NOVEL LINKERS FOR ANTIBODY-DRUG CONJUGATES AND RELATED COMPOUNDS, COMPOSITIONS, AND METHODS OF USE

Provided herein are antibody-cytotoxin antibody-drug conjugates and related compounds, such as linker-cytotoxin conjugates and the linkers used to make them, and intermediates in their synthesis; compositions; and methods, including methods of treating cancers

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Description
CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No. 14/895,893, filed on Dec. 3, 2015; which is a 35 U.S.C. § 371 national stage filing of International Patent Application No. PCT/US2014/041399, filed on Jun. 6, 2014; which claims the benefit of priority of U.S. Provisional Patent Application No. 61/832,130, filed on Jun. 6, 2013, U.S. Provisional Patent Application No. 61/891,390, filed on Oct. 15, 2013, and U.S. Provisional Patent Application No. 61/981,154, filed on Apr. 17, 2014, the content of each of which is incorporated herein by reference in its entirety. International Patent Application No. PCT/US2014/041399 also claims benefit of priority of International Patent Application No. PCT/US2013/064147, filed on Oct. 9, 2013, the content of which is incorporated herein by reference in its entirety.

FIELD

This invention relates to novel linkers for antibody-drug conjugates (ADCs) and related compounds, such as linkers used to make them, and intermediates in their synthesis; compositions; and methods, including methods of treating cancers.

BACKGROUND

Cancer is the second most prevalent cause of death in the U.S, yet there are few effective treatment options beyond surgical resection. Of the medical treatments for cancers, the use of monoclonal antibodies targeting antigens present on the cancer cells has become common. Anticancer antibodies approved for therapeutic use in the USA include alemtuzumab (CAMPATH®), a humanized anti-CD52 antibody used in the treatment of chronic lymphocytic leukemia; bevacizumab (AVASTIN®), a humanized anti-VEGF antibody used in colorectal cancer; cetuximab (ERBITUX®), a chimeric anti-epidermal growth factor antibody used in colorectal cancer, head and neck cancer, and squamous cell carcinoma; ipilimumab (YERVOY®), a human anti-CTLA-4 antibody used in melanoma; ofatumumab (ARZERRA®), a human anti-CD20 antibody used in chronic lymphocytic leukemia; panitumumab (VECTIBIX®), a human anti-epidermal growth factor receptor antibody used in colorectal cancer; rituximab (RITUXAN®), a chimeric anti-CD20 antibody used in non-Hodgkin lymphoma; tositumomab (BEXXAR®), a murine anti-CD20 antibody used in non-Hodgkin lymphoma; and trastuzumab (HERCEPTIN®), a humanized anti-HER2 antibody used in breast cancer. While these antibodies have proven useful in the treatments of the cancers for which they are indicated, they are rarely curative as single agents, and are generally used in combination with standard chemotherapy for the cancer.

As an example, trastuzumab is a recombinant DNA-derived humanized monoclonal antibody that selectively binds with high affinity to the extracellular domain of the human epidermal growth factor receptor2 protein, HER2 (ErbB2) (Coussens et al., Science 1985, 230, 1132-9; Salmon et al., Science 1989, 244, 707-12), thereby inhibiting the growth of HER2-positive cancerous cells.

Although HERCEPTIN is useful in treating patients with HER2-overexpressing breast cancers that have received extensive prior anti-cancer therapy, some patients in this population fail to respond or respond only poorly to HERCEPTIN treatment. Therefore, there is a significant clinical need for developing further HER2-directed cancer therapies for those patients with HER2-overexpressing tumors or other diseases associated with HER2 expression that do not respond, or respond poorly, to HERCEPTIN treatment.

Antibody drug conjugates (ADCs), a rapidly growing class of targeted therapeutics, represent a promising new approach toward improving both the selectivity and the cytotoxic activity of cancer drugs. See, for example, Trail et al., “Monoclonal antibody drug immunoconjugates for targeted treatment of cancer”, Cancer Immunol. Immunother. 2003, 52, 328-337; and Chari, “Targeted Cancer Therapy: Conferring Specificity to Cytotoxic Drugs”, Acc. Chem. Res., 2008, 41(1), 98-107. These ADCs have three components: (1) a monoclonal antibody conjugated through a (2) linker to a (3) cytotoxin. The cytotoxins are attached to either lysine or cysteine sidechains on the antibody through linkers that react selectively with primary amines on lysine or with sulfhydryl groups on cysteine. The maximum number of linkers/drugs that can be conjugated depends on the number of reactive amino or sulfhydryl groups that are present on the antibody. A typical antibody contains up to 90 lysines as potential conjugation sites; however, the optimal number of cytotoxins per antibody for most ADCs is typically between 2 and 4 due to aggregation of ADCs with higher numbers of cytotoxins. As a result, conventional lysine linked ADCs currently in clinical development are heterogeneous mixtures that contain from 0 to 10 cytotoxins per antibody conjugated to different amino groups on the antibody. Key factors in the success of an ADC include that the monoclonal antibody is cancer antigen specific, non-immunogenic, low toxicity, and internalized by cancer cells; the cytotoxin is highly potent and is suitable for linker attachment; while the linker may be specific for cysteine (S) or lysine (N) binding, is stable in circulation, may be protease cleavable and/or pH sensitive, and is suitable for attachment to the cytotoxin.

Anticancer ADCs approved for therapeutic use in the USA include brentuximab vedotin (ADCETRIS®), a chimeric anti-CD30 antibody conjugated to monomethylauristatin E used in anaplastic large cell lymphoma and Hodgkin lymphoma; and gemtuzumab ozogamicin (MYLOTARG®), a humanized anti-CD33 antibody conjugated to calicheamicin γ used in acute myelogeneous leukemia—though this was withdrawn in 2010 for lack of efficacy.

Although several ADCs have demonstrated recent clinical success, the utility of most ADCs currently in development may be limited by cumbersome synthetic processes resulting in high cost of goods, insufficient anti-tumor activity associated with limited potency of the cytotoxic drug, and questionable safety due to linker instability and ADC heterogeneity. See, for example, Ducry et al., “Antibody-Drug Conjugates: Linking Cytotoxic Payloads to Monoclonal Antibodies”, Bioconjugate Chem. 2010, 21, 5-13; Chari, “Targeted Cancer Therapy: Conferring Specificity to Cytotoxic Drugs”, Acc. Chem. Res. 2008, 41, 98-107; and Senter, “Recent advancements in the use of antibody drug conjugates for cancer therapy”, Biotechnol.: Pharma. Aspects, 2010, 11, 309-322.

As an example, trastuzumab has been conjugated to the maytansinoid drug mertansine to form the ADC trastuzumab emtansine, also called trastuzumab-DM1 or trastuzumab-MC-DM1, abbreviated T-DM1 (LoRusso et al., “Trastuzumab Emtansine: A Unique Antibody-Drug Conjugate in Development for Human Epidermal Growth Factor Receptor 2-Positive Cancer”, Clin. Cancer Res. 2011, 17, 6437-6447; Burris et al., “Trastuzumab emtansine: a novel antibody-drug conjugate for HER2-positive breast cancer”, Expert Opin. Biol. Ther. 2011, 11, 807-819). It is now in Phase III studies in the US for that indication. The mertansine is conjugated to the trastuzumab through a maleimidocaproyl (MC) linker which bonds at the maleimide to the 4-thiovaleric acid terminus of the mertansine side chain and forms an amide bond between the carboxyl group of the linker and a lysine basic amine of the trastuzumab. Trastuzumab has 88 lysines (and 32 cysteines). As a result, trastuzumab emtansine is highly heterogeneous, containing dozens of different molecules containing from 0 to 8 mertansine units per trastuzumab, with an average mertansine/trastuzumab ratio of 3.4.

Antibody cysteines can also be used for conjugation to cytotoxins through linkers that contain maleimides or other thiol specific functional groups. A typical antibody contains 4, or sometimes 5, interchain disulfide bonds (2 between the heavy chains and 2 between heavy and light chains) that covalently bond the heavy and light chains together and contribute to the stability of the antibodies in vivo. These interchain disulfides can be selectively reduced with dithiothreitol, tris(2-carboxyethyl)phosphine, or other mild reducing agents to afford 8 reactive sulfhydryl groups for conjugation. Cysteine linked ADCs are less heterogeneous than lysine linked ADCs because there are fewer potential conjugation sites; however, they also tend to be less stable due to partial loss of the interchain disulfide bonds during conjugation, since current cysteine linkers bond to only one sulfur atom. 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 monomethylauristatin E residues per antibody conjugated through cysteines.

Schumacher et al., “In Situ Maleimide Bridging of Disulfides and a New Approach to Protein PEGylation”, Bioconjugate Chem. 2011, 22, 132-136, disclose the synthesis of 3,4-disubstituted maleimides such as 3,4-bis(2-hydroxyethylsulfanyl)pyrrole-2,5-dione [referred to by Schumacher et al. as “dimercaptoethanolmaleimide” ] and 3,4-bis(phenylsulfanyl)pyrrole-2,5-dione [“dithiophenolmaleimide” ], and their N-PEGylated derivatives as PEGylating agents for somatostatin, where the substituted maleimide bonds to the two sulfur atoms of an opened cysteine-cysteine disulfide bond.

It would be desirable to develop linkers with improved in vivo stability as well as potent, homogeneous ADCs, compositions containing them and methods for their use in treating cancers, and methods and intermediates in their preparation.

SUMMARY

In one aspect, provided herein is a linker of the following formula (I):

or an enantiomer, diasteriomer, or mixtures thereof;
wherein:

  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-3 alkyl;
  • each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-3 alkyl;
  • Z is —CO2H, —NH2, —OH, —NH—R3a, or —CO2R3b; wherein R3a is an amino protecting group, and R3b is a carboxyl protecting group;
  • R is any chemical group; or R is absent;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each k and k′ is independently an integer of 0 or 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • each AA is independently an amino acid;
  • each r is 1 to 12; and
  • the bond represents a single or a double bond.

In certain embodiments of the linker of formula (I), the bond represents a single bond.

In certain embodiments of the linker of formula (I), each Y and Y′ is independently selected from the group consisting of a halo, a substituted thiol, and a substituted sulfonate. In certain embodiments, each Y and Y′ is independently selected from the group consisting of chloro, bromo, fluoro, and iodo. In certain embodiments, each Y and Y′ is independently selected from an optionally substituted thiophenyl, an optionally substituted thionaphthyl, an optionally substitued thiopyridyl, an optionally substituted isoquinolinyl, and an optionally substituted phenylsulfonate.

In certain embodiments of the linker of formula (I), each Y and Y′ is independently selected from the group consisting of:

wherein
RA is selected from the group consisting of hydroxyl, amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C1-6alkoxycarbonyl, C1-6 alkyl, C2-6 alkenyl, and C1-6 alkoxy.

In certain embodiments of the linker of formula (I), each Y and Y′ is independently selected from the group consisting of:

In certain embodiments of the linker of formula (I), each L1, L2 and La is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and -(AA)r-, where a, b and c are each independently 0, 1 or 2; and each p, q and r is independently 1, 2, 3 or 4.

In certain embodiments of the linker of formula (I), each AA is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. In certain embodiments, each AA is an amino acid selected from the group consisting of Gly, Arg, Val, Ala, Cys, Gin, Leu, Ile, Leu, Lys and Ser or their N-methylated analogues.

In certain embodiments of the linker of formula (I), R3a is selected from the group consisting of 9-fluorenylmethyloxycarbamate (FMOC), tert-butyloxycarbonyl (BOC), benzyl carbamate (Cbz), acetamide, trifluroacetamide, phthalimide, benzylamine, triphenylmethylamine, benzylideneamine, and p-toluenesulfonamide (p-TOS).

In certain embodiments of the linker of formula (I), R3b is selected from the group consisting of a methyl ester, a tert-butyl ester, a benzyl ester, an S-tert-butyl ester, and 2-alkyl-1,3-oxazoline.

In certain embodiments of the linker of formula (I), R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined above. In certain embodiments, R is a detectable probe.

In certain embodiments of the linker of formula (I), k and k′ are both 1.

In another aspect, provided herein is a linker-cytotoxin conjugate of the following formula (II):

or an enantiomer, diasteriomer, or mixtures thereof;
wherein:

  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-3 alkyl;
  • each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-3 alkyl;
  • CTX is a cytotoxin;
  • R is any chemical group; or R is absent;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each k and k′ is independently an integer of 0 or 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • m is an integer of 1 to 4;
  • each AA is independently an amino acid;
  • each r is 1 to 12; and
  • the bond represents a single or a double bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II), the bond represents a single bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II), CTX is bonded to (L1)a-(L2)b-(L3)c via an amide, an N—(C1-6 alkyl)amide, a carbamate, an N—(C1-6 alkyl)carbamate, an amine, an N—(C1-6 alkyl)amine, an ether, a thioether, an urea, an N—(C1-6 alkyl)urea, or an N,N-di(C1-6alkyl)urea bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II), CTX is bonded to (L1)a-(L2)b-(L3)c via a bond selected from the group consisting of:

wherein each RB is independently branched or unbranched C1-6 alkyl.

In certain embodiments of the linker-cytotoxin conjugate of formula (II), CTX is selected from a from the group consisting of a tubulin stabilizer, a tubulin destabilizer, a DNA alkylator, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an anti-metabolite. In certain embodiments, the CTX is selected from the group consisting of Actinomycin-D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade®, and Vincristine.

In certain embodiments of the linker-cytotoxin conjugate of formula (II), R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined above. In certain embodiments, R is a detectable probe. In certain embodiments, R is an antibody fragment.

In another aspect, provided herein antibody-drug conjugate of the following formula (III):

a pharmaceutically acceptable salt thereof;
wherein:

  • A is an antibody or antibody fragment;
  • the two depicted cysteine residues are from an opened cysteine-cysteine disulfide bond in A;
  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-3 alkyl;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or CH2CH(R2)—; wherein R1 and R2 are independently C1-3 alkyl;
  • CTX is a cytotoxin;
  • R is any chemical group; or R is absent;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each k and k′ is independently an integer of 0 or 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • each AA is independently an amino acid;
  • each r is 1 to 12;
  • m is an integer of 1 to 4;
  • n is an integer of 1 to 4; and
  • the bond represents a single or a double bond.

In certain embodiments of the antibody-drug conjugate of formula (III), the bond represents a single bond.

In certain embodiments of the antibody-drug conjugate of formula (III), A is an antibody that is specific to a cancer antigen. In certain embodiments, A is selected from the group consisting of alemtuzumab, anitumumab, bevacizumab, brentuximab, cetuximab, gemtuzumab, glembatumumab, inotuzumab, ipilimumab, lovortumumab, milatuzumab, ofatumumab, rituximab, tositumomab, and trastuzumab.

In certain embodiments of the antibody-drug conjugate of formula (III), CTX is selected from a from the group consisting of a tubulin stabilizer, a tubulin destabilizer, a DNA alkylator, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an anti-metabolite. In certain embodiments, the CTX is selected from the group consisting of Actinomycin D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade®, and Vincristine. In certain embodiments, the CTX is an auristatin, a calicheamicin, a maytansinoid, or a tubulysin. In certain embodiments, the CTX is monomethylauristatin E, monomethylauristatin F, calicheamicin γ, mertansine, tubulysin T3, or tubulysin T4.

In certain embodiments of the antibody-drug conjugate of formula (III), R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined above. In certain embodiments, R is a detectable probe.

In certain embodiments of the antibody-drug conjugate of formula (III), k and k′ are both 1.

In another aspect, provided herein is a pharmaceutical composition comprising the antibody-drug conjugate of formula (III) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluents, carrier or excipient.

In another aspect, provided herein is a method of treating a cancer by administering to a human suffering therefrom an effective amount of the antibody-drug conjugate of formula (III) or a pharmaceutical composition comprising such antibody-drug conjugate.

DETAILED DESCRIPTION Brief Description of the Drawings

FIG. 1: Human IgG Sub-types

FIG. 2: Forms of Antibodies and Antibody Fragments

FIG. 3: List of Antibodies in Clinical Trials

FIG. 4: Rapid Screening and Optimization of Bifunctional Linkers

FIG. 5: Mass Spectrometric Analysis of a Homogeneous ADC

 DEFINITIONS

An “antibody,” also known as an immunoglobulin, is a large Y-shaped protein that binds to an antigen. Antibodies are used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the antigen, because each tip of the “Y” of the antibody contains a site that is specific to a site on an antigen, allowing these two structures to bind with precision. An antibody may consist of four polypeptide chains, two heavy chains and two light chains connected by interchain cysteine disulfide bonds (see, e.g., FIG. 1). A “monoclonal antibody” is a monospecific antibody where all the antibody molecules are identical because they are made by identical immune cells that are all clones of a unique parent cell. Initially, monoclonal antibodies are typically prepared by fusing myeloma cells with the spleen cells from a mouse (or B-cells from a rabbit) that has been immunized with the desired antigen, then purifying the resulting hybridomas by such techniques as affinity purification. Recombinant monoclonal antibodies are prepared in viruses or yeast cells rather than in mice, through technologies referred to as repertoire cloning or phage display/yeast display, the cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities may be obtained. The resulting antibodies may be prepared on a large scale by fermentation. “Chimeric” or “humanized” antibodies are antibodies containing a combination of the original (usually mouse) and human DNA sequences used in the recombinant process, such as those in which mouse DNA encoding the binding portion of a monoclonal antibody is merged with human antibody-producing DNA to yield a partially-mouse, partially-human monoclonal antibody. Full-humanized antibodies are produced using transgenic mice (engineered to produce human antibodies) or phage display libraries. Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having similar structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of antibody-like molecules are produced at low levels by the lymph system and at increased levels by myelomas. The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity). Forms of antibodies and antibody fragments are shown schematically in FIG. 2. These antibodies may also include certain antibody fragments. An antibody can be chimeric, human, humanized and/or affinity matured. Exemplary antibodies are shown in FIG. 3. Antibodies of particular interest are those that are specific to cancer antigens, are non-immunogenic, have low toxicity, and are readily internalized by cancer cells; and suitable antibodies include alemtuzumab, bevacizumab, brentuximab, cetuximab, gemtuzumab, ipilimumab, ofatumumab, panitumumab, rituximab, tositumomab, inotuzumab, glembatumumab, lovortuzumab and trastuzumab. Additional antibodies include adecatumumab, afutuzumab, bavituximab, belimumab, bivatuzumab, cantuzumab, citatuzumab, cixutumumab, conatumumab, dacetuzumab, elotuzumab, etaracizumab, farletuzumab, figitumumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, necitumumab, nimotuzumab, olaratumab, oportuzumab, pertuzumab, pritumumab, ranibizumab, robatumumab, sibrotuzumab, siltuximab, tacatuzumab, tigatuzumab, tucotuzumab, veltuzumab votumumab, and zalutumumab.

The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, and are not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

“Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, two, three and as many as most or all of the functions normally associated with that portion when present in an intact antibody. In one aspect, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another aspect, an antibody fragment, such as an antibody fragment that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody. Such functions may include FcRn binding, antibody half life modulation, ADCC function and complement binding. In another aspect, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise on antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. The modifier term “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain aspects, such a monoclonal antibody may include an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. (See, Kohler et at, Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2.sup.nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, WO98/24893; WO96/34096; WO96/33735 and WO91/10741). The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567). “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one aspect, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In another aspect, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all the FRs are those of a human immunoglobulin sequence. The humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

“Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues. “Fc receptor” or “FcR” is a receptor that binds to the Fc region of an antibody. In certain embodiments, an FcR is a native human FcR. In one aspect, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses. (See Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).

The term “alkyl,” as used herein, means a straight, branched chain, or cyclic (in this case, it would also be known as “cycloalkyl”) hydrocarbon containing from 1-10 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylhexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In certain embodiments, such alkyl groups can optionally be substituted with 1 to 5 substituents selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —CO2H, —C(O)CH3, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe, and the like.

The term “C1-6alkyl,” as used herein, means a straight, branched chain, or cyclic (in this case, it would also be known as “cycloalkyl”) hydrocarbon containing from 1-6 carbon atoms.

The term “C1-3alkyl,” as used herein, means a straight or branched chain hydrocarbon containing from 1-3 carbon atoms.

The term “alkenyl,” as used herein, means a straight, branched chain, or cyclic (in which case, it would also be known as a “cycloalkenyl”) hydrocarbon containing from 2-10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. In some embodiments, depending on the structure, an alkenyl group is a monoradical or a diradical (i.e., an alkenylene group). In some embodiments, alkenyl groups are optionally substituted. Examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, and 2-methyl-1-heptenyl. In certain embodiments, such alkenyl groups can optionally be substituted with 1 to 5 substituents selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —CO2H, —C(O)CH3, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe, and the like.

