarbonate (3.8 g., 45.2 mmol) in meth ate (2 ml). After stirring for an additional 30 minutes, the ylene chloride (20 ml) was stirred for 2 h. The heterogeneous solution was extracted with ethyl acetate. The combined mixture was filtered and the filtrate was directly flash chro organic extracts were dried over anhydrous Sodium Sulfate, matographed on silica gel (12 g) with methylene chloride: filtered, and concentrated under reduced pressure. The resi ethylacetate as the eluent 100:0 to 80:20 over 10 minto afford due was flash chromatographed on silica gel (40 g) with 0.3 g (61% yield) of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- methylene chloride:ethylacetate as the eluent 100:0 to 0:100 yl)hexanal, 270, as a clear oil. US 2014/0363454 A1 Dec. 11, 2014

Example 18 min, the solution was blown dry with a stream of air. This product, ethyl (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2-((R)-1- 0288 6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanal (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperidine (270, 0.3 g 1.54 mmol) and (R)-piperidine-2-carboxylic acid 2-carboxamido)-N,3-dimethylpentanamido)-1-hydroxy-4- (271, 0.5 g., 3.87 mmol) in 1,2-dichloroethane (10 ml) was stirred for 20 min. Sodium triacetoxyborohydride (1.6 g., 7.55 methylpentyl)thiazole-4-carboxamido)-2-methyl-5- mmol) was added. After stirring for 1 h, the heterogeneous phenylpentanoate, 273, was used crude in the Subsequent mixture was filtered and the filtrate was directly flash chro step. matographed on silica gel (12 g) with methylene chloride: 0290 Acetic anhydride (0.20 ml, 2.12 mmol) was added to methanol as the eluent 100:0 to 80:20 over 10 minto afford a solution of ethyl (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2-((R)- 0.1 g (21% yield) of (R)-1-(6-((tert-butoxycarbonyeamino) 1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperi hexyl)piperidine-2-carboxylic acid, 272, as a white solid after dine-2-carboxamido)-N,3-dimethylpentanamido)-1-hy lyophilization. droxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl 0289 Diisopropylethylamine (0.05 ml, 287 umol) was 5-phenylpentanoate (273, material from GDP-150-039, ca. added to a heterogeneous mixture of (R)-1-(6-(2,5-dioxo-2, 27.2 umol) in pyridine (1 ml). After stirring for 2 h, ice was 5-dihydro-1H-pyrrol-1-yl)hexyl)piperidine-2-carboxylic added to the solution. Pyridine added to the maleimide so this acid (272, 12 mg, 38.9 umol) and HATU (24 mg. 63.1 mol) should be precooled prior to quenching. The Solution was in dimethylformamide (0.20 ml). The solution immediately directly purified by reverse phase HPLC to afford 3.1 mg became homogeneous. After standing for 15 min, the Solution (12% yield) of ethyl (2S,4R)-4-(2-((1R,3R)-1-acetoxy-3- was added to ethyl (2S,4R)-4-(2-((1R,3R)-3-((2S,3S)-2- ((2S,3S)-2-((R)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- amino-N.3-dimethylpentanamido)-1-hydroxy-4-methylpen yl)hexyl)piperidine-2-carboxamido)-N,3-dimethylpentana tyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate mido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl hydrochloride (255, 17 mg, 27.2 umol). After standing for 30 5-phenylpentanoate, 274, as a white solid. > - - -k CHCl2 HN Dess-Martin periodinane, NaHCO3, CHCl2 > 1, OH > 1, 275 276

