Dual-payload antibody-drug conjugates and uses thereof

Abstract

The present disclosure provides antibody-drug conjugate (ADC) structures, which include an antibody, a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker, and a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker. The disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates.

Description

Atty. Dkt: RDWD-053WO DUAL-PAYLOAD ANTIBODY-DRUG CONJUGATES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 63 / 538,962, filed September 18, 2023, the disclosure of which of which is incorporated herein by reference. INCORPORATION BYREFERENCE OFSEQUENCELISTINGXML

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “RDWD- 053WO_SEQ_LIST” created on September 16, 2024, and having a size of 24,245 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. INTRODUCTION

[0003] Introduced over two decades ago, antibody-drug conjugates (ADCs) remain a cutting-edge promising therapeutic drug format, produced by chemically attaching a cytotoxin or other bioactive compound to an antibody via a chemical linker (Figure 1). Owing to the distinctive affinity of antibodies towards their cognate antigens, ADCs are able to preferentially target malignant cells over healthy tissues, thus achieving selective drug delivery to tumors. One of the intended benefits of this new drug delivery mechanism was to more safely deliver cytotoxic chemotherapeutic-style drugs to tumors as compared to standard chemotherapy treatments. Indeed, ADCs were intended to be used to replace chemotherapies in treatment regimens, so that patients could benefit from good anti-tumor efficacy without the common (and commonly disabling) side-effects associated with chemotherapy treatments. However, most cancer patients are treated with a cocktail of chemotherapeutic drugs (typically small molecules) that work through different mechanisms of action to kill cancer cells and clear them from the body. The cocktail approach to chemotherapy, delivering multiple drugs in combination, was developed over decades of work and experimentation in research labs and through clinical trials. The improvement in patient response to treatment, including the ability to induce (long-term) complete responses, was marked for combination treatment over monotherapy (DeVita and Chu, Cancer Research, 68, 8643-8653 (2008)).Atty. Dkt: RDWD-053WO

[0004] By contrast, most ADCs deliver just a single type of payload to their target cell. The limitations of this approach relate back to the learnings from the early days of chemotherapy-based cancer treatment when monotherapies were more commonly used. What if a given tumor does not respond to a particular drug (e.g., there is refractory disease)? What if there is an initial response to the drug, but then the tumor begins to regrow (e.g., there is disease relapse) suggesting that the drug is no longer an effective treatment for that tumor? Both drug developers working in the ADC field and practicing physicians face these common problems associated with chemotherapeutic treatments. Quickly developing drug-resistance often leads to discontinuation of treatments due to futility. Tumor heterogeneity often causes tumor cells to respond in a non-uniform manner to a drug with particular mechanism of action, which ultimately renders the drug inefficacious.

[0005] Combination treatment strategies tend to improve outcomes by engaging more than one molecular mechanism to kill tumor cells (Mokhtari et al. Oncotarget, 8, 38022-38043 (2017)). However, before they are used in combination, the different drugs must first be proven safe and efficacious as monotherapies. Next, optimal dosing regimens must be established to deliver the maximum therapeutic benefit while limiting side effects to the extent possible. Another complication is that combination drugs may have different dosage forms and routes of administration and biodistribution. For example, intravenously-delivered biologics (e.g., monoclonal antibodies or conjugates) and oral small molecule drugs (e.g., kinase inhibitors) when dosed concurrently will have very different dose levels and rate of distribution-absorption as well as varying pharmacokinetic profiles and rates of clearance. These factors can complicate the implementation of combination therapies. SUMMARY

[0006] The present disclosure provides antibody-drug conjugate (ADC) structures, which include an antibody, a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker, and a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker. The disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates.

[0007] Aspects of the present disclosure include an antibody-drug conjugate comprising:Atty. Dkt: RDWD-053WO an antibody; a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker; and a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker.

[0008] In certain embodiments, the native amino acid residue of the antibody is a cysteine.

[0009] In certain embodiments, the cysteine is produced via reduction of an interchain disulfide bond of the antibody.

[0010] In certain embodiments, the site-specifically engineered conjugation site of the antibody comprises a formylglycine (fGly).

[0011] In certain embodiments, the antibody has a drug-antibody ratio (DAR) of 2 or more for the first drug.

[0012] In certain embodiments, two or more of the first drug are attached to the antibody through two or more of the first linker.