The term “alkoxy,” as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

The term “C2-6 alkenyl,” as used herein, means a straight, branched chain, or cyclic (in this case, it would also be known as “cycloalkyl”) hydrocarbon containing from 2-6 carbon atoms and at least one carbon-carbon double bond formed by the removal of two hydrogens.

The term “cycloalkyl,” as used herein, means a monocyclic or polycyclic radical that contains only carbon and hydrogen, and includes those that are saturated, partially unsaturated, or fully unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms.

The term “amino,” as used herein, refers to the radical —NH2.

The term “hydroxyl” or “hydroxy,” as used herein, refers to the radical —OH.

The term “carboxyl” as used herein, refers to the radical —CO2H.

The term “phenyl,” as used herein, refers to a C6H5 group as known in the art. “Phenylene,” as used herein, refeers to a divalent phenyl group, wherein the phenyl group is substituted at two positions on the phenyl ring that may be ortho (o-C6H4) or para (p-C6H4).

The term “thiol,” as used herein, refers to the radical —SH. The term “substituted thiol,” as used herein, refers to a radical such as —SR wherein R is any optionally substituted chemical group described herein. In certain embodiments, “substituted thiol” refers to a radical —SR where R is an alkyl, cycloalkyl, aryl or heteroaryl group as defined herein that may be optionally substituted as defined herein. Representative examples of substituted thiol include, but are not limited to, thiophenyl, thionaphthyl, thiopyridyl, thioisoquinolinyl, as depicted below:

The term “sulfonate,” as used herein, refers to the radical —OS(O2)H. “Substituted sulfonate” refers to a radical such as —OS(O2)R wherein R is an alkyl, cycloalkyl, aryl or heteroaryl group as defined herein that may be optionally substituted as defined herein. In certain embodiments, R is selected from lower alkyl, alkyl, aryl and heteroaryl. Representative examples of substituted sulfonate include, but are not limited to, tosylate, mesylate and triflate, as depicted below:

The term “aryl,” as used herein, refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, biphenyl, naphthyl and the like. Unless otherwise constrained by the definition for the individual substituent, such aryl groups can optionally be substituted with 1 or more substituents, for example, 1 to 5 substituents, such as, hydroxyl, amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkyl, C2-6 alkenyl, and C1-6 alkoxy, and the like. In certain embodiments, such aryl groups can optionally be substituted with 1 to 5 substituents selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —CO2H, —C(O)CH3, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe and C1-3alkyl.

The term “heteroaryl,” as used herein, refers to an aryl ring system having one to four heteroatoms as ring atoms in a heteroaromatic ring system, wherein the remainder of the atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur and nitrogen. Preferably, the heterocyclic ring system is monocyclic or bicyclic. Unless otherwise constrained by the definition for the individual substituent, such heteroaryl groups can optionally be substituted with 1 or more substituents, for example, 1 to 5 substituents, such as, hydroxyl, amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkyl, C2-6alkenyl, and C1-6 alkoxy, and the like. In certain embodiments, such heteroaryl groups can optionally be substituted with 1 to 5 substituents selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —CO2H, —C(O)CH3, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe and C1-3 alkyl. Examples of hetroaryl include, but are not limited to:

where Q is O, NR2 or S.

The term “chemical group,” as used herein, refers to two or more atoms bound together as a single unit and forming part of a molecule.

The term “protecting group,” as used herein, refers to a chemical group that is introduced into a molecule by chemical modification of a functional group in order to obtain chemoselectivity. Examples of protecting groups are disclosed, for example, in Greene, T. W. and Wuts, P. G. M., 1991, Protective Groups In Organic Synthesis, 3rd ed.; John Wiley & Sons: New York, and similar documents.

The term “amino protecting group,” as used herein, as used herein, refers to a protecting group that serves to protect an amino functional group. The term includes, without limitation, 9-fluorenylmethyloxycarbamate (FMOC), tert-butyloxycarbonyl (BOC), benzyl carbamate (Cbz), acetamide, trifluroacetamide, phthalimide, benzylamine, triphenylmethylamine, benzylideneamine, and p-toluenesulfonamide (p-TOS), and the like.

The term “carboxyl protecting group,” as used herein, refers to a protecting group that serves to protect a carboxylic acid functional group. The term includes, without limitation, a methyl ester, a tert-butyl ester, a benzyl ester, an S-tert-butyl ester, 2-alkyl-1,3-oxazoline, and the like.

An “amino acid” (or AA) or amino acid residue includes all 20 naturally occurring amino acids, commonly designated by three letter symbols (e.g., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val)). The amino acid residue of the present application also includes citrulline (Cit), 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, homocysteine, homoserine, omithine and methionine sulfone. The amino acid residue of the present application also includes the corresponding N-methyl amino acids, such as —N(CH3)CH2C(O)O—, —NHC(O)CH2CH2CH(NHCH3)C(O)O—, etc. The amino acids, dipeptides, tripeptides, oligomers and polypeptides designated as -(AA)r- of the present application may include the corresponding non-N-alkylated amino acids and peptides (such as non-N-methylated amino acids in the peptides), as well as a mixture of the non-N-alkylated amino acids and the N-alkylated amino acids of the peptides.

A “cytotoxin” (CTX) is a molecule that, when released within a cancer cell, is toxic to that cell.

A “linker” (noted as L or L1, L2 and L3) is a molecule with two reactive termini, one for conjugation to an antibody or to another linker and the other for conjugation to a cytotoxin. The antibody conjugation reactive terminus of the linker is typically a site that is capable of conjugation to the antibody through a cysteine thiol or lysine amine group on the antibody, and so is typically a thiol-reactive group such as a double bond (as in maleimide) or a leaving group such as a chloro, bromo or iodo or an R-sulfanyl group or sulfonyl group, or an amine-reactive group such as a carboxyl group or as defined herein; while the antibody conjugation reactive terminus of the linker is typically a site that is capable of conjugation to the cytotoxin through formation of an amide bond with a basic amine or carboxyl group on the cytotoxin, and so is typically a carboxyl or basic amine group. In one embodiment, when the term “linker” is used in describing the linker in conjugated form, one or both of the reactive termini will be absent (such as the leaving group of the thiol-reactive group) or incomplete (such as the being only the carbonyl of the carboxylic acid) because of the formation of the bonds between the linker and/or the cytotoxin.

The term “leaving group,” as used herein, refers to any group that leaves in the course of a chemical reaction involving the group as described herein and includes but is not limited to halogen, sulfonates (brosylate, mesylate, tosylate triflate etc. . . . ), p-nitrobenzoate and phosphonate groups, for example.

The term “electrophilic leaving group,” as used herein, refers to a leaving group that accepts an electron pair to make a covalent bond. In general, electrophiles are susceptible to attack by complementary nucleophiles, including the reduced thiols from the disulfide bond of an antibody.

The term “electrophilic leaving group that reacts selectively with thiols,” as used herein, refers to electrophilic leaving group that reacts selectively with thiols, over other nucleophiles. In certain embodiments, an electrophilic leaving group that reacts selectively with thiols reacts selectively with the reduced thiols from the disulfide bond of an antibody.

The term “detectable probe,” as used herein, refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

An “antibody-drug conjugate” (ADC) is an antibody that is conjugated to one or more cytotoxins, through one or more linkers. The antibody is typically a monoclonal antibody specific to a therapeutic target such as a cancer antigen.

A “cytotoxic agent” or “cytotoxin” is a molecule that has a cytotoxic effect on cells (e.g., when released within a cancer cell, is toxic to that cell).

“Tubulysin” includes both the natural products described as tubulysins, such as by Sasse et al. and other authors mentioned in the Description of the related art, and also the tubulysin analogs described in US Patent Application Publication No. US 2011/0021568 A1. Tubulysins disclosed in the present application are noted herein and may include the tubulysins of the formulae T3 and T4, and other tubulysins where the terminal N-methylpiperidine has been replaced by an unsubstituted piperidine, as illustrated below for T3 and T4:

allowing amide bond formation between the terminal piperidine and a linker.

The term “MMAF” generally refers to (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid. In some embodiments, MMAF may refer to ((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one aspect, the cell-proliferative disorder is cancer.

“Tumor,” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive. The terms “cancer” and “cancerous” refer to the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies.

A “basic amine”, such as the amine forming a part of the terminal piperidine group of the tubulysins, such as that of the formulae T3 and T4, is a primary or secondary amine that is not part of an amide.

A “therapeutically effective amount” means that amount of an ADC or composition disclosed herein which, when administered to a human suffering from a cancer, is sufficient to effect treatment for the cancer. “Treating” or “treatment” of the cancer includes one or more of:

(1) limiting/inhibiting growth of the cancer, e.g. limiting its development;
(2) reducing/preventing spread of the cancer, e.g. reducing/preventing metastases;
(3) relieving the cancer, e.g. causing regression of the cancer,
(4) reducing/preventing recurrence of the cancer; and
(5) palliating symptoms of the cancer.

As used herein, the term “pharmaceutically acceptable salt” refers to those salts of the ADCs formed by the process of the present application which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the ADC compounds, or separately by reacting the free base function or group of a compound with a suitable organic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, or salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc., or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, benzenesulfonate, benzoate, bisulfate, citrate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, 2-hydroxy-ethanesulfonate, lactate, laurate, malate, maleate, malonate, methanesulfonate, oleate, oxalate, palmitate, phosphate, propionate, stearate, succinate, sulfate, tartrate, p-toluenesulfonate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, or magnesium salts, and the like. Further pharmaceutically acceptable salts include, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl groups having from 1 to 6 carbon atoms (e.g., C1-6 alkyl), sulfonate and aryl sulfonate.

Cancers of interest for treatment include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer including, for example, HER2-positive breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CML), multiple myeloma and B-cell lymphoma, brain cancer, head and neck cancers and associated metastases.

Abbreviations/Acronyms

ADC: antibody-drug conjugate; BOC: tert-butyloxycarbonyl; BRA: bromoacrylamide; Cbz: benzyl carbamate; DAR: Drug-to-antibody ratio; DBB: dibromobenzyl; DBMA: dibromomaleamide; DBP: dibromopropanamide; DBSC: dibromosuccinamide; DEA: diethylamine; DEPC: diethylpyrocarbonate; DCC: 1,3-dicyclohexylcarbodiimide; DIAD: diisopropyl azodicarboxylate; DIPC: 1,3-diisopropylcarbodiimide; DIPEA: diisopropylethylamine; DMA: dimethyacetamide; DMF: N,N-dimethylformamide; DPBS: Dulbecco's phosphate-buffered saline; DTNB: 5,5′-dithiobis-(2-nitrobenzoic acid); DTPA: diethylenetriaminepentaacetic acid; DTT: dithiothreitol; EDC: ethyl 3-(3-dimethylaminopropyl)carbodiimide; EEDQ: ethoxycarbonyl-ethoxy-dihydroquinoline; Fmoc or FMOC: 9-fluorenylmethoxycarbonyl chloride; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; HIC: hydrophobic interaction chromatography; HOBT: N-hydroxybenzotriazole; HPLC: High Performance Liquid Chromatography; NHS: N-hydroxysuccinimide; NMM: N-methylmorpholine; MC: maleimido caproyl; MMAE: monomethylauristatin E; MMAF: monomethylauristatin F, monomethylauristatin phenylalanine; MC: maleimidocaproyl, 6-(2,5-dioxopyrrolyl)hexanoyl; PAB: para amino benzyl; PBD: pyrrolobenzodiazepine; PBS: phosphate-buffered saline; PEG: poly(ethyleneglycol); p-TOS; p-toluenesulfonamide; TBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TCEP: tris(2-carboxyethyl)phosphine; TFA: trifluoroacetic acid; TGI: tumor growth inhibition; TEA: triethanolamine; THF: tetrahydrofuran; VA: Valine-Alanine; VAP: Valine-Alanine-para amino benzyl; VA(PAB): Valine-Alanine-para amino benzyl.

Abbreviations/Acronyms (Antibodies)

ATZ: alemtuzumab; ATM: anitumumab; BCZ: bevacizumab; BTX: brentuximab; CTX: cetuximab; GTZ: gemtuzumab; GBT: glembatumumab; ITZ: inotuzumab; ILM: ipilimumab; LVT: lovortumumab; MTZ: milatuzumab; OTM: ofatumumab; RTX: rituximab; TTM: tositumomab; and TTZ: trastuzumab.

Linkers

The linkers disclosed herein are “opened ring” structures, such that the linker is capable of being chemically modified at two positions of the structure, the Z position, and the R position (see arrowed positions below):

Without intending to be bound by any particular theory or mechanism, it is believed that the linkers disclosed herein, which have “opened ring” structures, display improved in vivo stability with regard to the antibody-linker interface.

The “opened ring” structure of the linkers disclosed herein also allow greater flexibility in making ADCs capable of carrying two drug payloads or one drug payload and a detectable probe, as explained below.

For example, in certain embodiments, where R is selected from the group consisting of Z, (L1)a-(L2)b-(L3)c-Z, and W-(L1)-(L2)b-(L3)c-Z, two cytotoxin (CTX) molecules may be conjugated to the linker. Such linker may be used to make an ADC that is capable of carrying two drug payloads. Alternatively, in certain embodiments, where R is “capped off” with an unreactive side chain, for example, where R is W, (L1)a, (L2)b, (L3)c, or W-(L1)a-(L2)b-(L3)c, only one CTX may be conjugated to the linker. Such linker may be used to make an ADC that is capable of carrying one drug payload.

In certain embodiments, where R is a detectable probe (e.g., a fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment that provides a detectable signal via its activity), such a linker may be used to make an ADC capable of carrying one drug payload and a detectable probe. In certain embodiments, where R is a radiolabel, such a linker may be used to make an ADC that is capable of carrying one drug payload (CTX) and one radiolabel. The resultant radiolabeled ADC can be useful for imaging and/or therapy.

Antibody-Drug Conjugate (ADC)s

As mentioned in the Description of the related art, ADCs of the prior art that coordinate to cysteine thiols of the antibody have employed monofunctional linkers, of which the MC linker is an example. Reduction and opening of the cysteine-cysteine disulfide bonds to give free thiols for conjugation decreases the stability of the antibody, and the formation of the ADC by reaction of the reduced thiols does not re-form the interchain disulfide bond, as illustrated for an exemplary antibody depicted with four interchain disulfide bonds in the following Scheme A:

As a result, a heterogenous mixture of ADCs is produced, which may comprise all possible positional isomers of drug conjugate to cysteine thiol, and may comprise all possible drug antibody ratios (1, 2, 3, 4, . . . , and 8).

In contrast, the linkers disclosed herein contain two reactive functional groups (Y and Y′ in the scheme below) that selectively target the two sulfur atoms of an opened cysteine-cysteine disulfide bond (e.g., one or more opened interchain cysteine-cysteine disulfide bonds). Reaction of the bifunctional linker with the two cysteines of an opened cysteine-cysteine disulfide bond gives a “stapled” or “snapped” antibody conjugate with one linker per disulfide (e.g., one or more interchain disulfides) connected through two thioether bonds, as shown in the following exemplary Scheme B:

A homogenous ADC, for example, with an drug-to-antibody ratio of four (4) is produced. Scheme B depicts a homogenous ADC, where, for example, the four (4) interchain disulfide bonds of the antibody (2 H—H disulfide bonds, and 2 H-L disulfide bonds) are conjugated.

Unlike conventional methods for cysteine conjugation, the reaction re-forms a covalently bonded structure between the 2 cysteine sulfur atoms and therefore does not compromise the overall stability of the antibody. The method also enables conjugation of an optimal 4 drugs for an antibody to afford a homogeneous ADC in which the reactive cysteines are used. The overall result is replacement of a relatively labile disulfide with a stable “staple” or “snap” between the cysteines. The monosubstituted linkers (where one of Y and Y′ is hydrogen) are also effectively bifunctional in conjugation with the antibody because the double bond is capable of conjugation to one of the cysteine sulfur atoms and the Y group with the other.

Preparation of the Linkers

Linkers may be prepared by methods well known to a person of ordinary skill in the art, following procedures described in such references as Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodds Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

The linkers disclosed herein may be cleavable under normal physiological and/or intracellular conditions, or may remain stable (e.g., uncleaved or non-cleavable) under those same conditions.

For example, cleavable linkers may remain stable during systemic circulation but may be cleaved under certain intracellular conditions, such as in an acidic environment. For example, where an ADC is processed in a lysosome of a cell, the linker may be cleaved by the acidic environment and/or the enzymes in the lysosome, releasing the cytotoxin from the antibody. Examples of cleavable linkers are linkers which contain dipeptide moieties, where the peptide bond connecting the two peptides has the potential to be selectively cleaved by lysosomal proteases (e.g., cathepsin-B). Valine-citruline (Val-Cit) is a dipeptide moiety commonly used in cleavable linkers.

Noncleavable linkers may remain stable, both during systemic circulation and under certain intracellular conditions, such as in an acidic environment. Examples of stable linkers are linkers which do not contain dipeptide moieties, for example, alkyl and/or PEG linkers.

The following schemes a, b, c, and d illustrate general synthetic schemes for the stable linkers (e.g., uncleaved or non-cleavable) disclosed herein, which may be synthesized by the methods disclosed herein:

Illustrative General Synthetic Schemes for the Stable Linkers Disclosed Herein

The above schemes are merely illustrative, and not meant to be limiting.

The following schemes e and f illustrates a general synthetic schemes for the cleavable linkers disclosed herein, which may be synthesized by the methods disclosed herein:

Illustrative General Synthetic Schemes for the Cleavable Linkers Disclosed Herein

The above schemes are merely illustrative, and not meant to be limiting.

Exemplary stable and cleavable linkers which may be synthesized by the methods disclosed herein, are provided below:

Examples of Linkers (Stable and Cleavable)

Name Stable Name Cleavable BRA(C6) BRA- VAP DBP(C6) DBP- VAP DBMA(C6) DBMA- VAP BRA(C6) BRA- PEGn- VAP DBP(C6) DBP- PEGn- VAP DBMA(C6) DBMA- PEGn- VAP

In certain embodiments, stable linkers which may be synthesized by the methods disclosed herein are provided below:

6-(2-bromoacrylamido)hexanoic acid

6-(2,3-dibromopropanamido)hexanoic acid

(Z)-6-(4-amino-2,3-dibromo-4-oxobut-2-enamido) hexanoic acid

(Z)-6-(2,3-dibromo-3-carboxyacrylamido) hexanoic acid

1,2-dibromo-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid

18,19-dibromo-20-(methylamino)-17,20-dioxo-4,7,10,13-tetraoxa-16-azaicosan-1-oic acid

Preparation of the Linker-Cytotoxin Conjugates

Linker-cytotoxin conjugates may be prepared by methods analogous to those of Doronina et al., Bioconjugate Chem. 2006, 17, 114-124, and similar documents. The linker, 1 equivalent, and HATU, 1 equivalent, are dissolved in anhydrous DMF, followed by the addition of DIPEA, 2 equivalents. The resulting solution is added to the cytotoxin, 0.5 equivalents, dissolved in DMF, and the reaction stirred at ambient temperature for 3 hr. The linker-cytotoxin conjugate is purified by reverse phase HPLC on a C-18 column.

The following schemes illustrate various embodiments of the linker-cytotoxin conjugates disclosed herein, which may be synthesized by the methods disclosed herein:

Illustrative General Synthetic Schemes for the Linker-Cytotoxin Conjugates Disclosed Herein (e.g., Stable Linkers)

The following scheme illustrate an additional embodiment of the linker-cytotoxin conjugates disclosed herein, which may be synthesized by the methods disclosed herein:

Illustrative General Synthetic Schemes for the Linker-Cytotoxin Conjugates Disclosed Herein (e.g., Cleavable Linkers)

The above schemes are merely illustrative, and are not meant to be limiting. The linker-cytotoxin conjugates may be synthesized using any possible combination of linker and cytotoxin disclosed herein.