NaBH(OAc)3 1,2-dichloroethane ON1 ''', OH -e-

N O

277

HATU, DIPEA, DMF

278

COEt

255 INT-1 US 2014/0363454 A1 Dec. 11, 2014 72

-continued

O.N r NullJ. N C. 2N. O ul O ls 1.2

279

O 1s f Nulls N O 4N hydrogen chloride N r X- in 1,4-dioxane N-N-- Er $ / H s HN

28O

COEt

O H H N N HOAt, N N DIPEA,

N Pi H --DMF O O 1N O O rO

282

US 2014/0363454 A1 Dec. 11, 2014

0298 Fmoc-T4 was prepared by coupling Fmoc-D-2-pi 30 minutes to give T4, which was purified by preparative peridinecarboxylic acid to isoleucine in the presence of EDC HPLC on a C18 reverse phase column eluted with acetoni and Sodium bicarbonate, then coupling the resulting Fmoc trile/water. D-Pip-Ile-OH to the N-methylvaline intermediate 1 (pur chased from Concortis) by mixing with 1 equivalent of Example 21 HOBT and DIPC in DMF followed by addition of 2.5 equiva Synthesis of 6-(2,5-dioxopyrrolyl)hexanoyl-T4 MC lents of NMM. The reaction mixture was stirred overnight and purified by flash chromatography on silica gel using a T4 and 39-(3,4-dibromo-2,5-dioxopyrrolidinyl)-3,6, gradient of hexane and ethyl acetate. Evaporation of Solvent 9,12,15, 18.21,24,27.30,33,36-dodecaoxanonatria gave Fmoc-T4 as a yellow oil. The Fmoc-T4 was then depro contanoyl-T4 dBrPEG-T4 tected by treatment with 20% DEA in methylene chloride for 0299 --~~'sOH TBTU, DIPEA, DMF

T4 -- OR

O OH

N 1-e'--N O ~sO Br

O Br TBTU, DIPEA, DMF

O H O N. N w O ---. O

OH MC-T4 O

O H O NY N, N O ~s O . Br ^{ N1)- O w O Br OH

BrPEG-T4 US 2014/0363454 A1 Dec. 11, 2014 76

0300 Coupling of T4 to the MC or dBrPEG linkers 0303 39-(2,5-Dioxo-3,4-bis(pyridin-2-ylthio)-2,5-dihy described in Example 2 and 3 respectively was performed by dro-1H-pyrrol-1-yl)-3,6,9,12,15, 18.21,24,27.30,33,36 activating the linkers with 1 equivalent of TBTU in the pres dodecaoxanonatriacontanoic acid was added to a clean, ence of 2 equivalents of DIPEA in DMF, then coupling with flame-dried 50 mL round bottom flask, and the carboxylic the T4 for 72 hours at room temperature. Purification by acid was activated with NHS in 3 mL of DMF in the presence preparative C18 HPLC (acetonitrile-water gradient) gave of DCC. MMAF was predissolved in about 1 mL DMF and MC-T4 or dBrPEG-T4 suitable for conjugation to antibodies. transferred to the NHS-activated acid via 22 gauge needle. 0301 Similar syntheses using other linkers give the cor DIPEA was added to the reaction mixture and stirred over responding linker-T4 conjugates. Similar syntheses using T3, MMAF, or other cytotoxins with a basic amine give the cor night. The crude reaction mixture was purified by reverse responding linker-cytotoxin conjugates. Similar syntheses phase HPLC on a 21.2 mmx50 mm Agilent PREP-C18 col using amine-terminated linkers and cytotoxins with a car umn at a flow rate of 35 mL/min over 20 column volumes boxyl group, activating the cytotoxin in the same manner as (about 30 minutes of gradient time). Enriched fractions were the linker was activated in the above Example, give other identified, pooled and lyophilized to give the dPSPEG linker-cytotoxin conjugates. MMAF conjugate as a white semi-solid. 0304 Similar syntheses using other linkers give the cor Example 22 responding linker-MMAF conjugates. Similar syntheses Synthesis of 39-(2,5-dioxo-3,4-bis(2-pyridylsulfa using T3, T4 or other cytotoxins such as CTX-I", CTX-II", CTX-III', CTX-IV, CTX-V, CTX-VI", CTX-VII' and CTX nyl)pyrrolyl)-3,6,9,12,15, 18.21,24,27.30,33,36-dode VIII' with a basic amine give the corresponding linker-cyto caoxanonatriacontanoyl-MMAF dPSPEG-MMAF toxin conjugates, such as dPSPEG-T4. Similar syntheses 0302) using amine-terminated linkers and cytotoxins with a car

rn N-2 O O OH S O o1 O o1 o 's-1

N-(CH2CH2O)2CH2CH2CH3C(O)OH + Hull N N N-ulus N S O 1N O w Qu N2 S s DCC DIPEA

s N 21 O S O N CO O N N US 2014/0363454 A1 Dec. 11, 2014 77 boxyl group, activating the cytotoxin in the same manner as the linker was activated in the above Example, give other linker-cytotoxin conjugates. Synthesis of Antibody-Drug Conjugates Example 23 Synthesis of trastuzumab-dTSPEG-MMAF ADC 0305

n TCEP n o -e- c1N SH c1a S c1a SH

trastuzumab trastuzumab (only one (reduced) disulfide bond shown) rn Na2 O O OH S O o1 o o1 o Y