[0013] In certain embodiments, the antibody has a DAR of 8 for the first drug.

[0014] In certain embodiments, eight of the first drug are attached to the antibody through eight of the first linker.

[0015] In certain embodiments, the antibody has a DAR of 2 or more for the second drug.

[0016] In certain embodiments, two or more of the second drug are attached to the antibody through two or more of the second linker.

[0017] In certain embodiments, the antibody has a DAR of 2 for the second drug.

[0018] In certain embodiments, two of the second drug are attached to the antibody through two of the second linker.

[0019] In certain embodiments, the antibody has a DAR of 4 for the second drug.

[0020] In certain embodiments, four of the second drug are attached to the antibody through four of the second linker.

[0021] In certain embodiments, four of the second drug are attached to the antibody through two of the second linker, wherein each of the second linker is a branched linker.

[0022] In certain embodiments, the antibody has a DAR of 8 for the second drug.Atty. Dkt: RDWD-053WO

[0023] In certain embodiments, eight of the second drug are attached to the antibody through four of the second linker, wherein each of the second linker is a branched linker.

[0024] In certain embodiments, the antibody has a total DAR for the first drug and the second drug of 4 to 16.

[0025] Aspects of the present disclosure include a pharmaceutical composition comprising: a conjugate of the present disclosure; and a pharmaceutically-acceptable excipient.

[0026] Aspects of the present disclosure include a method comprising administering to a subject an amount of a conjugate of the present disclosure.

[0027] Aspects of the present disclosure include a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of the present disclosure, wherein the administering is effective to treat cancer in the subject.

[0028] Aspects of the present disclosure include a method of producing an antibody-drug conjugate, comprising: contacting an antibody comprising a site-specifically engineered conjugation site with: (1) a first drug-linker compound comprising a first reactive group configured to conjugate to a native amino acid residue of the antibody; and (2) a second drug- linker compound comprising a second reactive group configured to conjugate to the site- specifically engineered conjugation site.

[0029] In certain embodiments, the contacting comprises combining the antibody, the first drug-linker compound and the second drug-linker compound in a one-pot reaction.

[0030] In certain embodiments, the contacting comprises contacting the antibody with the first drug-linker compound and then contacting the antibody with the second drug-linker compound.

[0031] In certain embodiments, the contacting comprises contacting the antibody with the second drug-linker compound and then contacting the antibody with the first drug-linker compound.

[0032] In certain embodiments, prior to the contacting the method comprises reducing one or more interchain disulfide bonds of the antibody to produce two or more cysteine residues.

[0033] In certain embodiments, the first reactive group comprises a maleimide.

[0034] In certain embodiments, the site-specifically engineered conjugation site of the antibody comprises an fGly.Atty. Dkt: RDWD-053WO

[0035] In certain embodiments, the second reactive group comprises a hydrazinyl-indole or hydrazinyl-pyrrolo-pyridine group. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 shows a schematic illustrating the production of an antibody-drug conjugate (ADC) by chemically attaching a cytotoxin or other bioactive compound to an antibody via a chemical linker.

[0037] FIG. 2A shows a schematic illustrating the production of an ADC that includes a branched linker having a dual-payload with two different payloads.

[0038] FIG. 2B shows a schematic illustrating the production of an ADC using two different stochastic conjugation methods.

[0039] FIG. 2C shows a schematic illustrating the production of an ADC using two different site-specific orthogonal conjugation methods.

[0040] FIG. 3A shows a schematic illustrating the production of an ADC that includes 8 of a first drug conjugated to the antibody via maleimide conjugation methods and 2 of a second drug conjugated to the antibody via HIPS conjugation methods, according to embodiments of the present disclosure (LCTPSR [SEQ ID NO:1]).

[0041] FIG. 3B shows schematic illustrations of ADCs having DAR 2, DAR 4 and DAR 8, according to embodiments of the present disclosure.

[0042] FIG. 4 shows a scheme illustrating that a tandem-cleavage linker can involve two consecutive enzymatic steps in order to release the drug after internalization of ADC. The monosaccharide unit attached to the phenolic self-immolation moiety (PABA) serves as the protection element and is removed first by the corresponding lysosomal glycosidase to expose the peptide portion of the linker which is then readily cleaved by proteases to liberate the payload at the target location.