Exemplary linker-cytotoxin conjugates (stable or cleavable linkers), where CTX may be any cytotoxin disclosed herein, and which may be synthesized by the methods disclosed herein, are provided below:

Examples of Linker-Cytotoxin Conjugates

Name Stable Name Cleavable BRA(C6) BRA- VAP DBP(CG) DBP- VAP DBMA(C6) DBMA- VAP BRA(C6) BRA- PEGn- VAP DBP(CG) DBP- PEGn- VAP DBMA(CG) DBMA- PEGn- VAP

Further exemplary linker-cytotoxin conjugates disclosed herein (stable linkers), where CTX may be any cytotoxin disclosed herein, and which may be synthesized by the methods disclosed herein, are provided below:

It will be understood that for certain CTX (e.g., tubulysins or modified tubulysins disclosed herein), the amine or carboxyl group may be part of the CTX molecule itself, as illustrated below for the following molecule fragment:

Preparation of ADCs

Antibodies, typically monoclonal antibodies are raised against a specific cancer target (antigen), and purified and characterized. Therapeutic ADCs containing that antibody are prepared by standard methods for cysteine conjugation, such as by methods analogous to those of Hamblett et al., “Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate”, Clin. Cancer Res. 2004, 10, 7063-7070; Doronina et al., “Development of potent and highly efficacious monoclonal antibody auristatin conjugates for cancer therapy”, Nat. Biotechnol., 2003, 21(7), 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. Antibody-drug conjugates with four drugs per antibody are prepared by partial reduction of the antibody with an excess of a reducing reagent such as DTT or TCEP at 37° C. for 30 min, then the buffer exchanged by elution through SEPHADEX® G-25 resin with 1 mM DTPA in DPBS. The eluent is diluted with further DPBS, and the thiol concentration of the antibody may be measured using 5,5′-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, 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 resulting ADC mixture may be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC may then be then sterile filtered, for example, through a 0.2 μM filter, and lyophilized if desired for storage.

The formation of an ADC disclosed herein includes a homogenous ADC with a drug-to-antibody ratio of 4 (e.g., for an IgG1). Scheme B above depicts a “Y”-shaped structure denoting an antibody, for example, an IgG1, where all four (4) interchain disulfide bonds of the antibody (2 H—H disulfide bonds, and 2 H-L disulfide bonds) are conjugated with a drug-to-antibody ratio of 4.

The Linker:

In one aspect, provided herein is a linker of the following formula (I):

or an enantiomer, diasteriomer, or mixtures thereof:
wherein:

  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-6 alkyl;
  • each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-6 alkyl;
  • Z is —CO2H, —NH2, —OH, —NH—R3, or —CO2R3b; wherein R3 is an amino protecting group, and R3b is a carboxyl protecting group; or Z is any chemical group;
  • R is any chemical group; or R is absent;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each k and k′ is independently an integer of 0 or 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • each AA is independently an amino acid;
  • each r is 1 to 12; and
  • the bond represents a single or a double bond.

In certain embodiments of the linker of formula (I), k and k′ are both 1.

In another aspect, provided herein is a linker of the following formula (I′):

or an enantiomer, diasteriomer, or mixtures thereof;
wherein:

  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-6 alkyl;
  • each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-6 alkyl;
  • Z is —CO2H, —NH2, —OH, —NH—R3a, or —CO2R3b; wherein R3a is an amino protecting group, and R3b is a carboxyl protecting group;
  • R is any chemical group, provided that R is not OH;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • each AA is independently an amino acid;
  • each r is 1 to 12; and
  • the bond represents a single or a double bond.

In certain embodiments of the linker of formula (I) or (I′), the bond represents a single bond. In certain embodiments of the linker of formula (I) or (I′), the bond represents a double bond.

In certain embodiments of the linker of formula (I) or (I′), X and X′ are O.

In certain embodiments of the linker of formula (I) or (I′), W is —NH— or —N(R1)—, wherein R1 is C1-6 alkyl. In certain embodiments, W is —NH— or —N(R1)—, wherein R1 is C1-3 alkyl. In certain embodiments, W is —CH2NH— or —CH2N(R1)—, wherein R1 is C1-6 alkyl. In certain embodiments, W is —CH2NH— or —CH2N(R1)—, wherein R1 is C1-3alkyl. In certain embodiments, W is —CH2—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2), wherein R2 is C1-6 alkyl. In certain embodiments, W is —CH2—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2), wherein R2 is C1-3 alkyl.

In certain embodiments of the linker of formula (I) or (I′), X and X′ are O, and W is —NH—.

In certain embodiments of the linker of formula (I) or (I′), each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiol.

In certain embodiments of the linker of formula (I) or (I′), each Y and Y′ is independently selected from the group consisting of a halo, a substituted thiol, and a substituted sulfonate. In certain embodiments, each Y and Y′ is independently selected from the group consisting of chloro, bromo, fluoro, and iodo. In certain embodiments, each Y and Y′ is independently selected from an optionally substituted thiophenyl, an optionally substituted thionaphthyl, an optionally substitued thiopyridyl, an optionally substituted isoquinolinyl, and an optionally substituted phenylsulfonate.

In certain embodiments of the linker of formula (I) or (I′), when one or both of Y and Y′ is a substituted thiol, the substituent is selected from the group consisting of C6-10 aryl and C6-10 heteroaryl.

In certain embodiments of the linker of formula (I) or (I′), when one or both of Y and Y′ is a substituted thiol, the substituent is selected from the group consisting of C1-6alkyl optionally substituted with halo or hydroxyl; phenyl optionally substituted with halo, hydroxyl, carboxyl, C1-3alkoxycarbonyl, or C1-3alkyl; naphthyl optionally substituted with halo, hydroxyl, carboxyl, C1-3alkoxycarbonyl, or C1-3alkyl; and 2-pyridyl optionally substituted with halo, hydroxyl, carboxyl, C1-3alkoxycarbonyl or C1-3alkyl.

In certain embodiments of the linker of formula (I) or (I′), when one or both of Y and Y′ is a substituted sulfonate, the substituent is selected from the group consisting of C1-6alkyl, C2-10cycloalkyl, C6-10aryl, and C6-10heteroaryl.

In certain embodiments of the linker of formula (I) or (I′), each Y and Y′ is independently selected from the group consisting of:

wherein
RA is selected from the group consisting of hydroxyl, amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C1-6alkoxycarbonyl, C1-6 alkyl, C2-6 alkenyl, and C1-6 alkoxy. In certain embodiments, RA is selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH2, —OH, —NHCH3, —N(CH3)2 and C1-3alkyl.

In certain embodiments of the linker of formula (I) or (I′), each Y and Y′ is independently selected from the group consisting of:

In certain embodiments of the linker of formula (I) or (I′), Z is —CO2H, —NH2, —OH, —NH—R3a, or —CO2R3b; wherein R3a is an amino protecting group, and R3b is a carboxyl protecting group, as disclosed, for example, in Greene, T. W.; Wuts, P. G. M., 1991, Protective Groups In Organic Synthesis, 3rd ed.; John Wiley & Sons: New York, and similar documents. Those of ordinary skill in the art will be able to select appropriate amino or carboxyl protecting groups.

In certain embodiments of the linker of formula (I) or (I′), Z is —CO2H or —CO2R3b, and R3b is a carboxyl protecting group.

In certain embodiments of the linker of formula (I) or (I′), R3a is selected from the group consisting of 9-fluorenylmethyloxycarbamate (FMOC), tert-butyloxycarbonyl (BOC), benzyl carbamate (Cbz), acetamide, trifluroacetamide, phthalimide, benzylamine, nitrobenzene, triphenylmethylamine, benzylideneamine, and p-toluenesulfonamide (p-TOS).

In certain embodiments of the linker of formula (I) or (I′), R3b is selected from the group consisting of a methyl ester, a tert-butyl ester, a benzyl ester, an S-tert-butyl ester, and 2-alkyl-1,3-oxazoline.

In certain embodiments of the linker of formula (I) or (I′), Z is a detectable probe, a ligand, or an antibody. In certain embodiments, Z is a fluorophore, chromophore, radiolabel, enzyme, ligand, antibody or antibody fragment.

In certain embodiments of the linker of formula (I) or (I′), R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c—Z, as defined herein. In certain embodiments, R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, and W-(L1)a-(L2)b-(L3)c. In certain embodiments, R is selected from the group consisting of Z, (L1)a-(L2)b-(L3)c—Z, and W-(L1)a-(L2)b-(L3)c—Z.

In certain embodiments of the linker of formula (I) or (I′), R is a detectable probe. In certain embodiments, R is a fluorophore, chromophore, radiolabel, enzyme, ligand, antibody or antibody fragment. In certain embodiments, R is a ligand (e.g., a ligand specific for a receptor on a tumor cell, such as a prostate specific membrane antigen, or a virally infected cell, such as an HIV infected cell).

In certain embodiments of the linker of formula (I) or (I′), R is bonded to the rest of the linker molecule via an amide, an N—(C1-6 alkyl)amide, a carbamate, an N—(C1-6 alkyl)carbamate, an amine, an N—(C1-6 alkyl)amine, an ether, a thioether, an urea, an N—(C1-6alkyl)urea, or an N,N-di(C1-6alkyl)urea bond.

In certain embodiments of the linker of formula (I) or (I′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —C(O)NHCH2CH2—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —C(O)CH2CH2—, —(CH2CH2O)p—, —(OCH2CH2)p—, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH2(p-C6H4)—NH—, —OCH2(o-C6H4)—NH—, —NH-(p-C6H4)—CH2O—, —NH-(o-C6H4)—CH2O—, —OCH(CH2O—)2— and -(AA)r-; a, b and c are each independently 0, 1 or 2; each p, q and r is independently 1, 2, 3 or 4.

In certain embodiments of the linker of formula (I) or (I′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2 and -(AA)r-; a, b and c are each independently 0, 1 or 2; and each p, q and r is independently 1, 2, 3 or 4.

In certain embodiments of the linker of formula (I) or (I′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —OCH(CH2O—)2 and —C(O)NCH3—; a, b and c are each independently 0, 1 or 2; and each p and q is independently 1 or 2.

In certain embodiments of the linker of formula (I) or (I′), each L1, L2 and L3 is independently selected from the group consisting of —NH(CH2)2NH—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —OCH(CH2O—)2 and —C(O)NCH3—; and a, b and c are each independently 0 or 1.

In certain embodiments of the linker of formula (I) or (I′), each L1, L2 and L3 is independently selected from the group consisting of —NHC(O)—, —C(O)NH—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2 and -(AA)r-; a, b and c are each independently 0 or 1; and each p and r is independently 1, 2 or 3.

In certain embodiments of the linker of formula (I) or (I′), each L1, L2 and L3 is independently selected from the group consisting of —NHC(O)—, —C(O)NH—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl; where a, b and c are each independently 0 or 1; and each p and r is independently 1, 2 or 3. In certain embodiments, one or more of the L1, L2 and L3 is -(AA)r-, wherein -(AA)r is ValCit (e.g., the first amino acid is Valine, the second amino acid is Citrulline, and r is 1). In certain embodiments, one or more of the L1, L2 and L3 is -(AA)r-, wherein -(AA)r- is ValAla (e.g., the first amino acid is Valine, the second amino acid is Alanine, and r is 1). In certain embodiments, one or more of the L1, L2 and L3 is phenylenyl substituted by —C(O)OH and —NH2. In certain embodiments, one or more of the L1, L2 and L3 is phenylenyl substituted by —C(O)O— and —NH—. In certain embodiments, one or more of the L1, L2 and L3 is phenylenyl substituted by —OC(O)— and —NH—. In certain embodiments, one or more of the L1, L2 and L3 is phenylenyl substituted by —O— and —NH—. In certain embodiments, one or more of the L1, L2 and L3 is a moiety of the following structure:

In certain embodiments, one or more of the L1, L2 and L3 is a moiety of the following structure:

In certain embodiments, one or more of the L1, L2 and L3 is a moiety of the following structure:

In certain embodiments, one or more of the L1, L2 and L3 is a moiety of the following structure:

In certain embodiments, one or more of the L1, L2 and L3 is a moiety of the following structure:

In certain embodiments, one or more of the L1, L2 and L3 is a moiety of the following structure:

In certain embodiments, one or more of the L1, L2 and L3 is

In certain embodiments, one or more of the L1, L2 and L3 is para aminobenzyl (PAB), which is optionally substituted with —C(O)O—, —OC(O)— or —O—.

In certain embodiments of the linker of formula (I) or (I′), each AA is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cit, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. In one variation of the above, (AA)r is a single amino acid selected from the group consisting of Cit, Gly, Arg, Val, Ala, Cys, Gin, Leu, Ile, Leu, Lys and Ser or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Ala-Val, Val-Ala, Gly-Gly, Gly-Arg, Gly-Val, Gly-Ala, Gly-Cys, Gly-Gln, Gly-Ile, Leu, Lys-Leu, Gly-Lys, Val-Arg, Ala-Cit, Val-Cit, and Gly-Ser or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Gly-Gly-Gly, Gly-Arg-Gly, Gly-Val-Gly, Gly-Ala-Gly, Gly-Cys-Gly, Gly-Gln-Gly, Gly-lie-Gly, Lys-Leu-Gly, Gly-Lys-Gly and Gly-Ser-Gly or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Ala-Ala, Ala-Gly, Ala-Arg, Ala-Val, Ala-Ala, Ala-Cys, Ala-Gin, Ala-Ile, Ala-Leu, Ala-Lys, Ala-Cit, and Ala-Ser or their N-methylated analogues. In another variation of the above, (AA)r 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 or their N-methylated analogues.

In certain embodiments of the linker of formula (I) or (I′), L1 is —(CH2)q—, L2 is absent, L3 is absent, and Z is —CO2H.

In certain embodiments of the linker of formula (I) or (I′), L1 is —(CH2)q—, L2 is —(OCH2CH2)p—, L3 is absent, and Z is —CO2H.

In certain embodiments of the linker of formula (I) or (I′), L1 is —(CH2CH2O)p—, L2 is —(CH2)q—, L3 is absent, and Z is —CO2H.

In certain embodiments of the linker of formula (I) or (I′), each L1 is independently selected from the group consisting of —(CH2CH2O)pCH2CH2— and —CH2CH2—(CH2CH2O)p—, L2 is absent, L3 is absent, and Z is —CO2H.

In certain embodiments of the linker of formula (I) or (I′), each L1 is independently selected from the group consisting of —(CH2)q—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and —C(O)—, L2 is Val-Cit, L3 is PAB, and Z is —CO2H.

In certain embodiments of the linker of formula (I) or (I′), each L1 is independently selected from the group consisting of —(CH2)q—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and —C(O)—, L2 is Val-Cit, L3 is PAB, and Z is —CO2H.

In certain embodiments of the linker of formula (I) or (I′), each L1 is independently selected from the group consisting of —(CH2)q—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and —C(O)—, L2 is Val-Ala, L3 is PAB, and Z is —CO2H.

In certain embodiments, -(L1)a-(L2)b-(L3)c- is selected from the group consisting of —(CH2)1-5C(O)-Val-Ala-NH-(p-C6H4)—CH2OC(O)-(p-C6H4)—NO2, —(CH2CH2O)1-12—(CH2CH2)C(O)-Val-Ala-NH-(p-C6H4)—CH2OC(O)-(p-C6H4)—NO2, —(CH2)1-5C(O)-Val-Cit-NH-(p-C6H4)—CH2OC(O)-(p-C6H4)—NO2, —(CH2CH2O)1-12—(CH2CH2)C(O)-Val-Cit-NH-(p-CH4)—CH2OC(O)-(p-C6H4)—NO2.

The Cytotoxin (CTX):

In certain embodiments, disclosed herein are cytotoxins for use in conjugation to the linkers or ADCs disclosed herein. Those of ordinary skill in the art will be aware of appropriate chemotherapeutic agents as disclosed, for example, in Chu, E., DeVite, V. T., 2012, Physicians' Cancer Chemotherapy Drug Manual 2012 (Jones & Bartlett Learning Oncology), and similar documents.

In certain embodiments, the cytotoxin (CTX) may be any FDA-approved chemotherapeutic agent. In certain embodiments, the CTX may be any FDA-approved chemotherapeutic agent available for cancer treatment.

In certain embodiments, the CTX is selected from the group consisting of an alkylating agent, an anthracycline, a cytoskeletal disruptor (taxane), an epothilone, an histone deacetylase Inhibitor (HDAC), an inhibitor of Topoisomerase I, an Inhibitor of Topoisomerase II, a kinase inhibitor, a monoclonal antibody, a nucleotide analog, a peptide antibiotic, a platinum-based agent, a retinoid, a Vinca alkaloid or a derivative thereof, and a radioisotope.

In certain embodiments, the CTX is selected from the group consisting of: thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegal1 (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate: an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

In certain embodiments, the CTX is selected from the group consisting of: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RI VISor® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as ME inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAX1D®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH: and (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above of.

In certain embodiments, the CTX is selected from the group consisting of: alkylating agents such as chlorambucil, bendamustine hydrochloride or cyclophosphamide (CYTOXAN®); purine analogs such as fludurabine (FLUDARA®), pentostatin (NIPENT®), cladribine or nelarabine; pyrimidine analogs such as cytarabine; corticosteroids such as prednisone, prednisolone or methylprednisolone, immunomodulatory agents such as lenalidomide or thalidomide, synthetic flavons such as flavopiridol, Bcl2 antagonists such as oblimersen or ABT-263, antibiotics such as doxorubicin (ADRIAMYCIN®), daunorubicin, idarubicin, or mitoxentrone; anti-metabolites such as methotrexate and clofarabine; tyrosine kinase inhibitors such as imatinib mesylate (GLEEVEC®), bosutinib, dasatinib, and nilotinib; a hypomethylating agents such as azacytidine or decitabine, an FLT3 inhibitor such as midostaurin, sorafenib, or AC220; arsenic trioxide; all-trans retinoic acid; vincristine sulfate; and monoclonal antibodies such as rituximab (RITUXAN®), ofatumumab, obinutuzumab, veltuzumab, ocrelizumab, lumiliximab or alemtuzumab (CAMPATH®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above; as well as combinations of two or more of the above such as fludarabine plus cyclophosphamide (FC), cladribine plus cyclophosphamide (CC), fludarabine plus rituximab, fludarabine plus cyclophosphamide plus rituximab (FCR), and FCR plus alemtuzumab (CFAR). Chemotherapeutic agents may also include agents used in the treatment of multiple myeloma, including thalidomide, lenalidomide, bortezomib, dexamethesone, prednisone, and melphalan, as well as combinations of two or more of the above, such as thalidomide or lenalidomide plus dexamethasone, or bortezomib or lenalidomide plus melphalan and prednisone.

In certain embodiments, the CTX is selected from the group consisting of Actinomycin, all-trans retinoic acid, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine.

In certain embodiments, the CTX is selected from the group consisting of a tubulin stabilizer, a tubulin destabilizer, a DNA alkylator, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an anti-metabolite. In certain embodiments, the CTX is selected from the group consisting of Actinomycin D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade®, and Vincristine. In certain embodiments, the CTX is an auristatin, a calicheamicin, a maytansinoid, or a tubulysin.

In certain embodiments, the CTX is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), a pyrrolobenzodiazepine (PDB), calicheamicin γ, mertansine, or tubulysin T2. In certain embodiments, the CTX is MMAE or MMAF. In certain embodiments, the CTX is a PDB. In certain embodiments, the CTX is tubulysin T2. In certain embodiments, the CTX is tubulysin T3 or tubulysin T4, the structures for which are provided below:

The Cytotoxin (CTX): Modified Tubulysins

In certain embodiments, the CTX is of the formula:

wherein:

i is 0 or 1;

R4 is a C1-6 alkyl; R5 is a C1-6 alkyl; R6 is C1-6 alkyl;

R7 is selected from the group consisting of C1-6alkyl, —OC1-6alkyl, —OC(O)C1-6alkyl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; and

R8 is selected from the group consisting of —OH, —OC1-6alkyl, —CO2C1-6alkyl, —CO2C6-10aryl, —CH(C1-6alkyl)CO2Rc, —CH(C6-10aryl)CO2Rc, —NH—CH(C5H6)2, —NHC1-6alkyl, —NH(CH2)3—CO2Rc, —NH(CH2CH2)2C6-10aryl, —NHCH(CH2C6-10aryl)CH2CH(CH3)CO2Rc and —NHCH(CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl;

where each Rc is independently H or C1-6alkyl; and

R17 is selected from the group consisting of H, —CH3 and —C(O)CH3.

In certain embodiments, the CTX is of the formula:

wherein:

i is 0 or 1;

R4 is a C1-6alkyl; R5 is a C1-6alkyl;

R6 is selected from the group consisting of C1-6alkyl, C6-10aryl;

R7 is selected from the group consisting of C1-6alkyl, —OC1-6alkyl, —NHC(O)C1-6alkyl, —OC(O)C1-6alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; and

R8 is selected from the group consisting of —OH, —OC1-6alkyl, —CO2C1-6alkyl, —CO2C6-10aryl, —CH(C1-6alkyl)CO2Rc, —CH(C6-10aryl)CO2Rc, —NH—CH(C5H6)2, —NHC1-6alkyl, —NH(CH2)3—CO2Rc, —NH(CH2CH2)2C6-10aryl, —NHCH(CH2C6-10aryl)CH2CH(CH3)CO2Rc, —NHCH(CO2Rc)CH2-p-C6H4—NH2, —NHCH(CO2Rc)CH2-p-C6H4—NHC1-6alkyl and —NHCH(CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl;

where each Rc is independently selected from the group consisting of H, C1-6alkyl and C6-10aryl; and

R17 is selected from the group consisting of H, —CH3 and —C(O)CH3.