N-(CH2CH2O) CHCH co-sul N N N ulus N S E O 1N O ''' N21 N dPSPEG-MMAF | n O O OH

N-(CH2CH2O)2CH2CHC(O)-(CHCHO CHCH.CO-Null N N N- N cys1Ns E. H H O 1N O w . 4

trastuzumab-dTSPEG-MMAF

0306 Trastuzumab, 1 mL of a 20 mg/mL solution in pH 0307 Similar syntheses using other linker-cytotoxin con 7.4 PBS (Gibco Mg and Cafree) with 1 mM DTPA, is loaded jugates, such as dPSPEG-T4, and/or other antibodies, such as into a sterile 1.7 mL Eppendorf tube, then 2.75 equivalents of 18-2A (a murine IgG2a antibody), give the corresponding TCEP hydrochloride (Sigma ampule 0.5M concentration), is ADCs. 0308 As shown in the representative Figures, the ADCs added and the mixture incubated at 37°C. for 1 hour to give prepared from the method of the present application provides an average of 4 free thiol pairs per trastuzumab (this can be the products with significant homogeneity as shown by HIC verified by Ellman’s colorimetric assay see Ellman, “Tissue traces, when compared with the ADCs prepared by conven sulfhydryl groups', Arch. Biochem. Biophys, 1959, 82,70-77 tional methods that provide inhomogeneous ADCs with mul or later papers referring to this assay). The reduced antibody tiple products and positional isomers. solution is cooled in an ice-bathat about 0°C. for 15 minutes; then a solution of about 4 equivalents of dPSPEG-MMAF in Assays dimethylsulfoxide is added and the mixture incubated at 37° 0309 ADCs of this invention are tested for potency and C. for 2 hours (or at 4°C. for 20 hours). The resulting trastu selectivity in vitro by determining their cytotoxicity in cancer Zumab-dTSPEG-MMAF ADC is purified by size-exclusion cell lines of interest, such as those cancer cell lines expressing chromatography (GE AKTA pure chromatographic system) the antigen corresponding to the antibody portion of the ADC or PD10 desalting column. and similar cancer cell lines lacking the antigen. They are US 2014/0363454 A1 Dec. 11, 2014

tested for potency and safety in vivo in Such animal models as strated comparable affinity for F244 cells, indicating that the mouse Subcutaneous cancer Xenograft and mouse ortho conjugation of the drug payloads do not affect antigen bind topic cancer Xenograft models well known to those of skill in 1ng. the art of cancer research. 0313. The ADCs disclosed in Table 1 are found to provide comparable affinity for F244 cells, also Suggesting that con Example 24 jugation of the drug payloads with the antibody do not affect antigen binding. Cytotoxicity of Trastuzumab ADCs Compared to Trastuzumab Example 27 0310. The cytotoxicity of two ADCs where trastuzumab Potency of ADCs Against Antigen-Expressing Cells was conjugated to the currently used cytotoxin MMAF 0314. The potency of ADCs for inhibition of tumor cell through an MC linker trastuzumab-MC-MMAF was com growth was tested in cell proliferation assays. The Ramos pared to the cytotoxicity of trastuzumab alone in HER2 (B-cell lymphoma) and BT474 (HER2+ human breast carci positive and HER2-negative tumor cells. In the HER2-nega noma) cell lines were seeded into 96 well half-area plates the tive tumor cells, the ICs for both ADCs and for trastuzumab day before drug treatment at 3000 and 5000 cells per well itself was >500 nM; however, in the HER2-positive tumor respectively. ADCs and controls were serially diluted in a cells, while the ICs for trastuzumab itself was still