[0043] FIG. 6 shows the PLRP-HPLC analysis of Protocol 1, Step 2: Maleimide- conjugated, CH1-tagged trastuzumab, subjected to a HIPS reaction with compound 5 (cRW5675) yields a calculated DAR of 9.57.

[0044] FIG. 7 shows HIC-HPLC analysis of Protocol 1, Step 2: Maleimide-conjugated, CH1-tagged trastuzumab, subjected to a HIPS reaction with compound 5 (cRW5675) yields a calculated DAR of 9.47.Atty. Dkt: RDWD-053WO

[0045] FIG. 8 shows the SEC-HPLC analysis of Protocol 1, Step 2: Maleimide- conjugated, CH1-tagged trastuzumab, subjected to a HIPS reaction with compound 5 (cRW5675) yields a molecule with 9.0% high-molecular weight (HMW).

[0046] FIG. 9 shows the PLRP-HPLC analysis of Protocol 2, Step 1: HIPS reaction of compound 5 (cRW5676) on CH1-tagged trastuzumab yields a calculated DAR of 1.78.

[0047] FIG. 10 shows PLRP-HPLC analysis of Protocol 2, Step 2: HIPS-conjugated, CH1-tagged trastuzumab (cRW5676), subjected to a maleimide reaction with compound 15 (cRW5679) yields a calculated DAR of 9.61.

[0048] FIG. 11 shows the HIC-HPLC analysis of Protocol 2, Step 2: HIPS-conjugated, CH1-tagged trastuzumab (cRW5676), subjected to a maleimide reaction with compound 15 (cRW5679) yields a calculated DAR of 9.76.

[0049] FIG. 12 shows the SEC-HPLC analysis of Protocol 2, Step 2: HIPS-conjugated, CH1-tagged trastuzumab (cRW5676), subjected to a maleimide reaction with compound 15 (cRW5679) yields a molecule with 9.1% high-molecular weight (HMW).

[0050] FIG. 13 shows PLRP-the HPLC analysis of Protocol 3, (One-Pot Reaction): Maleimide reaction of compound 15 and HIPS reaction of compound 5 on CH1-tagged trastuzumab yields (cRW5704) a calculated DAR of 9.5.

[0051] FIG. 14 shows the HIC-HPLC analysis of Protocol 3, (One-Pot Reaction): Maleimide reaction of compound 15 and HIPS reaction of compound 5 on CH1-tagged trastuzumab yields (cRW5704) a calculated DAR of 9.0.

[0052] FIG. 15 shows the SEC-HPLC analysis of Protocol 3, (One-Pot Reaction): Maleimide reaction of compound 15 and HIPS reaction of compound 5 on CH1-tagged trastuzumab yields (cRW5704) a molecule with 12.75 % high-molecular weight (HMW).

[0053] FIG. 16 shows schemes illustrating different click-chemistry reactions that can be used to produce a linker according to the present disclosure. The click-chemistry reactions include azide-alkyne reactions, alkyne-DBCO (dibenzocyclooctyne) reactions, and TCO-Tz (trans-cycloctene tetrazine) reactions.

[0054] FIG. 17 provides a graph illustrating pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds according to embodiments of the present disclosure and sampled at the times shown.Atty. Dkt: RDWD-053WO

[0055] FIG. 18 provides a graph illustrating pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds according to embodiments of the present dislosure and sampled at the times shown.

[0056] FIG. 19 provides a graph illustrating pharmacokinetic data showing total antibody and total ADC concentrations as measured by ELISA in plasma samples from rats dosed with the indicated compounds according to embodiments of the present dislosure and sampled at the times shown.

[0057] FIG. 20 shows a schematic illustrating total antibody and total ADC (DAR- sensitive) quantification by ELISA.

[0058] FIG. 21 shows a graph illustrating efficacy of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 moderate JIMT-1 breast tumor xenograft model.

[0059] FIG. 22 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.

[0060] FIG. 23 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.

[0061] FIG. 24 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.

[0062] FIG. 25 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line SK-BR- 3.

[0063] FIG. 26 shows a graph illustrating in vitro potency of trastuzumab conjugates according to embodiments of the present disclosure against the HER2 positive cell line NCI- N87.