In certain embodiments, the CTX is of the formula:

wherein:

i is 0 or 1;

R4 is a C1-6alkyl or C6-10aryl;

R5 is a C1-6alkyl or C6-10aryl;

R6 is selected from the group consisting of C1-6alkyl, C6-10aryl, —CH2OCOC1-6alkyl, —CH2CO2C1-6alkyl, —CH2CONHC1-6alkyl, —CO2C1-6alkyl, —CH(C1-6alkyl)CO2H and —CH(C1-6alkyl)CO2C1-6alkyl;

R7 is selected from the group consisting of C1-6alkyl, —OC1-6alkyl, —NHC(O)C1-6alkyl, —OC(O)C1-6alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; and

R8 is selected from the group consisting of —OH, —OC1-6alkyl, —CO2C1-6alkyl, —CO2C6-10aryl, —CH(C1-6alkyl)CO2Rc, —CH(C6-10aryl)CO2Rc, —NH—CH(C5H6)2, —NHC1-6alkyl, —NH(CH2)3—CO2Rc, —NH(CH2CH2)2C6-10aryl, —NHCH(CH2C6-10aryl)CH2CH(CH3)CO2Rc, —NHCH(CH2CH(CH3)COORc)CH2-p-C6H4—NHC(O)CH(NHC(O)(CH2)5NHRc)(CH2)4NHRc, —NHCH(CO2Rc)CH2-p-C6H4—NH2, —NHCH(CO2Rc)CH2-p-C6H4—NHC1-6alkyl, —NHCH(CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl, —NHCH(CH2CH2CO2Rc)CH2p-C6H4—NHC1-6alkyl and —NHCH(CH2CH(CH3)CO2Rc)CH2-p-C6H4—NHC1-6alkyl;

where each Rc is independently selected from the group consisting of H, C1-6alkyl and C1-10aryl; and

R17 is selected from the group consisting of H, —CH3 and —C(O)CH3.

In certain embodiments, the CTX is of the formula:

wherein:

i is 0 or 1;

R4 is a C1-6alkyl or C6-10aryl;

R5 is a C1-6alkyl or C6-10aryl;

R6 is selected from the group consisting of C1-6alkyl, C6-10aryl, —CH2OCOC1-6alkyl, —CH2CO2C1-6alkyl, —CH2CONHC1-6alkyl, —CO2C1-6alkyl, —CH(C1-6alkyl)CO2H and —CH(C1-6alkyl)CO2C1-6alkyl;

R7 is a bond to the linker L1, L2 and/or L3; and

for CTX-III, R8 is selected from the group consisting of —OH, —OC1-6alkyl, —CO2C1-6alkyl, —CO2C6-10aryl, —CH(C1-6alkyl)CO2Rc, —CH(C6-10aryl)CO2Rc, —NH—CH(C5H6)2, —NHC1-6alkyl, —NH(CH2)3—CO2Rc, —NH(CH2CH2)2C6-10aryl, —NHCH(CH2C6-10aryl)CH2CH(CH3)CO2Rc, —NHCH(CH2CH(CH3)COORc)CH2-p-CH4—NHC(O)CH(NHC(O)(CH2)5NHRc)(CH2)4NHRc, —NHCH(CO2Rc)CH2-p-C6H4—NH2, —NHCH(CO2Rc)CH2-p-C6H4—NHC1-6alkyl, —NHCH(CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl, —NHCH(CH2CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl and —NHCH(CH2CH(CH3)CO2Rc)CH2-p-C6H4—NHC1-6alkyl;

where each Rc is independently selected from the group consisting of H, C1-6alkyl and C6-10aryl.

In certain embodiments, as provided herein, where R7 is a bond to the linker L (or L1-L2-L3), then the CTX is bonded to the linker from both at the squiggly line (˜) and at the bond that is R7; or the CTX is bonded to the linker only from the bond that is R7 and not on the squiggly line bond at the amine nitrogen of the CTX of Formula CTX-III.

In certain embodiments, the CTX is of the formula CTX-IIIa:

where i is 1;

R4 is a C1-6alkyl; R5 is a C1-3alkyl;

R6 is selected from the group consisting of C1-3alkyl, —CH2OCOC1-3alkyl, —CH2CO2C1-3alkyl, —CH2CONHC1-3alkyl, —CH(C1-3alkyl)CO2H and —CH(C1-3alkyl)CO2C1-6alkyl;

R7 is selected from the group consisting of —OC1-3alkyl, —NHC(O)C1-3alkyl, —OC(O)C1-3alkyl, —OC(O)-phenyl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; and

R8 is selected from the group consisting of —NH(CH2CH2)2-phenyl, —NHCH(CH2-phenyl)CH2CH(CH3)CO2Rc, —NHCH(CO2Rc)CH2-p-C6H4—NHC1-3alkyl, —NHCH(CH2CO2Rc)CH2-p-C6H4—NHC1-3alkyl, —NHCH(CH2CH2CO2Rc)CH2-p-C6H4—NHC1-3alkyl and —NHCH(CH2CH(CH3)CO2Rc)CH2-p-C6H4—NHC1-3alkyl; and wherein Rc is H or C1-3alkyl.

In certain embodiments, the CTX is of the formula CTX-IIIa:

where i is 1;

R4 is a C1-6alkyl; R5 is a C1-3alkyl;

R6 is selected from the group consisting of C1-3alkyl, —CH2CO2C1-3alkyl and —CH(C1-3alkyl)CO2C1-3alkyl;

R7 is selected from the group consisting of —OC1-3alkyl, —NHC(O)C1-3alkyl, —OC(O)C1-3alkyl, —OC(O)-phenyl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; and

R8 is selected from the group consisting of —NH(CH2CH2)2-phenyl, —NHCH(CO2Rc)CH2-p-C6H4—NHC1-3alkyl and —NHCH(CH2CH2CO2Rc)CH2-p-C6H4—NHC1-3alkyl; and wherein Rc is H or C1-3alkyl.

In certain embodiments of the formulas of CTX-I, CTX-II, CTX-IIIa, or CTX-IIIb, the CTX is not T3 or T4.

In certain embodiments, the CTX is of the formula:

where:

R4 is a C1-6alkyl or C6-10aryl; R5 is a C1-6alkyl or C6-10aryl;

R6 is selected from the group consisting of C1-6alkyl, C6-10aryl, —CH2OCOC1-6alkyl, —CH2CO2C1-6alkyl, —CH2CONHC1-6alkyl, —CO2C1-6alkyl, —CH(C1-6alkyl)CO2H and —CH(C1-6alkyl)CO2C1-6alkyl;

R7 is selected from the group consisting of halo, C1-6alkyl, —OC1-6alkyl, —NHC(O)C1-6alkyl, —OC(O)C1-6alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; or R7 is a bond to the linker L1, L2 and/or L3; and

R8 is selected from the group consisting of —OH, —OC1-6alkyl, —CH(C1-6alkyl)CO2Rc, —CH(C6-10aryl)CO2Rc, —NH—CH(C5H6)2, —NHC1-8alkyl, —NH(CH2)3—CO2Rc, —NH(CH2CH2)2C6-10aryl, —NHCH(CH2C6-10aryl)CH2CH(CH3)CO2Rc, —NHCH(CH2CH(CH3)CO2Rc)CH2-p-C6H4—NHC(O)CH(NHC(O)(CH2)5NHRc)(CH2)4NHRc, —NHCH(CO2Rc)CH2-p-C6H4, —NHCH(CO2Rc)CH2-p-C6H4—NH2, —NHCH(CH2CO2Rc)CH2-phenyl, —NHCH(CH2CO2Rc)CH2-p-C6H4—NH2, —NHCH(CH2CH2CO2Rc)CH2-phenyl, —NHCH(CH2CH2CO2Rc)CH2-p-C6H4—NH2, —NHCH(CH2CH(CH3)CO2Rc)CH2-phenyl, —NHCH(CH2CH(CH3)CO2Rc)CH2-p-C6H4—NH2, —NHCH(CO2Rc)CH2-p-C6H4—NH2, —NHCH(CO2Rc)CH2-p-C6H4—NHC1-6alkyl, —NHCH(CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl, —NHCH(CH2CH2CO2Rc)CH2-p-C6H4—NHC1-6alkyl and —NHCH(CH2CH(CH3)CO2Rc)CH2-p-C6H4—NHC1-6alkyl;

wherein each Rc is independently selected from the group consisting of H, C1-6alkyl and C6-10aryl; and

R18 is selected from the group consisting of H, —CH3 and —C(O)CH3.

In certain embodiments, the CTX is of the formula CTX-IV or CTX-IVa:

wherein: R4 is a C1-6alkyl; R5 is a C1-6alkyl;

R6 is selected from the group consisting of C1-3alkyl, —CH2OCOC1-3alkyl, —CH2CO2C1-3alkyl, —CH2CONHC1-3alkyl and —CH(C1-6alkyl)CO2C1-3alkyl;

R7 is selected from the group consisting of C1-6alkyl, —OC1-6alkyl, —NHC(O)C1-3alkyl, —OC(O)C1-3alkyl, —OC(O)phenyl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl; and

R8 is selected from the group consisting of —NH—CH(C5H6)2, —NHC1-6alkyl, —NH(CH2)3—CO2Rc, —NH(CH2CH2)2-phenyl, —NHCH(CH2-phenyl)CH2CH(CH3)CO2Rc, —NHCH(CO2Rc)CH2-phenyl, —NHCH(CH2CO2Rc)CH2-phenyl and —NHCH(CO2Rc)CH2-p-C6H4—NHC1-3alkyl; wherein each Rc is independently selected from the group consisting of H and C1-3alkyl.

In certain embodiments, the CTX is of the formula CTX-IV or CTX-IVa:

wherein: R4 is a C1-6alkyl; R5 is a C1-6alkyl; R6 is C1-3alkyl;

R7 is selected from the group consisting of C1-6alkyl, —OC1-6alkyl and —OC(O)C1-3alkyl; and

R8 is selected from the group consisting of —NH—CH(C5H6)2, —NH(CH2CH2)2-phenyl, —NHCH(CO2Rc)CH2-phenyl and —NHCH(CO2Rc)CH2p-C6H4—NHC1-3alkyl; wherein each Rc is independently selected from the group consisting of H and C1-3alkyl.

In certain embodiments, the CTX is of the formula:

wherein:

R4 is a C1-6alkyl or C6-10aryl; R5 is a C1-6alkyl or C6-10aryl;

R6 is H or is selected from the group consisting of C1-6alkyl, C6-10aryl, —CH2OCOC1-6alkyl, —CH2CO2C1-6alkyl, —CH2CONHC1-6alkyl, —CO2C1-6alkyl, —CH(C1-6alkyl)CO2H and —CH(C1-6alkyl)CO2C1-6alkyl;

R9 is selected from the group consisting C1-6alkyl, -phenyl, 1-naphthyl and 2-napthyl, wherein each -phenyl, 1-naphthyl and 2-naphthyl group is unsubstituted or substituted by 1 or 2 substituents selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —C(O)CH3, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe and C1-3alkyl; and

R10 is selected from the group consisting of C1-3alkyl, C2-6alkenyl, —O—C1-3alkyl and —OC6-10aryl;

R11 is H or C1-3alkyl;

wherein Rc is selected from the group consisting of H, C1-6alkyl and C6-10aryl; and

wherein * designates an R chiral center, an S chiral center or a mixture of R and S isomers.

In certain embodiments, the CTX is of the formula CTX-V or CTX-Va:

wherein: R4 is a C1-3alkyl; R5 is a C1-3alkyl;

R6 is selected from the group consisting of C1-3alkyl, —CH2OCOC1-3alkyl, —CH2CO2C1-3alkyl, —CO2C1-3alkyl and —CH(C1-3alkyl)CO2C1-3alkyl;

R9 is selected from the group consisting C1-6alkyl, -phenyl, 1-naphthyl and 2-napthyl, wherein each -phenyl, 1-naphthyl and 2-naphthyl is unsubstituted or substituted by 1 or 2 substituents selected from the group consisting of CF3—, CH3O—, —C(O)CH3, —NHCH3, —N(CH3)2 and C1-3alkyl; and

R10 is selected from the group consisting of C1-3alkyl, C2-6alkenyl, —O—C1-3alkyl and —O-phenyl; and

R17 is selected from the group consisting of H, —CH3 and —C(O)CH3.

In certain embodiments, the CTX is of the formula CTX-V:

wherein: R4 is a C1-3alkyl; R5 is a C1-3alkyl; R6 is C1-3alkyl;

R9 is selected from the group consisting C1-6alkyl, -phenyl, 1-naphthyl and 2-napthyl; and

R10 is selected from the group consisting of C1-3alkyl and C2-6alkenyl.

In certain embodiments, the CTX is of the formula:

wherein:

each R4 is independently a C1-6alkyl or C6-10aryl;

R5 is a C1-6alkyl or C6-10aryl;

each R6 is independently selected from the group consisting of H, C1-6alkyl, C6-10aryl, —CH2OCOC1-6alkyl, —CH2CO2C1-6alkyl, —CH2CONHC1-6alkyl, —CO2C1-6alkyl, —CH(C1-6alkyl)CO2H and —CH(C1-6alkyl)CO2C1-6alkyl;

each R7 is independently selected from the group consisting of —CN, —OC1-6alkyl, C1-6alkyl, —NHC(O)C1-6alkyl, —OC(O)C1-6alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl;

R11 is H or C1-6alkyl;

each R12 is independently selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —CO2H, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe, C1-3alkyl and C6-10aryl;

R13 is H or is selected from the group consisting of C1-3alkyl, —CF3, —C1-3alkyl-phenyl and C6-10aryl;

R18 is selected from the group consisting of H, —CH3 and —C(O)CH3; and

q is 0, 1 or 2.

In certain embodiments, the CTX is of the formula CTX-VI or CTX-VIa:

wherein: each R4 is independently a C1-3alkyl; R5 is a C1-3alkyl;

each R6 is independently selected from the group consisting of H, C1-6alkyl, —CH2OCOC1-6alkyl, —CH2CO2C1-3alkyl, —CH(C1-3alkyl)CO2H and —CH(C1-3alkyl)CO2C1-3alkyl;

each R7 is independently selected from the group consisting of —OC1-3alkyl, C1-3alkyl, —NHC(O)C1-3alkyl, —OC(O)C1-3alkyl and —OC(O)C6-10aryl;

R11 is H or C1-3alkyl;

each R12 is independently selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —NHCH3, —N(CH3)2, and C1-3alkyl;

R13 is H or is selected from the group consisting of C1-3alkyl, —CF3, —C1-5alkyl-phenyl.

In certain embodiments, the CTX is of the formula CTX-VI:

wherein: each R4 is independently a C1-3alkyl; R5 is a C1-3alkyl;

each R6 is independently H or C1-6alkyl;

each R7 is independently selected from the group consisting of —OC1-3alkyl, —OC(O)C1-3alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl;

R11 is H or C1-3alkyl;

each R12 is independently selected from the group consisting of CF3O—, CH3O— and C1-3alkyl; and

R13 is H or is selected from the group consisting of C1-3alkyl, —CF3, —C1-3alkyl-phenyl.

In certain embodiments, the CTX is of the formula:

wherein:

R11 is H or C1-3alkyl;

each R12 is independently selected from the group consisting of halo, cyano, nitro, CF3—, CF3O—, CH3O—, —CO2H, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe, C1-3alkyl and C6-10aryl;

R13 is H or is selected from the group consisting of C1-3alkyl, —CF3, —C1-2alkyl-phenyl and C6-10aryl; and

q is 0, 1 or 2.

In certain embodiments, the CTX is of the formula CTX-VII:

wherein: R11 is H;

R12 is selected from the group consisting of CF3—, CF3O—, CH3O—, —CO2H, —NHCH3, —N(CH3)2, —C1-3alkyl and phenyl;

R13 is H or is selected from the group consisting of C1-3alkyl, —C1-2alkyl-phenyl and phenyl;

R18 is selected from the group consisting of H, —CH3 and —C(O)CH3;

and q is 1.

In certain embodiments, the CTX is of the formula CTX-VII:

wherein: R11 is H and

R13 is H, C1-3alkyl or —C1-2alkyl-phenyl; and q is 0.

In certain embodiments, the CTX is of the formula:

wherein:

each R4 is independently a C1-6alkyl or C6-10aryl;

R5 is a C1-6alkyl or C6-10aryl;

each R6 is independently selected from the group consisting of H, C1-3alkyl, C6-10aryl, —CH2OCOC1-6alkyl, —CH2CO2C1-6alkyl, —CH2CONHC1-6alkyl, —CO2C1-8alkyl, —CH(C1-6alkyl)CO2H and —CH(C1-6alkyl)CO2C1-6alkyl;

each R7 is independently selected from the group consisting of —CN, —OC1-6alkyl, C1-6alkyl, —NHC(O)C1-6alkyl, —OC(O)C1-6alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl;

R11 is H or C1-3alkyl;

R14 is selected from the group consisting of C1-3alkyl and C6-10aryl;

R15 is H or is selected from the group consisting of —OH, NH2, —NHCH3, C1-3alkyl, —OC1-3alkyl and —OC6-10aryl; and

R16 is selected from the group consisting of C1-6alkyl, C6-10aryl and heteroaryl; and R18 is selected from the group consisting of H, —CH3 and —C(O)CH3.

In certain embodiments, the CTX is of the formula CTX-VIII or CTX-VIIIa:

wherein: each R4 is independently a C1-3alkyl; R5 is a C1-3alkyl;

each R6 is independently selected from the group consisting of H, C14alkyl, —CH2OCOC1-6alkyl, —CH2CO2C1-3alkyl, —CH(C1-3alkyl)CO2H and —CH(C1-3alkyl)CO2C1-3alkyl;

each R7 is independently selected from the group consisting of —OC1-3alkyl, C1-3alkyl, —NHC(O)C1-3alkyl, —OC(O)C1-3alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl;

R11 is H or C1-6alkyl;

R14 is C1-3alkyl;

R15 is H or is selected from the group consisting of —OH, NH2, —NHCH3 and —OC1-3alkyl; and

R16 is C6-10aryl.

In certain embodiments, the CTX is of the formula CTX-VIII or CTX-VIIIa:

wherein: each R4 is independently a C1-3alkyl; R5 is a C1-3alkyl;

each R6 is independently H or C1-6alkyl;

each R7 is independently selected from the group consisting of —OC1-3alkyl, —OC(O)C1-3alkyl, —OC(O)C6-10aryl, —OC(O)NHC1-6alkyl and —OC(O)NHC6-10aryl;

R11 is H or C1-3alkyl;

R14 is C1-3alkyl;

R15 is selected from the group consisting of —OH, NH2 and —NHCH3; and

R16 is C6-10aryl.

In certain embodiments of the above variables, any designated aryl group, such as a C6-10aryl, may be a phenyl group, a 1-naphthyl or 2-naphthyl group, and the aryl group is unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of halo, cyano, nitro, CF3—CF3O—, CH3O—, —CO2H, —C(O)CH3, —NH2, —OH, —SH, —NHCH3, —N(CH3)2, —SMe and C1-3 alkyl.

The Linker-Cytotoxin Conjugate:

In another aspect, provided herein is a linker-cytotoxin conjugate of the following formula (II):

or an enantiomer, diasteriomer, or mixtures thereof;
wherein:

  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-6 alkyl;
  • each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-6 alkyl;
  • CTX is a cytotoxin;
  • R is any chemical group; or R is absent;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each k and k′ is independently an integer of 0 or 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • m is an integer of 1 to 4;
  • each AA is independently an amino acid;
  • each r is 1 to 12; and
  • the bond represents a single or a double bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II), k and k′ are both 1.

In another aspect, provided herein is a linker-cytotoxin conjugate of the following formula (II′):

or an enantiomer, diasteriomer, or mixtures thereof;
wherein:

  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-6 alkyl;
  • each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-6 alkyl;
  • CTX is a cytotoxin;
  • R is any chemical group, provided that R is not OH;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • m is an integer of 1 to 4;
  • each AA is independently an amino acid;
  • each r is 1 to 12; and
  • the bond represents a single or a double bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), the bond represents a single bond. In certain embodiments of the linker of formula (I) or (I′), the bond represents a double bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), m is 1.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), X and X′ are O.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), W is —NH— or —N(R1)—, wherein R1 is C1-6 alkyl. In certain embodiments, W is —NH— or —N(R1)—, wherein R1 is C1-3 alkyl. In certain embodiments, W is —CH2NH— or —CH2N(R1)—, wherein R1 is C1-3 alkyl. In certain embodiments, W is —CH2NH— or —CH2N(R1)—, wherein R1 is C1-3 alkyl. In certain embodiments, W is —CH2—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2), wherein R2 is C1-6alkyl. In certain embodiments, W is —CH2—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2), wherein R2 is C1-3alkyl.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), X and X′ are O, and W is —NH—.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiol.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each Y and Y′ is independently selected from the group consisting of a halo, a substituted thiol, and a substituted sulfonate. In certain embodiments, each Y and Y′ is independently selected from the group consisting of chloro, bromo, fluoro, and iodo. In certain embodiments, each Y and Y′ is independently selected from an optionally substituted thiophenyl, an optionally substituted thionaphthyl, an optionally substitued thiopyridyl, an optionally substituted isoquinolinyl, and an optionally substituted phenylsulfonate.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), when one or both of Y and Y′ is a substituted thiol, the substituent is selected from the group consisting of C6-10 aryl and C6-10 heteroaryl.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), when one or both of Y and Y′ is a substituted thiol, the substituent is selected from the group consisting of C1-6alkyl optionally substituted with halo or hydroxyl; phenyl optionally substituted with halo, hydroxyl, carboxyl, C1-3alkoxycarbonyl, or C1-3alkyl; naphthyl optionally substituted with halo, hydroxyl, carboxyl, C1-3alkoxycarbonyl, or C1-3alkyl; and 2-pyridyl optionally substituted with halo, hydroxyl, carboxyl, C1-3alkoxycarbonyl or C1-3alkyl.

In certain embodiments of linker-cytotoxin conjugate of formula (II) or (II′), when one or both of Y and Y′ is a substituted sulfonate, the substituent is selected from the group consisting of C1-6alkyl, C2-10cycloalkyl, C6-10aryl, and C6-10heteroaryl.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each Y and Y′ is independently selected from the group consisting of:

wherein
RA is selected from the group consisting of hydroxyl, amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkyl, C2-6 alkenyl, and C1-6 alkoxy. In certain embodiments, RA is selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3alkyl, —C(O)CH3, —CN, —NH2, —OH, —NHCH3, —N(CH3)2 and C1-3alkyl.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each Y and Y′ is independently selected from the group consisting of:

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)a-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined herein. In certain embodiments, R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, and W-(L1)a-(L2)b-(L3)c. In certain embodiments, R is selected from the group consisting of Z, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), R is a detectable probe. In certain embodiments, R is a fluorophore, chromophore, radiolabel, enzyme, ligand, antibody or antibody fragment. In certain embodiments, R is a ligand (e.g., a ligand specific for a receptor on a tumor cell, such as a prostate specific membrane antigen, or a virally infected cell, such as an HIV infected cell).

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), R is bonded to the rest of the linker molecule via an amide, an N—(C1-6 alkyl)amide, a carbamate, an N—(C1-6 alkyl)carbamate, an amine, an N—(C1-6 alkyl)amine, an ether, a thioether, an urea, an N—(C1-6 alkyl)urea, or an N,N-di(C1-6 alkyl)urea bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2 and -(AA)-; a, b and c are each independently 0, 1 or 2; and each p, q and r is independently 1, 2, 3 or 4.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —OCH(CH2O—)2 and —C(O)NCH3—; a, b and c are each independently 0, 1 or 2; and each p and q is independently 1 or 2.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1, L2 and L3 is independently selected from the group consisting of —NH(CH2)2NH—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —OCH(CH2O—)2 and —C(O)NCH3—; and a, b and c are each independently 0 or 1.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1, L2 and L3 is independently selected from the group consisting of —NHC(O)—, —C(O)NH—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2 and -(AA)r; a, b and c are each independently 0 or 1; and each p and r is independently 1, 2 or 3.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each AA is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. In one variation of the above, (AA)r is a single amino acid selected from the group consisting of Gly, Arg, Val, Ala, Cys, Gin, Leu, Ile, Leu, Lys and Ser or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Gly-Gly, Gly-Arg, Gly-Val, Gly-Ala, Gly-Cys, Gly-Gin, Gly-Ile, Leu, Lys-Leu, Gly-Lys and Gly-Ser or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Gly-Gly-Gly, Gly-Arg-Gly, 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 N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Ala-Ala, Ala-Gly, Ala-Arg, Ala-Val, Ala-Ala, Ala-Cys, Ala-Gin, Ala-Ile, Ala-Leu, Ala-Lys and Ala-Ser or their N-methylated analogues. In another variation of the above, (AA)r 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 or their N-methylated analogues.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), L1 is —(CH2)q—, L2 is absent, L3 is absent, and the CTX is bonded to (L)a-(L2)b-(L)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), L1 is —(CH2)q—, L2 is —(OCH2CH2)p—, L3 is absent, and the CTX is bonded to (L1)a-(L2)b-(L3)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), L1 is —(CH2CH2O)p—, L2 is —(CH2)q—, L3 is absent, and the CTX is bonded to (L1)a-(L2)b-(L3)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1 is independently selected from the group consisting of —(CH2CH2O)pCH2CH2— and —CH2CH2—(CH2CH2O)p—, L2 is absent, L3 is absent, and the CTX is bonded to (L1)a-(L2)b-(L3)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1 is independently selected from the group consisting of —(CH2)q—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and —C(O)—, L2 is Val-Cit, L3 is PAB, and the CTX is bonded to (L)a-(L2)b-(L3)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1 is independently selected from the group consisting of —(CH2)q—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and —C(O)—, L2 is Val-Cit, L3 is PAB, and the CTX is bonded to (L1)a-(L2)b-(L3)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), each L1 is independently selected from the group consisting of —(CH2)q—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and —C(O)—, L2 is Val-Ala, L3 is PAB, and the CTX is bonded to (L)a-(L2)b-(L3)c via an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), CTX is bonded to (L1)a-(L2)b-(L3)c via an amide, an N—(C1-6 alkyl)amide, a carbamate, an N—(C1-6alkyl)carbamate, an amine, an N—(C1-6 alkyl)amine, an ether, a thioether, an urea, an N—(C1-6 alkyl)urea, or an N,N-di(C1-6 alkyl)urea bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), CTX is bonded to (L1)a-(L2)b-(L3)c via a group selected from —NHC(O)—, —NHC(O)O—, —N(C1-3alkyl)C(O)O—, —NH—, —N(C1-3alkyl)-, —N(C1-3alkyl)C(O)NH— and —N(C1-3alkyl)C(O)N(C1-3alkyl)-.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), when (L1)a-(L2)b-(L3)c together is —(CH2)1-12— or —(CH2CH2O)1-12CH2CH2— then L1, L2 and L3 are not bonded to CTX by an amide bond.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), CTX is bonded to (L1)a-(L2)b-(L3)c via a bond selected from the group consisting of:

wherein each RB is independently branched or unbranched C1-6 alkyl.

In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), the CTX is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), a pyrrolobenzodiazepine (PDB), calicheamicin γ, mertansine, or tubulysin T2. In certain embodiments of the linker-cytotoxin conjugate of formula (II) or (II′), the CTX is MMAE or MMAF.

In certain embodiments, the CTX is a PDB. In certain embodiments, the CTX is tubulysin T2. In certain embodiments, the CTX is tubulysin T3 or tubulysin T4.

The Antibody (A):

In certain embodiments, disclosed herein are antibodies or antibody fragments (A) for use in the ADCs disclosed herein.

In certain embodiments, A is an antibody or an antibody fragment. In certain embodiments, A is a monoclonal antibody or monoclonal antibody fragment.

In certain embodiments, the antibody (A) is a monoclonal antibody or a humanized antibody. In certain embodiments, the antibody is specific to a cancer antigen. In another embodiment, the antibody employed in the ADC of the present application is selected from the group consisting of alemtuzumab, bevacizumab, cetuximab, ipilimumab, ofatumumab, anitumumab, rituximab, tositumomab, inotuzumab, glembatumumab, lovortuzumab, milatuzumab and trastuzumab.

The Antibody-Drug Conjugate (ADC):

In another aspect, provided herein is antibody-drug conjugate of the following formula (III):

or pharmaceutically acceptable salt thereof,
wherein:

  • A is an antibody or antibody fragment;
  • the two depicted cysteine residues are from an opened cysteine-cysteine disulfide bond in A;
  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-6 alkyl;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-6 alkyl;
  • CTX is a cytotoxin;
  • R is any chemical group; or R is absent;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each k and k′ is independently an integer of 0 or 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • each AA is independently an amino acid;
  • each r is 1 to 12;
  • m is an integer of 1 to 4;
  • n is an integer of 1 to 4; and
  • the bond represents a single or a double bond.

In certain embodiments of the antibody-drug conjugate of formula (III), k and k′ are both 1.

In another aspect, provided herein is antibody-drug conjugate of the following formula (III′):

wherein:

  • A is an antibody or antibody fragment;
  • the two depicted cysteine residues are from an opened cysteine-cysteine disulfide bond in A;
  • each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-6 alkyl;
  • W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-6alkyl;
  • CTX is a cytotoxin;
  • R is any chemical group, provided that R is not OH;
  • each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl;
  • a, b and c are each independently 0, 1, 2 or 3, provided that at least one of a, b or c is 1;
  • each p is independently an integer of 1 to 14;
  • each q is independently an integer or 1 to 12;
  • each AA is independently an amino acid;
  • each r is 1 to 12;
  • m is an integer of 1 to 4;
  • n is an integer of 1 to 4; and
  • the bond represents a single or a double bond.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), the bond represents a single bond. In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), the bond represents a double bond.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), m is 1.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), X and X′ are O.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), W is —NH— or —N(R1)—, wherein R1 is C1-6alkyl. In certain embodiments, W is —NH— or —N(R1)—, wherein R1 is C1-3alkyl. In certain embodiments, W is —CH2NH— or —CH2N(R1)—, wherein R1 is C1-6 alkyl. In certain embodiments, W is —CH2NH— or —CH2N(R1)—, wherein R1 is C1-6alkyl. In certain embodiments, W is —CH2—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2), wherein R2 is C1-6 alkyl. In certain embodiments, W is —CH2—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2), wherein R2 is C1-3 alkyl.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), X and X′ are O, and W is —NH—.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined herein. In certain embodiments, R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, and W-(L1)a-(L2)b-(L3)c. In certain embodiments, R is selected from the group consisting of Z, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), R is selected from the group consisting of W-(L1)a-(L2)b-(L3)c-(CTX)m.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), R is a detectable probe. In certain embodiments, R is a fluorophore, chromophore, radiolabel, enzyme, ligand, antibody or antibody fragment. In certain embodiments, R is a ligand (e.g., a ligand specific for a receptor on a tumor cell, such as a prostate specific membrane antigen, or a virally infected cell, such as an HIV infected cell).

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), R is bonded to the rest of the linker molecule via an amide, an N—(C1-6 alkyl)amide, a carbamate, an N—(C1-6 alkyl)carbamate, an amine, an N—(C1-6 alkyl)amine, an ether, a thioether, an urea, an N—(C1-6 alkyl)urea, or an N,N-di(C1-6 alkyl)urea bond.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2 and -(AA)r-; a, b and c are each independently 0, 1 or 2; each p, q and r is independently 1, 2, 3 or 4; and n is an integer of 1 to 4.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —OCH(CH2O—)2 and —C(O)NCH3—; a, b and c are each independently 0, 1 or 2; each p and q is independently 1 or 2; and n is an integer of 1 to 4.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), each L1, L2 and L3 is independently selected from the group consisting of —NH(CH2)2NH—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —OCH(CH2O—)2 and —C(O)NCH3—; a, b and c are each independently 0 or 1; and n is an integer of 1 to 4.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), each L1, L2 and L3 is independently selected from the group consisting of —NHC(O)—, —C(O)NH—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2 and -(AA)r-; a, b and c are each independently 0 or 1; each p and r is independently 1, 2 or 3; and n is an integer of 1 to 4.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), each AA is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. In one variation of the above, (AA)r is a single amino acid selected from the group consisting of Gly, Arg, Val, Ala, Cys, Gln, Leu, Ile, Leu, Lys and Ser or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Gly-Gly, Gly-Arg, Gly-Val, Gly-Ala, Gly-Cys, Gly-Gln, Gly-Ile, Leu, Lys-Leu, Gly-Lys and Gly-Ser or their N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Gly-Gly-Gly, Gly-Arg-Gly, 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 N-methylated analogues. In another variation of the above, (AA)r is selected from the group consisting of Ala-Ala, Ala-Gly, Ala-Arg, Ala-Val, Ala-Ala, Ala-Cys, Ala-Gln, Ala-Ile, Ala-Leu, Ala-Lys and Ala-Ser or their N-methylated analogues. In another variation of the above, (AA)r 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 or their N-methylated analogues.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), CTX is bonded to (L1)a-(L2)b-(L3)c via a group selected from —NHC(O)—, —NHC(O)O—, —N(C1-3alkyl)C(O)O—, —NH—, —N(C1-3alkyl)-, —N(C1-3alkyl)C(O)NH— and —N(C1-3alkyl)C(O)N(C1-3alkyl)-.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), when (L1)a-(L2)b-(L3)c together is —(CH2)1-12— or —(CH2CH2O)1-12CH2CH2— then L1, L2 and L3 are not bonded to CTX by an amide bond.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), CTX is bonded to (L1)a-(L2)b-(L3)c via a bond selected from the group consisting of:

wherein each RB is independently branched or unbranched C1-6 alkyl.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), A is an antibody that is specific to a cancer antigen. In certain embodiments, A is selected from the group consisting of alemtuzumab, anitumumab, bevacizumab, brentuximab, cetuximab, gemtuzumab, glembatumumab, inotuzumab, ipilimumab, lovortumumab, milatuzumab, ofatumumab, rituximab, tositumomab, and trastuzumab.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), CTX is selected from a from the group consisting of a tubulin stabilizer, a tubulin destabilizer, a DNA alkylator, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an anti-metabolite. In certain embodiments, the CTX is selected from the group consisting of Actinomycin D, Amonafide, an aurstatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade®, and Vincristine. In certain embodiments, the CTX is an auristatin, a calicheamicin, a maytansinoid, or a tubulysin. In certain embodiments, the CTX is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), a pyrrolobenzodiazepine (PDB), calicheamicin γ, mertansine, or tubulysin T2.

In certain embodiments of the antibody-drug conjugate of formula (III) or (III′), the CTX is MMAE or MMAF. In certain embodiments, the CTX is a PDB. In certain embodiments, the CTX is tubulysin T2. In certain embodiments, the CTX is tubulysin T3 or tubulysin T4.

The following tables illustrate embodiments of the linkers (Tables 1 and 4), linker-conjugates (Tables 2 and 5) and the ADCs (Tables 3 and 6) disclosed herein.

TABLE 1 Linkers (I) Entry Y, Y′ X, X′ W L1 L2 L3 Z 1 single halo O or S —NH— or — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 2 single halo O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 3 single halo O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 4 single halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 5 single halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 6 single halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 7 single halo O or S —NH— or — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 8 single halo O or S —NH— or — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 9 single halo O or S —NH— or — —(CH2CH2O)— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 10 single halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 11 single halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 12 single halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 13 single halo O or S —NH— or — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 14 single halo O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 15 single halo O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 16 single halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 17 single halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 18 single halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 19 single halo O or S —CH2—, — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 20 single halo O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 21 single halo O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 22 single halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 23 single halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 24 single halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 25 single halo O or S —CH2—, — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 26 single halo O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 27 single halo O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 28 single halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 29 single halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 30 single halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 31 single halo O or S —CH2—, — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 32 single halo O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 33 single halo O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 34 single halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 35 single halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 36 single halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 37 single substituted O or S —NH— or — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 38 single substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 39 single substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 40 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 41 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 42 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 43 single substituted O or S —NH— or — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 44 single substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 45 single substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 46 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 47 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 48 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 49 single substituted O or S —NH— or — —(CH2)2— —NCHsC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 50 single substituted O or S —NH— or — —(CH2CH2O)12— —NCHsC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 51 single substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 52 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCHsC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 53 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 54 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCHsC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 55 single substituted O or S —CH2—, — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 56 single substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 57 single substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 58 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 59 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 60 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 61 single substituted O or S —CH2—, — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 62 single substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 63 single substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 64 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 65 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 66 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 67 single substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 68 single substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 69 single substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 70 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 71 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 72 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 73 single substituted O or S —NH— or — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 74 single substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 75 single substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 76 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 77 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 78 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 79 single substituted O or S —NH— or — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 80 single substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 81 single substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 82 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 83 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 84 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 85 single substituted O or S —NH— or — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 86 single substituted O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 87 single substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 88 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 89 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 90 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 91 single substituted O or S —CH2—, — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 92 single substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 93 single substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 94 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 95 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 96 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 97 single substituted O or S —CH2—, — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 98 single substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 99 single substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 100 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 101 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 102 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 103 single substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 104 single substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 105 single substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 106 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 107 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 108 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 109 double halo O or S —NH— or — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 110 double halo O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 111 double halo O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 112 double halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 113 double halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 114 double halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 115 double halo O or S —NH— or — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 116 double halo O or S —NH— or — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 117 double halo O or S —NH— or — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 118 double halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 119 double halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 120 double halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 121 double halo O or S —NH— or — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 122 double halo O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 123 double halo O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 124 double halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 125 double halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 126 double halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —N(R1)— —NH—R3a, or —CO2R3b 127 double halo O or S —CH2—, — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 128 double halo O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 129 double halo O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 130 double halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 131 double halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 132 double halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 133 double halo O or S —CH2—, — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 134 double halo O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 135 double halo O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 136 double halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 137 double halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 138 double halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 139 double halo O or S —CH2—, — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 140 double halo O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 141 double halo O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 142 double halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 143 double halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 144 double halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 145 double substituted O or S —NH— or — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 146 double substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 147 double substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 148 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 149 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 150 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 151 double substituted O or S —NH— or — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 152 double substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 153 double substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 154 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 155 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 156 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 157 double substituted O or S —NH— or — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 158 double substituted O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 159 double substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 160 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 161 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 162 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —N(R1)— —NH—R3a, or —CO2R3b 163 double substituted O or S —CH2—, — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 164 double substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 165 double substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 166 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 167 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 168 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 169 double substituted O or S —CH2—, — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 170 double substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 171 double substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 172 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 173 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 174 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 175 double substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 176 double substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 177 double substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 178 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 179 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 180 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, thiol —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 181 double substituted O or S —NH— or — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 182 double substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 183 double substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 184 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 185 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 186 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 187 double substituted O or S —NH— or — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 188 double substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 189 double substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 190 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 191 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 192 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 193 double substituted O or S —NH— or — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 194 double substituted O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 195 double substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 196 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 197 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 198 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —N(R1)— —NH—R3a, or —CO2R3b 199 double substituted O or S —CH2—, — —(CH2)2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 200 double substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 201 double substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 202 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 203 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 204 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 205 double substituted O or S —CH2—, — —(CH2)2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 206 double substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 207 double substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 208 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 209 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 210 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 211 double substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 212 double substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 213 double substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 214 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 215 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— 216 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— —CO2H, —NH2, —OH, sulfonate —CH2CH2—, —NH—R3a, or —CO2R3b —CH(R2)—, or —CH2CH(R2)— For all Entries 1-216 provided in Table 1 above, R, k, k′, a, b and c may be defined as follows: R: W, (L1)a, (L2)b, (L3)c, Z, W—(L1)a—(L2)b—(L3)c, (L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c—Z, a detectable probe, a ligand, or an antibody fragment k, k′: each independently 0 or 1 a, b and c: each independently 0, 1,2 or 3, provided that at least one of a, b or c is 1