[0064] FIG. 27 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to CH1-tagged Trastuzumab yields a DAR of 1.83 (HIPS) and 7.8 (maleimide) as determined by reduced PLRP.Atty. Dkt: RDWD-053WO

[0065] FIG. 28 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to CH1-tagged Trastuzumab is 97.7% monomeric as determined by analytical size exclusion chromatography (SEC).

[0066] FIG. 29 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to CH1-tagged Trastuzumab yields a DAR of 1.83 (HIPS) and 7.5 (maleimide) as determined by reduced PLRP.

[0067] FIG. 30 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to CH1-tagged Trastuzumab is 95.7% monomeric as determined by analytical SEC.

[0068] FIG. 31 shows an HPLC trace demonstrating that Compound 26 and Compound 15 conjugated to CH1-tagged Trastuzumab yields a DAR of 1.8 (HIPS) and 7.59 (maleimide) as determined by reduced PLRP.

[0069] FIG. 32 shows an HPLC trace demonstrating that Compound 26 and Compound 15 conjugated to CH1-tagged Trastuzumab is 97.7% monomeric as determined by analytical SEC.

[0070] FIG. 33 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to 58Q-tagged Trastuzumab yields a DAR of 1.7 (HIPS) and 7.34 (maleimide) as determined by reduced PLRP.

[0071] FIG. 34 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to 58Q-tagged Trastuzumab is 98.1% monomeric as determined by analytical SEC.

[0072] FIG. 35 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to 288F-tagged Trastuzumab yields a DAR of 1.83 (HIPS) and 7.95 (maleimide) as determined by reduced PLRP.

[0073] FIG. 36 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to 288F-tagged Trastuzumab is 96.7% monomeric as determined by analytical SEC.

[0074] FIG. 37 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to 22A-tagged Trastuzumab yields a DAR of 1.78 (HIPS) and 7.9 (maleimide) as determined by reduced PLRP.

[0075] FIG. 38 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to 22A-tagged Trastuzumab is 97.5% monomeric as determined by analytical SEC.Atty. Dkt: RDWD-053WO

[0076] FIG. 39 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to CH1-tagged Trastuzumab yields a DAR of 1.67 (HIPS) and 7.85 (maleimide) as determined by reduced PLRP.

[0077] FIG. 40 shows an HPLC trace demonstrating that Compound 5 and Compound 15 conjugated to CH1-tagged Trastuzumab is 97.0% monomeric as determined by analytical SEC.

[0078] FIG. 41 shows an HPLC trace demonstrating that Compound 5 and Compound 40 conjugated to CH1-tagged Trastuzumab yields a DAR of 1.83 (HIPS) and 7.8 (maleimide) as determined by reduced PLRP.

[0079] FIG. 42 shows an HPLC trace demonstrating that Compound 5 and Compound 40 conjugated to CH1-tagged Trastuzumab is 96.5% monomeric as determined by analytical SEC.

[0080] FIG. 43 shows an HPLC trace demonstrating that Compound 17 and Compound 40 conjugated to CH1-tagged Upifitamab yields a DAR of 1.05 (Tz) and 7.64 (maleimide) as determined by reduced PLRP.

[0081] FIG. 44 shows an HPLC trace demonstrating that Compound 17 and Compound 40 conjugated to CH1-tagged Upifitamab is 97.6% monomeric as determined by analytical SEC.

[0082] FIG. 45 shows an HPLC trace demonstrating that Compound 5 and Compound 40 conjugated to CH1-tagged Trastuzumab yields a DAR of 1.7 (HIPS) and 7.85 (maleimide) as determined by reduced PLRP.

[0083] FIG. 46 shows an HPLC trace demonstrating that Compound 5 and Compound 40 conjugated to CH1-tagged Trastuzumab is 96.2% monomeric as determined by analytical SEC.

[0084] FIG. 47 shows an HPLC trace demonstrating that Compound 5 and Compound 40 conjugated to 225Q-tagged Upifitamab yields a DAR of 1.37 (HIPS) and 5.76 (maleimide) as determined by reduced PLRP.

[0085] FIG. 48 shows an HPLC trace demonstrating that Compound 5 and Compound 40 conjugated to 225Q-tagged Upifitamab is 93.5% monomeric as determined by analytical SEC.

[0086] FIG. 49 shows an HPLC trace demonstrating that Compound 17 and Compound 44 conjugated to CH1-tagged Upifitamab yields a DAR of 0.7 (Tz) and 7.56 (maleimide) as determined by reduced PLRP.