TABLE 2 Linker-Cytotoxin Conjugates (II) Entry Y, Y′ X, X′ W L1 L2 L3 Bond to CTX 1 single halo O or S —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 2 single halo O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 3 single halo O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 4 single halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 5 single halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 6 single halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 7 single halo O or S —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 8 single halo O or S —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 9 single halo O or S —NH— or — —(CH2CH2O)— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 10 single halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 11 single halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 12 single halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 13 single halo O or S —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 14 single halo O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 15 single halo O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 16 single halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 17 single halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 18 single halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 19 single halo O or S —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 20 single halo O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 21 single halo O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 22 single halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 23 single halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 24 single halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 25 single halo O or S —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 26 single halo O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 27 single halo O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 28 single halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 29 single halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 30 single halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 31 single halo O or S —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 32 single halo O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 33 single halo O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 34 single halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 35 single halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 36 single halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 37 single substituted O or S —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 38 single substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 39 single substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 40 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 41 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 42 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 43 single substituted O or S —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 44 single substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 45 single substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 46 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 47 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 48 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 49 single substituted O or S —NH— or — —(CH2)2— —NCHsC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 50 single substituted O or S —NH— or — —(CH2CH2O)12— —NCHsC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 51 single substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 52 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCHsC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 53 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 54 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCHsC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 55 single substituted O or S —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 56 single substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 57 single substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 58 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 59 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 60 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 61 single substituted O or S —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 62 single substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 63 single substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 64 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 65 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 66 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 67 single substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 68 single substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 69 single substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 70 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 71 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 72 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 73 single substituted O or S —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 74 single substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 75 single substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 76 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 77 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 78 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 79 single substituted O or S —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 80 single substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 81 single substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 82 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 83 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 84 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 85 single substituted O or S —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 86 single substituted O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 87 single substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 88 single substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 89 single substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 90 single substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 91 single substituted O or S —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 92 single substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 93 single substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 94 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 95 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 96 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 97 single substituted O or S —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 98 single substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 99 single substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 100 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 101 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 102 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 103 single substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 104 single substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 105 single substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 106 single substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 107 single substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 108 single substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 109 double halo O or S —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 110 double halo O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 111 double halo O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 112 double halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 113 double halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 114 double halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 115 double halo O or S —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 116 double halo O or S —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 117 double halo O or S —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 118 double halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 119 double halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 120 double halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 121 double halo O or S —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 122 double halo O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 123 double halo O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 124 double halo O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 125 double halo O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 126 double halo O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 127 double halo O or S —CH2—, —(CH2)2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 128 double halo O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 129 double halo O or S —CH2—, —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 130 double halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 131 double halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 132 double halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 133 double halo O or S —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 134 double halo O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 135 double halo O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 136 double halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 137 double halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 138 double halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 139 double halo O or S —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 140 double halo O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 141 double halo O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 142 double halo O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 143 double halo O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 144 double halo O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 145 double substituted O or S —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamides thoil —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 146 double substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 147 double substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 148 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 149 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 150 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 151 double substituted O or S —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 152 double substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 153 double substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 154 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 155 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 156 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 157 double substituted O or S —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 158 double substituted O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 159 double substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 160 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 161 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 162 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 163 double substituted O or S —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 164 double substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 165 double substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 166 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 167 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 168 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 169 double substituted O or S —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 170 double substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 171 double substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 172 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 173 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 174 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 175 double substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 176 double substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 177 double substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 178 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 179 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 180 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides thiol —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 181 double substituted O or S —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 182 double substituted O or S —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 183 double substituted O or S —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 184 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 185 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 186 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 187 double substituted O or S —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 188 double substituted O or S —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 189 double substituted O or S —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 190 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 191 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 192 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 193 double substituted O or S —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 194 double substituted O or S —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 195 double substituted O or S —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 196 double substituted O or S —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 197 double substituted O or S —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 198 double substituted O or S —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonte —N(R1)— carbamate, N-alkylcarbamate, amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 199 double substituted O or S —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 200 double substituted O or S —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 201 double substituted O or S —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 202 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 203 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 204 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 205 double substituted O or S —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 206 double substituted O or S —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 207 double substituted O or S —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 208 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 209 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 210 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 211 double substituted O or S —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 212 double substituted O or S —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 213 double substituted O or S —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 214 double substituted O or S —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 215 double substituted O or S —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea 216 double substituted O or S —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamides sulfonate —CH2CH2—, carbamate, —CH(R2)—, or N-alkylcarbamate, —CH2CH(R2)— amine, N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea For all Entries 1-216 provided in Table 2 above, CTX, R, k, k′, a, b, and c may be defined as follows: CTX: Actinomycin D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Corobretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade ® , or Vincristine R: W, (L1)a, (L2)b, (L3)c, Z, W—(L1)a—(L2)b—(L3)c, (L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c—CTX, a detectable probe, a ligand, or an antibody fragment k, k′: each independently 0 or 1 a, b and c: each independently 0, 1,2 or 3, provided that at least one of a, b or c is 1 m: an integer of 1 to 4

TABLE 3 Antibody-Drug Conjugates (III) En- X, try A X′ W L1 L2 L3 Bond to CTX  1 single antibody or O —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  2 single antibody or O —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  3 single antibody or O —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  4 single antibody or O —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  5 single antibody or O —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  6 single antibody or O —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  7 single antibody or O —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  8 single antibody or O —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ)  9 single antibody or O —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 10 single antibody or O —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 11 single antibody or O —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 12 single antibody or O —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 13 single antibody or O —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 14 single antibody or O —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 15 single antibody or O —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 16 single antibody or O —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 17 single antibody or O —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 18 single antibody or O —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 19 single antibody or O —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 20 single antibody or O —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 21 single antibody or O —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 22 single antibody or O —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 23 single antibody or O —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 24 single antibody or O —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 25 single antibody or O —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 26 single antibody or O —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 27 single antibody or O —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 28 single antibody or O —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 29 single antibody or O —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 30 single antibody or O —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 31 single antibody or O —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 32 single antibody or O —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 33 single antibody or O —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 34 single antibody or O —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 35 single antibody or O —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 36 single antibody or O —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 37 double antibody or O —NH— or — —(CH2)2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 38 double antibody or O —NH— or — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 39 double antibody or O —NH— or — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 40 double antibody or O —NH— or —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 41 double antibody or O —NH— or —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 42 double antibody or O —NH— or —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 43 double antibody or O —NH— or — —(CH2)2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 44 double antibody or O —NH— or — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 45 double antibody or O —NH— or — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 46 double antibody or O —NH— or —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 47 double antibody or O —NH— or —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 48 double antibody or O —NH— or —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 49 double antibody or O —NH— or — —(CH2)2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 50 double antibody or O —NH— or — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 51 double antibody or O —NH— or — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 52 double antibody or O —NH— or —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 53 double antibody or O —NH— or —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 54 double antibody or O —NH— or —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —N(R1)— carbamate, fragment (e.g., S N-alkylcarbamate, ATZ, ATM, amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 55 double antibody or O —CH2—, — —(CH2)2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 56 double antibody or O —CH2—, — —(CH2CH2O)12— —CH2CH2C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 57 double antibody or O —CH2—, — —(CH2CH2O)6— —CH2CH2C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 58 double antibody or O —CH2—, —(CH2)2CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 59 double antibody or O —CH2—, —(CH2)4CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 60 double antibody or O —CH2—, —(CH2)5CO— —NHCH2CH2— —OC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 61 double antibody or O —CH2—, — —(CH2)2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 62 double antibody or O —CH2—, — —(CH2CH2O)12— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 63 double antibody or O —CH2—, — —(CH2CH2O)6— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 64 double antibody or O —CH2—, —(CH2)2CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 65 double antibody or O —CH2—, —(CH2)4CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 66 double antibody or O —CH2—, —(CH2)5CO— —NHCH2CH2— —NHC(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 67 double antibody or O —CH2—, — —(CH2)2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 68 double antibody or O —CH2—, — —(CH2CH2O)12— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 69 double antibody or O —CH2—, — —(CH2CH2O)6— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 70 double antibody or O —CH2—, —(CH2)2CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 71 double antibody or O —CH2—, —(CH2)4CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) 72 double antibody or O —CH2—, —(CH2)5CO— —NHCH2CH2— —NCH3C(O)— amide, N-alkylamide, antibody or —CH2CH2—, carbamate, fragment (e.g., S —CH(R2)—, or N-alkylcarbamate, ATZ, ATM, —CH2CH(R2)— amine, N-alkylamine, BCZ, BTX, ether, thioether, CTX, GTZ, urea, N-alkylurea, or GBT, ITZ, ILM, N,N-dialkylurea LVT, MTZ, OTM, RTX, TTM, or TTZ) For all Entries 1-72 provided in Table 3 above, CTX, R, k, k′, a, b, c, and n may be defined as follows: CTX: Actinomycin D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade ®, or Vincristine R: W, (L1)a, (L2)b, (L3)c, Z, W—(L1)a—(L2)b—(L3)c, (L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c—CTX, a detectable probe, a ligand, or an antibody fragment k, k′: each independently 0 or 1 a, b and c: each independently 0, 1, 2 or 3, provided that at least one of a, b or c is 1 m: an integer of 1 to 4 n: an integer of 1 to 4 Abbreviations for “A” are as follows: alemtuzumab (ATZ), anitumumab (ATM), bevacizumab (BCZ), brentuximab (BTX), cetuximab (CTX), gemtuzumab (GTZ), glembatumumab (GBT), inotuzumab (ITZ), ipilimumab (ILM), lovortumumab (LVT), milatuzumab (MTZ), ofatumumab (OTM), rituximab (RTX), tositumomab (TTM), and trastuzumab (TTZ)

TABLE 4 Linkers (I) En- X, try Y, Y′ X′ W (L1)a L2 L3 Z 1 single halo O or —NH— or —N(R1)— —(CH2)n— — — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 2 single halo O or —NH— or —N(R1)— —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 3 single halo O or —NH— or —N(R1)— —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 4 single halo O or —NH— or —N(R1)— —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 5 single halo O or —NH— or —N(R1)— —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b S -Val-Cit- 6 single halo O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S (OCH2CH2)n— -Val-Cit- CO— 7 single halo O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S (CH2CH2O)n— -Val-Cit- CO— 8 single halo O or —NH— or —N(R1)— —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b S (CH2)n—CO— -Val-Cit- 9 single halo O or —CH2—, —CH2CH2—, —(CH2)n— — — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 10 single halo O or —CH2—, —CH2CH2—, —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 11 single halo O or —CH2—, —CH2CH2—, —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 12 single halo O or —CH2—, —CH2CH2—, —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 13 single halo O or —CH2—, —CH2CH2—, —(OCH2CH2)n— —(CH2)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 14 single halo O or —CH2—, —CH2CH2—, —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, -Val-Cit- or —CH2CH(R2)— 15 single halo O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (OCH2CH2)n— -Val-Cit- or —CH2CH(R2)— CO— 16 single halo O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (CH2CH2O)n— -Val-Cit- or —CH2CH(R2)— CO— 17 single halo O or —CH2—, —CH2CH2—, —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 18 single halo O or —CH2—, —CH2CH2—, —(OCH2CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 19 single substituted O or —NH— or —N(R1)— —(CH2)n— — — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 20 single substituted O or —NH— or —N(R1)— —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 21 single substituted O or —NH— or —N(R1)— —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 22 single substituted O or —NH— or —N(R1)— —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 23 single substituted O or —NH— or —N(R1)— —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S -Val-Cit- 24 single substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S (OCH2CH2)n— -Val-Cit- CO— 25 single substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S (CH2CH2O)n— -Val-Cit- CO— 26 single substituted O or —NH— or —N(R1)— —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S (CH2)n—CO— -Val-Cit- 27 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— — — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 28 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 29 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 30 single substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 31 single substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— —(CH2)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or or —CH2CH(R2)— —CO2R3b 32 single substituted O or —CH2—, —CH2CH2—, —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, -Val-Cit- or —CH2CH(R2)— 33 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (OCH2CH2)n— -Val-Cit- or —CH2CH(R2)— CO— 34 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (CH2CH2O)n— -Val-Cit- or —CH2CH(R2)— CO— 35 single substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 36 single substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 37 single substituted O or —NH— or —N(R1)— —(CH2)n— — — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 38 single substituted O or —NH— or —N(R1)— —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 39 single substituted O or —NH— or —N(R1)— —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 40 single substituted O or —NH— or —N(R1)— —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 41 single substituted O or —NH— or —N(R1)— —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S -Val-Cit- 42 single substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S (OCH2CH2)n— -Val-Cit- CO— 43 single substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S (CH2CH2O)n— -Val-Cit- CO— 44 single substituted O or —NH— or —N(R1)— —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S (CH2)n—CO— -Val-Cit- 45 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— — — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 46 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 47 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 48 single substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 49 single substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— —(CH2)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 50 single substituted O or —CH2—, —CH2CH2—, —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, -Val-Cit- or —CH2CH(R2)— 51 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (OCH2CH2)n— -Val-Cit- or —CH2CH(R2)— CO— 52 single substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (CH2CH2O)n— -Val-Cit- or —CH2CH(R2)— CO— 53 single substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 54 single substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 55 double halo O or —NH— or —N(R1)— —(CH2)n— — — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 56 double halo O or —NH— or —N(R1)— —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 57 double halo O or —NH— or —N(R1)— —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 58 double halo O or —NH— or —N(R1)— —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, S —NH—R3a, or —CO2R3b 59 double halo O or —NH— or —N(R1)— —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b S -Val-Cit- 60 double halo O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S (OCH2CH2)n— -Val-Cit- CO— 61 double halo O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S (CH2CH2O)n— -Val-Cit- CO— 62 double halo O or —NH— or —N(R1)— —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b S (CH2)n—CO— -Val-Cit- 63 double halo O or —CH2—, —CH2CH2—, —(CH2)n— — — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 64 double halo O or —CH2—, —CH2CH2—, —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 65 double halo O or —CH2—, —CH2CH2—, —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 66 double halo O or —CH2—, —CH2CH2—, —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 67 double halo O or —CH2—, —CH2CH2—, —(OCH2CH2)n— —(CH2)n— — —CO2H, —NH2, —OH, S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 68 double halo O or —CH2—, —CH2CH2—, —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, -Val-Cit- or —CH2CH(R2)— 69 double halo O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (OCH2CH2)n— -Val-Cit- or —CH2CH(R2)— CO— 70 double halo O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (CH2CH2O)n— -Val-Cit- or —CH2CH(R2)— CO— 71 double halo O or —CH2—, —CH2CH2—, —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 72 double halo O or —CH2—, —CH2CH2—, —(OCH2CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 73 double substituted O or —NH— or —N(R1)— —(CH2)n— — — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 74 double substituted O or —NH— or —N(R1)— —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 75 double substituted O or —NH— or —N(R1)— —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 76 double substituted O or —NH— or —N(R1)— —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, thiol S —NH—R3a, or —CO2R3b 77 double substituted O or —NH— or —N(R1)— —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S -Val-Cit- 78 double substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S (OCH2CH2)n— -Val-Cit- CO— 79 double substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S (CH2CH2O)n— -Val-Cit- CO— 80 double substituted O or —NH— or —N(R1)— —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S (CH2)n—CO— -Val-Cit- 81 double substituted O or —CH2—, —CH2CH2—, —(CH2)n—, — — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 82 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 83 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 84 double substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH2(R2)— 85 double substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— —(CH2)n— — —CO2H, —NH2, —OH, thiol S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 86 double substituted O or —CH2—, —CH2CH2—, —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, -Val-Cit- or —CH2CH(R2)— 87 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (OCH2CH2)n— -Val-Cit- or —CH2CH(R2)— CO— 88 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (CH2CH2O)n— -Val-Cit- or —CH2CH(R2)— CO— 89 double substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 90 double substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b thiol S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 91 double substituted O or —NH— or —N(R1)— —(CH2)n— — — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 92 double substituted O or —NH— or —N(R1)— —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 93 double substituted O or —NH— or —N(R1)— —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 94 double substituted O or —NH— or —N(R1)— —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, sulfonate S —NH—R3a, or —CO2R3b 95 double substituted O or —NH— or —N(R1)— —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S -Val-Cit- 96 double substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S (OCH2CH2)n— -Val-Cit- CO— 97 double substituted O or —NH— or —N(R1)— —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S (CH2CH2O)n— -Val-Cit- CO— 98 double substituted O or —NH— or —N(R1)— —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S (CH2)n—CO— -Val-Cit- 99 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— — — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 100 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(CH2CH2O)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 101 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— —(OCH2CH2)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 102 double substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— —(CH2)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 103 double substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— —(CH2)n— — —CO2H, —NH2, —OH, sulfonate S —CH(R2)—, —NH—R3a, or —CO2R3b or —CH2CH(R2)— 104 double substituted O or —CH2—, —CH2CH2—, —(CH2)nCO— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, -Val-Cit- or —CH2CH(R2)— 105 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (OCH2CH2)n— -Val-Cit- or —CH2CH(R2)— CO— 106 double substituted O or —CH2—, —CH2CH2—, —(CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (CH2CH2O)n— -Val-Cit- or —CH2CH(R2)— CO— 107 double substituted O or —CH2—, —CH2CH2—, —(CH2CH2O)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— 108 double substituted O or —CH2—, —CH2CH2—, —(OCH2CH2)n— -Val-Ala- or PAB —CO2H or —CO2R3b sulfonate S —CH(R2)—, (CH2)n—CO— -Val-Cit- or —CH2CH(R2)— For all Entries 1-108 provided in Table 1 above, R, k, k′, a, b and c may be defined as follows: R: W, (L1)a, (L2)b, (L3)c, Z, W—(L1)a—(L2)b—(L3)c, (L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c—Z, a detectable probe, a ligand, or an antibody fragment k, k′: each independently 0 or 1 a, b and c: each independently 0, 1, 2 or 3, provided that at least one of a, b or c is 1

TABLE 5 Linker-Cytotoxin Conjugates (II) En- X, try Y, Y′ X′ W (L1)a L2 L3 Bond to CTX  1 single halo O —NH— or —(CH2)n— — — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea  2 single halo O —NH— or —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea  3 single halo O —NH— or —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea  4 single halo O —NH— or —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea  5 single halo O —NH— or —(CH2)nCO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  6 single halo O —NH— —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  7 single halo O —NH— —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  8 single halo O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala or PAB amide or —N(R1)— -Val-Cit- S  9 single halo O —CH2—, —(CH2)n— — — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, —CH2CH(R2)— thioether, urea, N-alkylurea, or N,N-dialkylurea  10 single halo O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, —CH2CH(R2)— thioether, urea, N-alkylurea, or N,N-dialkylurea  11 single halo O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, —CH2CH(R2)— ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  12 single halo O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, —CH2CH(R2)— thioether, urea, N-alkylurea, or N,N-dialkylurea  13 single halo O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, —CH2CH(R2)— ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  14 single halo O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  15 single halo O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  16 single halo O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  17 single halo O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  18 single halo O —CH2—, —(OCH2CH2)n—(CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  19 single substituted O —NH— or —(CH2)n— — — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  20 single substituted O —NH— or —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  21 single substituted O —NH— or —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  22 single substituted O —NH— or —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  23 single substituted O —NH— —(CH2)nCO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  24 single substituted O —NH— —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  25 single substituted O —NH— —(CH)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide thio or —N(R1)— -Val-Cit- S  26 single substituted O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  27 single substituted O —CH2—, —(CH2)n— — — amide, N-alkylamide, carbamate, thio or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  28 single substituted O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, thio or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  29 single substituted O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, thio or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  30 single substituted O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, thio or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  31 single substituted O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, thio or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  32 single substituted O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  33 single substituted O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  34 single substituted O —CH2—, —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  35 single substituted O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  36 single substituted O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  37 single substituted O —NH— —(CH2)n— — — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  38 single substituted O —NH— —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  39 single substituted O —NH— —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  40 single substituted O —NH— —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  41 single substituted O —NH— or —(CH2)nCO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  42 single substituted O —NH— or —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  43 single substituted O —NH— or —(CH2)n—(CH2CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  44 single substituted O —NH— or —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  45 single substituted O —CH2—, —(CH2)n— — — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  46 single substituted O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  47 single substituted O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  48 single substituted O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  49 single substituted O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  50 single substituted O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  51 single substituted O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  52 single substituted O —CH2—, —(CH2)n—(CH2CH2O)nCO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  53 single substituted O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  54 single substituted O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  55 double halo O —NH— —(CH2)n— — — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  56 double halo O —NH— —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  57 double halo O —NH— —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  58 double halo O —NH— —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  59 double halo O —NH— —(CH2)nCO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  60 double halo O —NH— —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  61 double halo O —NH— —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  62 double halo O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide or —N(R1)— -Val-Cit- S  63 double halo O —CH2—, —(CH2)n— — — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  64 double halo O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  65 double halo O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  66 double halo O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  67 double halo O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  68 double halo O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  69 double halo O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  70 double halo O —CH2—, —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  71 double halo O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  72 double halo O —CH2—, —(OCH2CH2)n—(CH2)n—CO— -Val-Ala- or PAB amide or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  73 double substituted O —NH— —(CH2)n— — — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  74 double substituted O —NH— —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  75 double substituted O —NH— —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  76 double substituted O —NH— —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, thiol or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  77 double substituted O —NH— —(CH2)nCO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  78 double substituted O —NH— —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  79 double substituted O —NH— —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  80 double substituted O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide thiol or —N(R1)— -Val-Cit- S  81 double substituted O —CH2—, —(CH2)n— — — amide, N-alkylamide, carbamate, thiol or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  82 double substituted O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, thiol or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  83 double substituted O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, thiol or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  84 double substituted O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, thiol or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  85 double substituted O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, thiol or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea  86 double substituted O —CH2—, —(CH2)nCO— -Val-Ala- PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  87 double substituted O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  88 double substituted O —CH2—, —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  89 double substituted O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  90 double substituted O —CH2—, —(CH2)nCO— -Val-Ala- PAB amide thiol or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)—  91 double substituted O —NH— —(CH2)n— — — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  92 double substituted O —NH— —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  93 double substituted O —NH— —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  94 double substituted O —NH— —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —N(R1)— N-alkylcarbamate, amine, S N-alkylamine, ether, thioether, urea, N-alkylurea, or N,N-dialkylurea  95 double substituted O —NH— —(CH2)nCO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  96 double substituted O —NH— —(CH2)n—(OCH2CH2)n— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  97 double substituted O —NH— —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  98 double substituted O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —N(R1)— -Val-Cit- S  99 double substituted O —CH2—, —(CH2)n— — — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea 100 double substituted O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea 101 double substituted O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea 102 double substituted O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea 103 double substituted O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, carbamate, sulfonate or —CH2CH2—, N-alkylcarbamate, amine, S —CH(R2)—, or N-alkylamine, ether, thioether, urea, —CH2CH(R2)— N-alkylurea, or N,N-dialkylurea 104 double substituted O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)— 105 double substituted O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)— 106 double substituted O —CH2—, —(CH2)n—(CH2CH2O)n— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)— 107 double substituted O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)— 108 double substituted O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide sulfonate or —CH2CH2—, -Val-Cit- S —CH(R2)—, or —CH2CH(R2)— For all Entries 1-108 provided in Table 2 above, CTX, R, k, k′, a, b, and c may be defined as follows: CTX: Actinomycin D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC (298223), Cemadotin, Colchicine, Combrestatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade ®, or Vincristine R: W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a—(L2)b—(L3)c, (L1)a—(L2)b—(L3)c—Z, W-(L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c-CTX, a detectable probe, a ligand, or an antibody fragment k,k′: each independently 0 or 1 a, b, c: each independently 0, 1, 2 or 3, provided that at least one of a, b or c is 1 m: an integer of 1 to 4