[0087] FIG. 50 shows an HPLC trace demonstrating that Compound 17 and Compound 44 conjugated to CH1-tagged Upifitamab is 90.7% monomeric as determined by analytical SEC.Atty. Dkt: RDWD-053WO

[0088] FIG. 51 shows an HPLC trace demonstrating that Compound 17 and Compound 48 conjugated to CH1-tagged Upifitamab yields a DAR of 1.07 (Tz) and 7.58 (maleimide) as determined by reduced PLRP.

[0089] FIG. 52 shows an HPLC trace demonstrating that Compound 17 and Compound 48 conjugated to CH1-tagged Upifitamab is 98.1% monomeric as determined by analytical SEC.

[0090] FIG. 53 shows an HPLC trace demonstrating that Compound 31 and Compound 52 conjugated to CH1 / CT-tagged Trastuzumab yields a DAR of 7.16 (HIPS) and 7.32 (maleimide) as determined by reduced PLRP.

[0091] FIG. 54 shows an HPLC trace demonstrating that Compound 31 and Compound 52 conjugated to CH1 / CT-tagged Trastuzumab is 98.5% monomeric as determined by analytical SEC.

[0092] FIG. 55 shows an HPLC trace demonstrating that Compound 31 and Compound 52 conjugated to CH1 / CT-tagged anti-FITC yields a DAR of 6.97 (HIPS) and 7.26 (maleimide) as determined by reduced PLRP.

[0093] FIG. 56 shows an HPLC trace demonstrating that Compound 31 and Compound 52 conjugated to CH1 / CT-tagged anti-FITC is 96.9% monomeric as determined by analytical SEC.

[0094] FIG. 57 shows an HPLC trace demonstrating that Compound 26 and Compound 54 conjugated to 58Q-tagged Trastuzumab yields a DAR of 1.58 (HIPS) and 7.76 (maleimide) as determined by reduced PLRP.

[0095] FIG. 58 shows an HPLC trace demonstrating that Compound 26 and Compound 54 conjugated to 58Q-tagged Trastuzumab is 97.6% monomeric as determined by analytical SEC.

[0096] FIG. 59 shows an HPLC trace demonstrating that Compound 39 and Compound 54 conjugated to 58Q-tagged Trastuzumab yields a DAR of 1.73 (HIPS) and 7.92 (maleimide) as determined by reduced PLRP.

[0097] FIG. 60 shows an HPLC trace demonstrating that Compound 39 and Compound 54 conjugated to 58Q-tagged Trastuzumab is 97.5% monomeric as determined by analytical SEC.Atty. Dkt: RDWD-053WO

[0098] FIG. 61 shows an HPLC trace demonstrating that Compound 26 and Compound 66 conjugated to 58Q-tagged Trastuzumab yields a DAR of 1.7 (HIPS) and 8.0 (maleimide) as determined by reduced PLRP.

[0099] FIG. 62 shows an HPLC trace demonstrating that Compound 39 and Compound 54 conjugated to 58Q-tagged Trastuzumab is 97.2% monomeric as determined by analytical SEC.

[0100] FIG. 63 shows an HPLC trace demonstrating that Compound 26 and Compound 62 conjugated to 58Q-tagged Trastuzumab yields a DAR of 1.7 (HIPS) and 5.78 (bromoacetamide) as determined by reduced PLRP.

[0101] FIG. 64 shows an HPLC trace demonstrating that Compound 26 and Compound 62 conjugated to 58Q-tagged Trastuzumab is 97.2% monomeric as determined by analytical SEC.

[0102] FIG. 65 shows an HPLC trace demonstrating that Compound 26 and Compound 63 conjugated to 58Q-tagged Trastuzumab yields a DAR of 1.7 (HIPS) and 7.97 (bromoacetamide) as determined by reduced PLRP.

[0103] FIG. 66 shows an HPLC trace demonstrating that Compound 26 and Compound 63 conjugated to 58Q-tagged Trastuzumab is 97.6% monomeric as determined by analytical SEC. DEFINITIONS

[0104] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0105] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0106] The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1carbon atom) have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogenAtty. Dkt: RDWD-053WO or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NRaRb, wherein R’and R”may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0107] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -O-, -NR10-, -NR10C(O)-, -C(O)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like.

[0108] “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below.

[0109] The term “alkane” refers to alkyl group and alkylene group, as defined herein.

[0110] The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R’NHR”- where R’ is alkyl group as defined herein and R” is alkylene, alkenylene or alkynylene group as defined herein.

[0111] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0112] “Alkoxy” refers to the group –O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec- butoxy, n-pentoxy, and the like. The term “alkoxy” also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0113] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-Atty. Dkt: RDWD-053WO where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0114] The term “alkoxyamino” refers to the group –NH-alkoxy, wherein alkoxy is defined herein.

[0115] The term “haloalkoxy” refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.

[0116] The term “haloalkyl” refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group. Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.

[0117] The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0118] The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S- substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0119] “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.

[0120] The term “substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and - SO2-heteroaryl.Atty. Dkt: RDWD-053WO

[0121] “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH2C≡CH).

[0122] The term “substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and - SO2-heteroaryl.

[0123] “Alkynyloxy” refers to the group –O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0124] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl- C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-

[0125] “Acylamino” refers to the groups –NR20C(O)alkyl, -NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, -NR20C(O)substituted cycloalkyl, - NR20C(O)cycloalkenyl, -NR20C(O)substituted cycloalkenyl, -NR20C(O)alkenyl, - NR20C(O)substituted alkenyl, -NR20C(O)alkynyl, -NR20C(O)substituted alkynyl, -NR20C(O)aryl, -NR20C(O)substituted aryl, -NR20C(O)heteroaryl, -NR20C(O)substituted heteroaryl, -NR20C(O)heterocyclic, and -NR20C(O)substituted heterocyclic, wherein R20is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,Atty. Dkt: RDWD-053WO substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0126] “Aminocarbonyl” or the term “aminoacyl” refers to the group -C(O)NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0127] “Aminocarbonylamino” refers to the group –NR21C(O)NR22R23where R21, R22, and R23are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.

[0128] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0129] The term “acyloxy” refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0130] “Aminosulfonyl” refers to the group –SO2NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,Atty. Dkt: RDWD-053WO substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0131] “Sulfonylamino” refers to the group –NR21SO2R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0132] “Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0133] “Aryloxy” refers to the group –O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.

[0134] “Amino” refers to the group –NH2.Atty. Dkt: RDWD-053WO

[0135] The term “substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.

[0136] The term “azido” refers to the group –N3.

[0137] “Carboxyl,” “carboxy” or “carboxylate” refers to –CO2H or salts thereof.

[0138] “Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0139] “(Carboxyl ester)oxy” or “carbonate” refers to the groups –O-C(O)O- alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O- C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O- C(O)O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O- substituted cycloalkenyl, -O-C(O)O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O- heterocyclic, and -O-C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0140] “Cyano” or “nitrile” refers to the group –CN.

[0141] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ringAtty. Dkt: RDWD-053WO structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0142] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0143] “Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.

[0144] The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0145] “Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.

[0146] “Cycloalkoxy” refers to –O-cycloalkyl.

[0147] “Cycloalkenyloxy” refers to –O-cycloalkenyl.

[0148] “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

[0149] “Hydroxy” or “hydroxyl” refers to the group –OH.

[0150] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as,Atty. Dkt: RDWD-053WO pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N- oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0151] The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0152] “Heteroaryloxy” refers to –O-heteroaryl.

[0153] “Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, where, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or – SO2- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein.Atty. Dkt: RDWD-053WO

[0154] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1- dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0155] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, - SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0156] “Heterocyclyloxy” refers to the group –O-heterocyclyl.

[0157] The term “heterocyclylthio” refers to the group heterocyclic-S-.

[0158] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.

[0159] The term “hydroxyamino” refers to the group -NHOH.

[0160] “Nitro” refers to the group –NO2.

[0161] “Oxo” refers to the atom (=O).

[0162] “Sulfonyl” refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, - SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cylcoalkyl, -SO2-cycloalkenyl, -SO2- substituted cylcoalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,Atty. Dkt: RDWD-053WO heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0163] “Sulfonyloxy” refers to the group -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2- alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cylcoalkyl, -OSO2- cycloalkenyl, -OSO2-substituted cylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2- heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, and -OSO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0164] “Sulfate” or “sulfate ester” refers the group -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O- substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O- SO2-O-substituted cylcoalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cylcoalkenyl, -O- SO2-O-aryl, -O-SO2-O-substituted aryl, -O-SO2-O-heteroaryl, -O-SO2-O-substituted heteroaryl, - O-SO2-O-heterocyclic, and -O-SO2-O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0165] The term “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0166] “Thiol” refers to the group -SH.