TABLE 6 Antibody-Drug Conjugates (III) En- X, try A X′ W (L1)a L2 L3 Bond to CTX 1 single antibody or antibody O —NH— or —(CH2)n— — — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 2 single antibody or antibody O —NH— or —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 3 single antibody or antibody O —NH— or —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 4 single antibody or antibody O —NH— or —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 5 single antibody or antibody O —NH— —(CH2)nCO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 6 single antibody or antibody O —NH— —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 7 single antibody or antibody O —NH— —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 8 single antibody or antibody O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 9 single antibody or antibody O —CH2—, —(CH2)n— — — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 10 single antibody or antibody O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 11 single antibody or antibody O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 12 single antibody or antibody O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 13 single antibody or antibody O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 14 single antibody or antibody O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, TTZ) 15 single antibody or antibody O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, TTZ) 16 single antibody or antibody O —CH2—, —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, TTZ) 17 single antibody or antibody O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, TTZ) 18 single antibody or antibody O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, TTZ) 19 double antibody or antibody O —NH— —(CH2)n— — — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 20 double antibody or antibody O —NH— —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 21 double antibody or antibody O —NH— —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 22 double antibody or antibody O —NH— —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —N(R1)— carbamate, BCZ, BTX, CTX, GTZ, S N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 23 double antibody or antibody O —NH— —(CH2)nCO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 24 double antibody or antibody O —NH— —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 25 double antibody or antibody O —NH— —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 26 double antibody or antibody O —NH— —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —N(R1)— -Val-Cit- BCZ, BTX, CTX, GTZ, S GBT, ITZ, ILM, LVT, MTZ, OTM, RTX, TTM, or TTZ) 27 double antibody or antibody O —CH2—, —(CH2)n— — — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 28 double antibody or antibody O —CH2—, —(CH2)n— —(CH2CH2O)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 29 double antibody or antibody O —CH2—, —(CH2)n— —(OCH2CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 30 double antibody or antibody O —CH2—, —(CH2CH2O)n— —(CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 31 double antibody or antibody O —CH2—, —(OCH2CH2)n— —(CH2)n— — amide, N-alkylamide, fragment (e.g., ATZ, ATM, or —CH2CH2—, carbamate, BCZ, BTX, CTX, GTZ, S —CH(R2)—, or N-alkylcarbamate, GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— amine, N-alkyl- OTM, RTX, TTM, or TTZ) amine, ether, thioether, urea, N-alkylurea, or N,N- dialkylurea 32 double antibody or antibody O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, or TTZ) 33 double antibody or antibody O —CH2—, —(CH2)n—(OCH2CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, or TTZ) 34 double antibody or antibody O —CH2—, —(CH2)n—(CH2CH2O)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, or TTZ) 35 double antibody or antibody O —CH2—, —(CH2CH2O)n—(CH2)n—CO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, or TTZ) 36 double antibody or antibody O —CH2—, —(CH2)nCO— -Val-Ala- or PAB amide fragment (e.g., ATZ, ATM, or —CH2CH2—, -Val-Cit- BCZ, BTX, CTX, GTZ, S —CH(R2)—, or GBT, ITZ, ILM, LVT, MTZ, —CH2CH(R2)— OTM, RTX, TTM, or TTZ) For all entries 1-108 provided in Table 3 above, CTX, R, k, k′, a, b, c, and n may be defined as follows: CTX: Actinomycin D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC (298223), Cemadotin, Colchicine, Combrestatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade ®, Vincristine R: W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a—(L2)b—(L3)c, (L1)a—(L2)b—(L3)c—Z, W-(L1)a—(L2)b—(L3)c—Z, W—(L1)a—(L2)b—(L3)c-CTX, a detectable probe, a ligand, or an antibody fragment k,k′: each independently 0 or 1 a, b, c: each independently 0, 1, 2 or 3, provided that at least one of a, b or c is 1 m: an integer of 1 to 4 Abbreviations for “A” are as follows: alemtuzumab (ATZ), anitumumab (ATM), bevacizumab (BCZ), brentuximab (BTX), cetuximab (CTX), gemtuzumab (GTZ), glembatumumab (GBT), inotuzumab (ITZ), ipilimumab (ILM), Iovortumumab (LVT), milatuzumab (MTZ), ofatumumab (OTM), rifuximab (RTX), tositumomab (TTM), and trastuzumb (TTZ)

Assays

The ADCs disclosed herein may be assayed for binding affinity to and specificity for the desired antigen by any of the methods conventionally used for the assay of antibodies; and they may be assayed for efficacy as anticancer agents by any of the methods conventionally used for the assay of cytostatic/cytotoxic agents, such as assays for potency against cell cultures, xenograft assays, and the like. A person of ordinary skill in the art will have no difficulty, considering that skill and the literature available, in determining suitable assay techniques: from the results of those assays, in determining suitable doses to test in humans as anticancer agents, and, from the results of those tests, in determining suitable doses to use to treat cancers in humans.

Formulation and Administration

The ADCs disclosed herein will typically be formulated as solutions for intravenous administration, or as lyophilized concentrates for reconstitution to prepare intravenous solutions (to be reconstituted, e.g., with normal saline, 5% dextrose, or similar isotonic solutions). They will typically be administered by intravenous injection or infusion. A person of ordinary skill in the art of pharmaceutical formulation, especially the formulation of anticancer antibodies, will have no difficulty, considering that skill and the literature available, in developing suitable formulations.

EXAMPLES Synthesis of Linkers

The following procedures may be employed for the preparation of the compounds of the present invention. The starting materials and reagents used in preparing these compounds are either 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 Fiesers Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.: and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

In some cases, protective groups may be introduced and finally removed. Suitable protective groups for amino, hydroxy and carboxy groups are described in Greene et al., Protective Groups in Organic Synthesis, Second Edition, John Wiley and Sons, New York, 1991. Standard organic chemical reactions can be achieved by using a number of different reagents, for examples, as described in Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

While a number of exemplary embodiments, aspects and variations have been provided herein, those of skill in the art will recognize certain modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations. It is intended that the following claims are interpreted to include all such modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations are within their scope.

Example 1—Synthesis of R-substituted 6-(2,3-dibromo-4-(amino)-4-oxobutanamido)hexanoic acid (5)

3,4-dibromodihydrofuran-2,5-dione (1) is reacted with tert-butyl 6-aminohexanoate (2) in anhydrous THF in the presence of TEA to yield 2,3-dibromo-4-((6-(tert-butoxy)-6-oxohexyl)amino)-4-oxobutanoic acid (3).

The purified product (3) is then added to a clean, flame-dried 50 mL round bottom flask, and the free carboxylic acid is coupled to reactant, H2N—R, in 3 mL of CH2Cl2 in the presence of DCC to yield R-substituted tert-butyl 6-(2,3-dibromo-4-(amino)-4-oxobutanamido)hexanoate (4).

To fully deprotect (4) to the free acid, R-substituted 6-(2,3-dibromo-4-(amino)-4-oxobutanamido)hexanoic acid (5), the lyophilized material is treated with 5% TFA in CH2Cl2, concentrated to dryness and lyophilized in acetonitrile:water (50:50) overnight.

tert-butyl 6-aminohexanoate (2) may be substituted in Example 1 with tert-butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate to produce R-substituted 20-amino-18,19-dibromo-17,20-dioxo-4,7,10,13-tetraoxa-16-azaicosan-1-oic acid (6):

Example 2—Synthesis of 6-(2,3-dibromopropanamido)hexanoic acid (10)

2,3-dibromopropanoic acid (7) is added to a clean, flame-dried 50 mL round bottom flask, and the free carboxylic acid is coupled to the free amine of tert-butyl 6-aminohexanoate (8) in 3 mL of CH2Cl2 in the presence of DIPC/DIPEA to yield tert-butyl 6-(2,3-dibromopropanamido)hexanoate (9).

To fully deprotect (9) to the free acid, 6-(2,3-dibromopropanamido)hexanoic acid (10), the lyophilized material is treated with 5% TFA in CH2Cl2, concentrated to dryness and lyophilized in acetonitrile:water (50:50) overnight.

tert-butyl 6-aminohexanoate (8) may be substituted in Example 2 with tert-butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate to produce 1,2-dibromo-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid (11):

Example 3- Synthesis of 7,8-dibromooctanoic acid (13)

Oct-7-enoic acid (12) is treated with 0.5 equivalents of bromine in chloroform followed by refluxing overnight to give 7,8-dibromooctanoic acid (13) after flash purification on silica gel.

Example 4—Synthesis of L1, L2 and L3

General procedures for the synthesis of L1, L2 and L3 may be performed using standard synthetic procedures as described in Larock, above, or Modern Synthetic Reactions, Second Edition, H. O. House, The Benjamin/Cummings Publishing Company, Menlo Park, Calif. 1972; the chemistry of amino acids and peptide synthesis described in The Chemistry of the Amino Acids, J. P. Greenstein, M. Winitz, Robert E. Krieger Publishing Company, Malabar, Fla. 1986, Volumes 1, 2 and 3.

Example 5—Synthesis of 4-((S)-2-((S)-2-(6-(2-bromoacrylamido)hexanamido)-3-methylbutanamido)propanamido)benzyl (4-nitrophenyl) carbonate (23)

(S)-2,5-dioxopyrrolidin-1-yl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanoate (14) was reacted with (S)-tert-butyl 2-aminopropanoate (15) in the presence of 2 equivalents of DIPEA in THF to yield (S)-tert-butyl 2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoate (16). To fully deprotect (16) to the free acid, (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoic acid (17), the lyophilized material was treated with 5% TFA in CH2Cl2. The free carboxylic acid of the purified product (17) was then coupled to (4-aminophenyl)methanol (18), in the presence of 2 equivalents of EEDQ in THE to yield (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (19). The product (19) was treated with 20% piperidine in DMA to yield (S)-2-amino-N—((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (20). Coupling of the product (20) with 6-(2-bromoacrylamido)hexanoic acid (21) was performed by activation with 1 equivalent of TBTU in the presence of 2 equivalents of NMM in DMF for 72 hours at room temperature to produce 6-(2-bromoacrylamido)-N—((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (22). The product (22) was then reacted with 4-nitrophenyl carbonochloridate to produce 4-((S)-2-((S)-2-(6-(2-bromoacrylamido)hexanamido)-3-methylbutanamido)propanamido)benzyl (4-nitrophenyl) carbonate (23).

Synthesis of Linker-Cytotoxin Conjugate Example 6—Synthesis of T4

Fmoc-T4 was prepared by coupling Fmoc-D-2-piperidinecarboxylic acid (24) to isoleucine in the presence of EDC and sodium bicarbonate, then coupling the resulting Fmoc-D-Pip-Ile-OH (25) to the N-methylvaline intermediate (26) (purchased from Concortis) by mixing with 1 equivalent of HOBT and DIPC in DMF followed by addition of 2.5 equivalents of NMM. The reaction mixture was stirred overnight and purified by flash chromatography on silica gel using a gradient of hexane and ethyl acetate. Evaporation of solvent gave Fmoc-T4 (27) as a yellow oil. The Fmoc-T4 was then deprotected by treatment with 20% DEA in methylene chloride for 30 minutes to give T4, which was purified by preparative HPLC on a C18 reverse phase column eluted with acetonitrile/water.

Example 7—Synthesis of R-substituted (2S,4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-2-((2R)-1-(6-(2,3-dibromo-4-(amino)-4-oxobutanamido)hexanoyl)piperidine-2-carboxamido)-N,3-dimethylpentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (28)

Coupling of T4 to the linker described in Example 1 (R-substituted 6-(2,3-dibromo-4-(amino)-4-oxobutanamido)hexanoic acid (5)) was performed by activating the linker with 1 equivalent of HATU in the presence of 2 equivalents of DIPEA in DMF, then coupling with the T4 for 72 hours at room temperature. Another coupling reagent, for example, TBTU may be substituted for HATU to activate the linker. Purification by preparative C18 HPLC (acetonitrile-water gradient) gave a linker-T4 conjugate suitable for conjugation to antibodies.

Similar syntheses using other linkers give the corresponding linker-T4 conjugates. Similar syntheses using T3, MMAF, or other cytotoxins with a basic amine give the corresponding linker-cytotoxin conjugates. Similar syntheses using amine-terminated linkers and cytotoxins with a carboxyl group, activating the cytotoxin in the same manner as the linker was activated in the above Example, give other linker-cytotoxin conjugates.

Example 8—Synthesis of (2S)-2-((2R,3R)-3-((2S)-1-((12S,15S,18S,19R)-1,2-dibromo-18-((R)-sec-butyl)-12,15-diisopropyl-19-methoxy-11,17-dimethyl-3,10,13,16-tetraoxo-4,11,14,17-tetraazahenicosan-21-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (29)

Coupling of MMAF to the linker described in Example 2 (6-(2,3-dibromopropanamido)hexanoic acid (10)) was performed by activating the linker with 1 equivalent of HATU in the presence of 2 equivalents of DIPEA in DMF, then coupling with the MMAF for 72 hours at room temperature. Another coupling reagent, for example, TBTU may be substituted for HATU to activate the linker. Purification by preparative C18 HPLC (acetonitrile-water gradient) gave the linker-MMAF conjugate suitable for conjugation to antibodies.

MMAF generally refers to (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid. Pictured above in Scheme 7 is ((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine.

Similar syntheses using other linkers give the corresponding linker-MMAF conjugates. Similar syntheses using T3, T4, or other cytotoxins with a basic amine give the corresponding linker-cytotoxin conjugates. Similar syntheses using amine-terminated linkers and cytotoxins with a carboxyl group, activating the cytotoxin in the same manner as the linker was activated in the above Example, give other linker-cytotoxin conjugates.

Example 9—Synthesis of (2S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5R)-4-((2S)-2-((2S)-2-(7,8-dibromo-N-methyloctanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (30)

Coupling of MMAF to the linker described in Example 3 (7,8-dibromooctanoic acid (13)) was performed by activating the linker with 1 equivalent of HATU in the presence of 2 equivalents of DIPEA in DMF, then coupling with the MMAF for 72 hours at room temperature. Another coupling reagent, for example, TBTU may be substituted for HATU to activate the linker. Purification by preparative C18 HPLC (acetonitrile-water gradient) gave the linker-MMAF conjugate, (2S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5R)-4-((2S)-2-((2S)-2-(7,8-dibromo-N-methyloctanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (30) suitable for conjugation to antibodies.

Similar syntheses using other linkers give the corresponding linker-MMAF conjugates. Similar syntheses using T3, T4, or other cytotoxins with a basic amine give the corresponding linker-cytotoxin conjugates. Similar syntheses using amine-terminated linkers and cytotoxins with a carboxyl group, activating the cytotoxin in the same manner as the linker was activated in the above Example, give other linker-cytotoxin conjugates.

Example 10—Synthesis of (S)-2-((2R,3R)-3-((S)-1-((12S,15S,18S,19R)-2-bromo-18-((S)-sec-butyl)-12,15-diisopropyl-19-methoxy-11,17-dimethyl-3,10,13,16-tetraoxo-4,11,14,17-tetraazahenicos-1-en-21-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (36)

2,3-dibromopropanoic acid (7) was treated with N-methylmorpholine in CH2Cl2 at room temperature to produce 2-bromoacrylic acid (31), which following treatment with oxalyl chloride in catalytic DMF produced 2-bromoacryloyl chloride (32). The product (32) was reacted with 6-aminohexanoic acid (33) in THF and sodium bicarbonate, followed by absorption on silicate gel in the presence of MeOH, to produce methyl 6-(2-bromoacrylamido)hexanoate (34). Deprotection of (34) with 1N aqueous NaOH in THE produced the free acid, 6-(2-bromoacrylamido)hexanoic acid (35).

Coupling of MMAF to (35) was performed by activation with 2 equivalents of HATU/Hunig's Base in DMF, then coupling with the MMAF for 72 hours at −10° C. to room temperature. Purification by preparative C18 HPLC (acetonitrile-water gradient) gave the linker-MMAF conjugate, (S)-2-((2R,3R)-3-((S)-1-((12S,15S,18S,19R)-2-bromo-18-((S)-sec-butyl)-12,15-diisopropyl-19-methoxy-11,17-dimethyl-3,10,13,16-tetraoxo-4,11,14,17-tetraazahenicos-1-en-21-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (36) suitable for conjugation to antibodies.

Example 11—Synthesis of (S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-1-(4-((R)-2-((R)-2-(6-(2-bromoacrylamido)hexanamido)-3-methylbutanamido)propanamido)phenyl)-11-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecan-14-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (37)

Coupling of MMAF to the linker described in Example 5 (4-((S)-2-((S)-2-(6-(2-bromoacrylamido)hexanamido)-3-methylbutanamido)propanamido)benzyl (4-nitrophenyl) carbonate (23)) was performed by activating the linker with 1 equivalent of DEPC in CH2Cl2, then coupling with the MMAF for 72 hours at room temperature. Purification by preparative C18 HPLC (acetonitrile-water gradient) gave the linker-MMAF conjugate, (S)-2-((2R,3R)-3-((S)-1-((5S,8S,11S,12R)-1-(4-((R)-2-((R)-2-(6-(2-bromoacrylamido)hexanamido)-3-methylbutanamido)propanamido)phenyl)-11-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecan-14-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (37) suitable for conjugation to antibodies.

Example 12—Screening and Optimization of Bifunctional Linkers for ADCs

Antibody-drug conjugates were prepared comprising a linker and cytotoxin as described in Examples 7-11. In some experiments, Herceptin was used in the preparation of ADCs. Exemplary conjugates were analyzed by SDS-PAGE, HIC and LC-MS, as shown in FIGS. 4 and 5.

Bifunctional linkers may be rapidly screened and/or optimized by SDS-PAGE, as illustrated in FIG. 4, for optimal properties (e.g., resulting in 1 drug/payload per 1 disulfide bond).

In some experiments, for SDS-PAGE under non-reducing conditions, sample preparation were as follows: In a 1 ml eppendorf vial was pipetted Novex Tris-Glycine SDS sample buffer (2×) (300 μL) (Life Technologies catalog #LC2676) and deionized water (150 ul). 30 μL of the sample buffer was pipetted into a 1 ml eppendorf vial and 10 μL of the sample was added.

In some experiments, for SDS-PAGE under reducing conditions, sample preparation were as follows: In a 1 ml eppendorf vial was pipetted Novex Tris-Glycine SDS sample buffer (2×) (300 μL) (Life Technologies catalog #LC2676), deionized water (120 μl), and 1 M dithiothreitol (Sigma catalog #D0632). 30 μL of the sample buffer was pipetted into a 1 ml eppendorf vial and 10 μL of the sample was added. The vials were placed in a sand bath at 100° C. for 2 minutes and cooled to ambient temperature.