[0167] “Thioxo” or the term “thioketo” refers to the atom (=S).

[0168] “Alkylthio” or the term “thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

[0169] The term “substituted thioalkoxy” refers to the group -S-substituted alkyl.

[0170] The term “thioaryloxy” refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.

[0171] The term “thioheteroaryloxy” refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.Atty. Dkt: RDWD-053WO

[0172] The term “thioheterocyclooxy” refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.

[0173] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0174] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)O -M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant toAtty. Dkt: RDWD-053WO include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N- morpholinyl.

[0175] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3–M+, -SO3R70, -OSO2R70, -OSO3–M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2–M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2–M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+.

[0176] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2 O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, - NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0177] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0178] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serialAtty. Dkt: RDWD-053WO substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0179] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.

[0180] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0181] The term “pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[0182] The term “salt thereof” means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0183] “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to,Atty. Dkt: RDWD-053WO methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0184] “Stereoisomer” and “stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0185] “Tautomer” refers to alternate forms of a molecule that differ only in electronic bonding of atoms and / or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.

[0186] It will be appreciated that the term “or a salt or solvate or stereoisomer thereof” is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound.

[0187] “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[0188] “Patient” refers to human and non-human subjects, especially mammalian subjects.

[0189] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.

[0190] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless specifically indicatedAtty. Dkt: RDWD-053WO otherwise, “polypeptide,” “peptide,” and “protein” can include genetically coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, proteins which contain at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant host cell); immunologically tagged proteins; and the like. In certain embodiments, a polypeptide is an antibody.

[0191] “Native amino acid sequence” or “parent amino acid sequence” are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include at least one modified amino acid residue.

[0192] The terms “amino acid analog,” “unnatural amino acid,” and the like may be used interchangeably, and include amino acid-like compounds that are similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and / or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α- hydroxy acids, and α-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.

[0193] The terms “amino acid side chain” or “side chain of an amino acid” and the like may be used to refer to the substituent attached to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. An amino acid side chain can also include an amino acid side chain as described in the context of the modified amino acids and / or conjugates described herein.Atty. Dkt: RDWD-053WO

[0194] The term “carbohydrate” and the like may be used to refer to monomers units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to the smaller carbohydrates, such as monosaccharides, disaccharides. The term “carbohydrate derivative” includes compounds where one or more functional groups of a carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemistry) or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates.

[0195] The term “glycoside” or “glycosyl” refers to a sugar molecule or group bound to a moiety via a glycosidic bond. For example, the moiety that the glycoside is bound to can be a cleavable linker as described herein. A glycosidic bond can link the glycoside to the other moiety through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O- glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). In some cases, glycosides can be cleaved from the moiety they are attached to, such as by chemically-mediated hydrolysis or enzymatically- mediated hydrolysis.

[0196] The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like. An antibody is capable of binding a target antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen can have one or more binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) formed by one or more variable regions of an antibody.

[0197] The term “natural antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies.

[0198] The term “humanized antibody” or “humanized immunoglobulin” refers to a non- human (e.g., mouse or rabbit) antibody containing one or more amino acids (in a frameworkAtty. Dkt: RDWD-053WO region, a constant region or a CDR, for example) that have been substituted with a correspondingly positioned amino acid from a human antibody. In general, humanized antibodies produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO 98 / 45331 and WO 98 / 45332. In particular embodiments, a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art.

[0199] The term “chimeric antibodies” refer to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although domains from other mammalian species may be used.

[0200] An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, 1991). The framework region of anAtty. Dkt: RDWD-053WO antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen.

[0201] A “parent Ig polypeptide” is a polypeptide comprising an amino acid sequence which lacks an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions and / or substitutions).

[0202] As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and / or in which the compound of interest is partially or substantially purified.

[0203] As used herein, the term “substantially purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated.