In some experiments, for SDS-PAGE, running buffer preparation were as follows: Novex Tris-Glycine SDS running buffer (10×) (100 ml) (Life Technologies catalog #LC2675-4) was added to deionized water (900 ml).

In some experiments, denaturing electrophoresis gel was as follows: Life Technologies Novex 4-20% Tris-Glycine midi gel (Life Technologies catalog #WT4201BX10) was placed into a gel box. The running buffer was poured into the gel box until full. To the outermost lanes on the left and right side was added Novex Mark 12™ unstained standard (Life Technologies catalog #LC5677). To each of the interior lanes was added either a non-reducing or reducing sample. The lid was placed onto the gel box and the electric current was started (125 V). Bubbling was observed. The starting amps were ca. 90 mA then falling to ca. 50 mA at the end of the run over ca. a 90 min period.

In some experiments, staining of the gels was as follows: The gels were carefully removed from their enclosures and placed directly into a plastic container for staining. A staining solution consisting of SYPRO Orange protein stain (20 μl) (Life Technologies catalog #S6651) in a 7.5% aqueous glacial acetic acid solution (100 ml) was gently poured over the gel(s) and gently rocked for 30 min. The staining solution was decanted and the gels were gently washed with deionized water and decanted (3×). 7.5% aqueous glacial acetic acid solution (100 ml) was gently poured over the gel(s) and gently rocked for an additional 30 min. The destaining solution was decanted and the gels were gently washed with deionized water and decanted (3×). The gels were imaged using a GE Healthcare Typhoon Trio+ scanner.

Exemplary SDS-PAGE screening of a bifunctional linker as described in Example 12 is shown in FIG. 4. Additional analysis by HIC and LC-MS is shown in FIGS. 4 and 5. Lane 7 in FIG. 4 corresponds to Herceptin conjugated to a a bromocrylamide (BRA) linker, as described herein.

Synthesis of ADC Example 13: Antibody Disulfide Reduction and Linker-Cytotoxin Conjugation to Antibody

This example provides an exemplary protocol for reduction of the disulfides of the antibodies disclosed herein, and conjugation of the reduced antibodies to the linker-cytotoxin conjugates disclosed herein.

Protocol:

Step 1: Antibody Disulfide Reduction

A) Dilute antibody to 15 mg/mL (0.1 mM IgG) in PBS, pH 7.4.

B) Prepare a fresh 20 mM (5.7 mg/mL) stock solution of TCEP in H2O.

C) Add 25 μL of TCEP stock solution from B to 1 mL of antibody from A (0.5 mM final concentration TCEP).

D) Incubate at 37° C. for 2 hours (check for free thiols using DTNB test).

E) Aliquat the reduced antibody into 4 tubes (250 μL each).

Step 1: Linker-Cytotoxin Conjugation to Antibody

A) Prepare 10 mM stock solution of linker-cytotoxin conjugate in DMSO (DMA, DMF or CH3CN are also acceptable).

B) Add 5 equivalents of 12.5 μL stock solution from A to each tube of reduced antibody (0.5 mM final concentration linker-cytotoxin conjugate stock solution).

C) Incubate overnight at 4° C. for 4 hours at room temperature; check for free thiols using DTNB test.

D) Run analytical HIC to determine DAR and homogeneity.

Example 14: Reduction and Purification of Herceptin for Conjugation to Linker-Cytotoxin Conjugate

This example provides an exemplary protocol for reduction and purification of herceptin for conjugation to the linker-cytotoxin conjugates disclosed herein.

Purpose: Determine the effect of purifying reduced antibody on conjugation efficiency.

Protocol:

Purge all buffers and DMSO stock solutions with Argon for 1 h prior to use.

1) Aliquat 1 mL of Herceptin or IGN 523 from 10 mg/mL stock into a 2 mL eppendorf tube.

2) Dilute with 1 mL 100 mM Borate (pH 8.4) to afford a 10 mg/mL stock solution (67 μM).

3) Prepare a 50 mM stock solution of TCEP in water.

4) Add 20 mL of TCEP to 2 mL of Herceptin and incubate at 37° C. for 3 hours.

5) Aliquat into 4×0.5 mL eppendorf tubes and place 3 tubes in storage at −20° C.

6) Purify one 0.5 mL aliquat (approx. 5 mg) via SEC on Biorad using degassed PBS.

7) Collect monomeric antibody peak in a sealed tube (approx. 4 mL total volume) at 4° C.

8) Aliquat into 4 equal 1 mL eppendorf tubes (1 mg/mL).

9) Add 6 equivalents of the cytotoxin-linker conjugate from 2 mM stock solutions in DMSO to each tube.

10) Incubate at 4° C. for 48 hours.

11) Analyze by HIC, SDS-PAGE and LC/MS, and compare against control.

Example 15: Synthesis of ADC

Trastuzumab, 1 mL of a 20 mg/mL solution in pH 7.4 PBS (Gibco Mg and Ca free) with 1 mM DTPA, is loaded into a sterile 1.7 mL Eppendorf tube, then 2.75 equivalents of TCEP hydrochloride (Sigma ampule 0.5M concentration), is added and the mixture incubated at 37° C. for 1 hour to give an average of 4 free thiol pairs per trastuzumab (this can be verified by Ellman's colorimetric assay—see Ellman, “Tissue sulfhydryl groups”, Arch. Biochem. Biophys, 1959, 82, 70-77 or later papers referring to this assay). The reduced antibody solution is cooled in an ice-bath at about 0° C. for 15 minutes; then a solution of about 4.5 equivalents of (2S)-2-((2R,3R)-3-((2S)-1-((12S,15S,18S,19R)-1,2-dibromo-18-((R)-sec-butyl)-12,15-diisopropyl-19-methoxy-11,17-dimethyl-3,10,13,16-tetraoxo-4,11,14,17-tetraazahenicosan-21-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (19) of Example 7 in dimethylsulfoxide is added and the mixture incubated at 37° C. for 2 hours (or at 4° C. for 20 hours). The resulting ADC is purified by size-exclusion chromatography (GE ÄKTA pure chromatographic system) or PD10 desalting column.

Similar syntheses using other linker-cytotoxin conjugates, and/or other antibodies, give the corresponding ADCs.

In certain embodiments, the ADCs prepared from the method of the present application provides the products with significant homogeneity as shown by HIC traces, when compared with the ADCs prepared by conventional methods that provide inhomogeneous ADCs with multiple products and positional isomers.

Assays

The ADCs disclosed herein are tested for potency and selectivity in vitro by determining their cytotoxicity in cancer cell lines of interest, such as those cancer cell lines expressing the antigen corresponding to the antibody portion of the ADC and similar cancer cell lines lacking the antigen. The ADCs disclosed herein are tested for potency and safety in vivo in such animal models as the mouse subcutaneous cancer xenograft and mouse orthotopic cancer xenograft models well known to those of skill in the art of cancer research.

Example 16: Cytotoxicity of ADCs Compared to Antibody

The cytotoxicity of the ADCs disclosed herein, where the antibody is conjugated to a cytotoxin through one of the linkers disclosed herein, is compared to the cytotoxicity of antibody alone in HER2-positive and HER2-negative tumor cells. In certain embodiments, the ADCs are considerably more potent than their parental antibodies.

Example 17: Binding Affinity of ADCs for Antigen-Expressing Cells

Binding of the antibodies and ADCs to antigen-expressing cells are measured using a cell ELISA. Sarcoma cells transduced to express the target (F279 cells for HER2, F244 cells for CD98) are plated the day at 5000 cells per well in a 384-well plate. The following day, antibodies are serially diluted in a separate plate, and then transferred to the cell plate, which has previously had media removed by aspiration. After a 2 hour incubation at room temperature, the plate is washed with wash buffer (DPBS at pH7.4 with 0.1% bovine serum albumin) and then 25 μL horseradish peroxidase-labeled secondary antibody diluted in media is added and incubated for 30 minutes at room temperature. The plate is then washed and 15 μL of a chemiluminescent substrate (Pierce catalog #37069) is added; and the plate is read in a plate-based luminescence reader. Antibodies and ADCs demonstrating comparable affinity for F244 cells indicate that conjugation of the drug payloads does not affect antigen binding.

Example 18: Potency of ADCs Against Antigen-Expressing Cells

The potency of ADCs disclosed herein for inhibition of tumor cell growth is tested in cell proliferation assays. The Ramos (B-cell lymphoma) and BT474 (HER2+ human breast carcinoma) cell lines are seeded into 96 well half-area plates the day before drug treatment at 3000 and 5000 cells per well respectively. ADCs and controls are serially diluted in a master plate, and then transferred to the cell plates, which are incubated at 37° C. and 5% CO2 for 3 days. The cells are quantitated by measuring the level of ATP in the wells using the ATPLite 1Step kit (Perkin Elmer catalog #50-904-9883) as described by the manufacturer.

Example 19: Efficacy of ADCs in Murine Xenograft Models

The Ramos cell xenograft model.

The Ramos cell line is obtained from ATCC and cultured according to the supplier's protocols. 4-6 Week-old immunodeficient female mice (Taconic C.B-17 scid) are subcutaneously injected on the right flank with 1×107 viable cells in a mixture of PBS (without magnesium or calcium) and BD Matrigel (BD Biosciences) at a 1:1 ratio. The injected total volume per mouse is 200 μL with 50% being Matrigel. Once the tumor reaches a size of 65-200 mm3, mice are randomized. ADCs are formulated in PBS and administered once intravenously at a dose of 1 mg/Kg into the lateral tail vein, and body weights and tumors are measured twice weekly. Tumor volume are calculated as described in van der Horst et al., “Discovery of Fully Human Anti-MET Monoclonal Antibodies with Antitumor Activity against Colon Cancer Tumor Models In Vivo”, Neoplasia, 2009, 11, 355-364. The experiments are performed on groups of 8 animals per experimental point. The negative control group receives HB121 (an IgG2a-negative antibody) and free MMAF or T4, as appropriate, at a concentration equimolar to the concentration that would be released by the ADC, while the positive control group receives 18-2A.

The BT474 cell xenograft model.

The BT474 cell line is obtained from ATCC and cultured according to the supplier's protocols. 4-6 Week-old immunodeficient female mice (Taconic C.B-17 scid) are implanted with a β-estradiol pellet 3 days before being subcutaneously injected on the right flank with 1×107 viable cells in a mixture of PBS (without magnesium or calcium) and BD Matrigel (BD Biosciences) at a 1:1 ratio. The injected total volume per mouse is 200 μL with 50% being Matrigel. Once the tumor reaches a size of 100-150 mm3, mice are randomized. ADCs are formulated in PBS and administered once intravenously at a dose of 1 mg/Kg into the lateral tail vein, and body weights and tumors are measured twice weekly. Tumor volume is calculated as described in van der Horst et al., cited above. The experiments are performed on groups of 8 animals per experimental point. The negative control group receives HB121 and free MMAF or T4, as appropriate, at a concentration equimolar to the concentration that would be released by the ADCs, while the positive control group receives trastuzumab at 1 mg/Kg.

Similar tests are conducted with other cancers (those expressing different antigens) and ADCs where the antibody corresponds to the antigen expressed by the cancer.

While a number of exemplary embodiments, aspects and variations have been provided herein, those of skill in the art will recognize certain modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations. It is intended that the following claims are interpreted to include all such modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations are within their scope.

Claims

1. A linker of the following formula (I):

or an enantiomer, diasteriomer, or mixtures thereof,
wherein: each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-3 alkyl; each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group; W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-3 alkyl; Z is —CO2H, —NH2, —OH, —NH—R3a, or —CO2R3b; wherein R3a is an amino protecting group, and R3b is a carboxyl protecting group; R is any chemical group; or R is absent; each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl; a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1; each k and k′ is independently an integer of 0 or 1; each p is independently an integer of 1 to 14; each q is independently an integer or 1 to 12; each AA is independently an amino acid; each r is 1 to 12; and the bond represents a single or a double bond.

2. The linker of claim 1, wherein the bond represents a single bond.

3. The linker of claim 1, wherein each Y and Y′ is independently selected from the group consisting of a halo, a substituted thiol, and a substituted sulfonate.

4. (canceled)

5. (canceled)

6. The linker of claim 3, wherein each Y and Y′ is independently selected from the group consisting of:

wherein RA is selected from the group consisting of hydroxyl, amino, nitro, cyano, chloro, bromo, fluoro, iodo, oxo, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkyl, C2-6 alkenyl, and C1-6 alkoxy; or
wherein each Y and Y′ is independently selected from the group consisting of:

7. (canceled)

8. The linker of claim 1, wherein:

each L1, L2 and L3 is independently selected from the group consisting of —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, and -(AA)r-;
a, b and c are each independently 0, 1 or 2; and
each p, q and r is independently 1, 2, 3 or 4; or
wherein each AA is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val; or
wherein each AA is an amino acid selected from the group consisting of Gly, Arg, Val, Ala, Cys, Gln, Leu, Ile, Leu, Lys and Ser or their N-methylated analogues.

9. (canceled)

10. (canceled)

11. The linker of claim 1, wherein R3a is selected from the group consisting of 9-fluorenylmethyloxycarbamate (FMOC), t-butyl carbamate (BOC), benzyl carbamate (Cbz), acetamide, trifluroacetamide, phthalimide, benzylamine, triphenylmethylamine, benzylideneamine, and p-toluenesulfonamide (p-TOS); or

wherein R3b is selected from the group consisting of a methyl ester, a t-butyl ester, a benzyl ester, an S-t-butyl ester, and 2-alkyl-1,3-oxazoline; or
wherein R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined in claim 1; or
wherein R is a detectable probe.

12. (canceled)

13. (canceled)

14. (canceled)

15. The linker of claim 1, wherein k and k′ are both 1.

16. A linker-cytotoxin conjugate of the following formula (II):

or an enantiomer, diasteriomer, or mixtures thereof;
wherein: each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-3 alkyl; each Y and Y′ is independently hydrogen or an electrophilic leaving group that reacts selectively with thiols, provided if one of Y and Y′ is hydrogen, the other is the electrophilic leaving group; W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-3 alkyl; CTX is a cytotoxin; R is any chemical group; or R is absent; each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl; a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1; each k and k′ is independently an integer of 0 or 1; each p is independently an integer of 1 to 14; each q is independently an integer or 1 to 12; m is an integer of 1 to 4; each AA is independently an amino acid; each r is 1 to 12; and the bond represents a single or a double bond.

17. The linker-cytotoxin conjugate of claim 16, wherein the bond represents a single bond.

18. The linker-cytotoxin conjugate of claim 16, wherein CTX is bonded to (L1)a-(L2)b-(L3)c via an amide, an N—(C1-6 alkyl)amide, a carbamate, an N—(C1-6 alkyl)carbamate, an amine, an N—(C1-6 alkyl)amine, an ether, a thioether, an urea, an N—(C1-6 alkyl)urea, or an N,N-di(C1-6 alkyl)urea bond; or

CTX is bonded to (L1)a-(L2)b-(L3)c via a bond selected from the group consisting of:
 wherein each RB is independently branched or unbranched C1-6 alkyl.

19. (canceled)

20. The linker-cytotoxin conjugate of claim 16, wherein the CTX is selected from a from the group consisting of a tubulin stabilizer, a tubulin destabilizer, a DNA alkylator, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an anti-metabolite; or

wherein the CTX is selected from the group consisting of Actinomycin-D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade®, and Vincristine.

21. (canceled)

22. The linker-cytotoxin conjugate of claim 16, wherein R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, W-(L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-CTX, as defined in claim 16; or

wherein R is a detectable probe.

23. (canceled)

24. The linker-cytotoxin conjugate of claim 16, wherein k and k′ are both 1.

25. An antibody-drug conjugate of the following formula (III):

or a pharmaceutically acceptable salt thereof,
wherein: A is an antibody or antibody fragment; the two depicted cysteine residues are from an opened cysteine-cysteine disulfide bond in A; each X and X′ is independently O, S, NH or NR1 wherein R1 is C1-3 alkyl; W is —NH—, —N(R1)—, —CH2—, —CH2—NH—, —CH2—N(R1)—, —CH2CH2—, —CH(R2)—, or —CH2CH(R2)—; wherein R1 and R2 are independently C1-3 alkyl; CTX is a cytotoxin; R is any chemical group; or R is absent; each L1, L2 and L3 is independently a linker selected from the group consisting of —O—, —C(O)—, —S—, —S(O)—, —S(O)2—, —NH—, —NCH3—, —(CH2)q—, —NH(CH2)2NH—, —OC(O)—, —CO2—, —NHCH2CH2C(O)—, —C(O)NHCH2CH2NH—, —NHCH2C(O)—, —NHC(O)—, —C(O)NH—, —NCH3C(O)—, —C(O)NCH3—, —(CH2CH2O)p, —(CH2CH2O)pCH2CH2—, —CH2CH2—(CH2CH2O)p—, —OCH(CH2O—)2, -(AA)r-, cyclopentanyl, cyclohexanyl, unsubstituted phenylenyl, and phenylenyl substituted by 1 or 2 substituents selected from the group consisting of halo, CF3—, CF3O—, CH3O—, —C(O)OH, —C(O)OC1-3 alkyl, —C(O)CH3, —CN, —NH—, —NH2, —O—, —OH, —NHCH3, —N(CH3)2, and C1-3 alkyl; a, b and c are each independently an integer of 0, 1, 2 or 3, provided that at least one of a, b or c is 1; each k and k′ is independently an integer of 0 or 1; each p is independently an integer of 1 to 14; each q is independently an integer or 1 to 12; each AA is independently an amino acid; each r is 1 to 12; m is an integer of 1 to 4; n is an integer of 1 to 4; and the bond represents a single or a double bond.

26. The antibody-drug conjugate or pharmaceutically acceptable salt of claim 25, wherein the bond represents a single bond.

27. The antibody-drug conjugate or pharmaceutically acceptable salt of claim 25, wherein A is an antibody that is specific to a cancer antigen; or

wherein A is selected from the group consisting of alemtuzumab, anitumumab, bevacizumab, brentuximab, cetuximab, gemtuzumab, glembatumumab, inotuzumab, ipilimumab, lovortumumab, milatuzumab, ofatumumab, rituximab, tositumomab, and trastuzumab.

28. (canceled)

29. The antibody-drug conjugate or pharmaceutically acceptable salt of claim 25, wherein the CTX is selected from a from the group consisting of a tubulin stabilizer, a tubulin destabilizer, a DNA alkylator, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an anti-metabolite; or

wherein the CTX is selected from the group consisting of Actinomycin-D, Amonafide, an auristatin, benzophenone, benzothiazole, a calicheamicin, Camptothecin, CC-1065 (NSC 298223), Cemadotin, Colchicine, Combretastatin A4, Dolastatin, Doxorubicin, Elinafide, Emtansine (DM1), Etoposide, KF-12347 (Leinamycin), a maytansinoid, Methotrexate, Mitoxantrone, Nocodazole, Proteosome Inhibitor 1 (PSI 1), Roridin A, T-2 Toxin (trichothecene analog), Taxol, a tubulysin, Velcade®, and Vincristine.

30. (canceled)

31. The antibody-drug conjugate or pharmaceutically acceptable salt of claim 30, wherein the CTX is an auristatin, a calicheamicin, a maytansinoid, or a tubulysin; or

wherein the CTX is monomethylauristatin E, monomethylauristatin F, calicheamicin Y, a pyrrolobenzodiazepine, mertansine, tubulysin T2, tubulysin T3, or tubulysin T4.

32. (canceled)

33. The antibody-drug conjugate or pharmaceutically acceptable salt of claim 25, wherein R is selected from the group consisting of W, (L1)a, (L2)b, (L3)c, Z, W-(L1)a-(L2)b-(L3)c, (L1)a-(L2)b-(L3)c-Z, and W-(L1)a-(L2)b-(L3)c-Z, as defined in claim 22; or

wherein R is a detectable probe.

34. (canceled)

35. The antibody-drug conjugate or pharmaceutically acceptable salt of claim 25, wherein k and k′ are both 1.

36. A pharmaceutical composition comprising the antibody-drug conjugate or pharmaceutically acceptable salt of claim 25, and a pharmaceutically acceptable diluent, carrier or excipient.

37. A method of treating a cancer by administering to a human suffering therefrom an effective amount of the antibody-drug conjugate of claim 25 or the pharmaceutical composition of claim 36.

Patent History
Publication number: 20230109312
Type: Application
Filed: Dec 8, 2021
Publication Date: Apr 6, 2023
Inventors: David Y. Jackson (Belmont, CA), Edward Ha (Solano Beach, CA)
Application Number: 17/545,562
Classifications
International Classification: A61K 47/68 (20060101);