[0204] The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

[0205] By “reactive partner” is meant a molecule or molecular moiety that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include a cysteine or serine of a sulfatase motif and Formylglycine Generating Enzyme (FGE), which react to form a reaction product of a converted aldehyde tag containing a formylglycine (fGly) in lieu of cysteine or serine in the motif. Other exemplary reactive partners include an aldehyde of an fGly residue of a converted aldehyde tag (e.g., a reactive aldehyde group) and an “aldehyde-reactive reactive partner”, which comprises an aldehyde-reactive group and a moiety of interest, and which reacts to form a reaction product of a modified aldehyde tagged polypeptide having the moiety of interest conjugated to the modified polypeptide through a the fGly residue.Atty. Dkt: RDWD-053WO

[0206] “N-terminus” refers to the terminal amino acid residue of a polypeptide having a free amine group, which amine group in non-N-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.

[0207] “C-terminus” refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, which carboxyl group in non-C-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.

[0208] By “internal site” as used in referenced to a polypeptide or an amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or at the C-terminus.

[0209] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0210] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0211] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e.,Atty. Dkt: RDWD-053WO compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.

[0212] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0213] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0214] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0215] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Atty. Dkt: RDWD-053WO DETAILED DESCRIPTION

[0216] The present disclosure provides antibody-drug conjugate (ADC) structures, which include an antibody, a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker, and a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker. The disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates. ANTIBODY-DRUG CONJUGATES

[0217] The present disclosure provides a conjugate, e.g., an antibody-drug conjugate (ADC). By “conjugate” is meant a polypeptide (e.g., an antibody) is covalently attached to one or more other moieties (e.g., drugs or active agents). For example, an antibody-drug conjugate according to the present disclosure includes one or more drugs or active agents covalently attached to an antibody. In certain embodiments, the polypeptide (e.g., antibody) and the one or more drugs or active agents are bound to each other through one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds can include a linker as described herein.

[0218] In certain embodiments, the conjugate is a polypeptide conjugate, which includes a polypeptide (e.g., an antibody) conjugated to one or more other moieties. In certain embodiments, the one or more moieties conjugated to the polypeptide can each independently be any of a variety of moieties of interest such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilization of the polypeptide to a membrane or a surface. In certain embodiments, the conjugate is a drug conjugate, where a polypeptide is an antibody, thus providing an antibody-drug conjugate. For instance, the conjugate can be a drug conjugate, where a polypeptide is conjugated to one or more drugs or active agents. Various types of drugs or active agents may be used in the conjugates and are described in more detail herein.

[0219] The one or more drugs or active agents can be conjugated to the polypeptide (e.g., antibody) at any desired site of the polypeptide. Thus, the present disclosure provides, for example, a polypeptide having a drug or active agent conjugated at a site at or near the C- terminus of the polypeptide. Other examples include a polypeptide having a drug or active agent conjugated at a position at or near the N-terminus of the polypeptide. Examples also include aAtty. Dkt: RDWD-053WO polypeptide having a drug or active agent conjugated at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above are also possible where the polypeptide is conjugated to two or more drugs or active agents.

[0220] In certain embodiments, a conjugate of the present disclosure includes one or more drugs or active agents conjugated to an amino acid residue of a polypeptide at the α-carbon of an amino acid residue. Stated another way, a conjugate includes a polypeptide where the side chain of one or more amino acid residues in the polypeptide has been modified and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents through a linker as described herein). For example, a conjugate includes a polypeptide where the α-carbon of one or more amino acid residues in the polypeptide has been modified and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents through a linker as described herein).

[0221] Embodiments of the present disclosure include conjugates where a polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, 10 moieties, 11 moieties, 12 moieties, 13 moieties, 14 moieties, 15 moieties, 16 moieties, 17 moieties, 18 moieties, 19 moieties, or 20 or more moieties. The moieties may be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example where a polypeptide is conjugated to a first moiety at a first amino acid residue and conjugated to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to first and second moieties at a first amino acid residue and conjugated to third and fourth moieties at a second amino acid residue, etc. In some cases, two or more amino acid residues in the polypeptide are each conjugated to a pair of moieties (i.e., two moieties), where each pair of moieties is conjugated to the polypeptide through a branched linker as described herein. In some cases, one amino acid residue in the polypeptide isAtty. Dkt: RDWD-053WO conjugated to a pair of moieties through a branched linker as described herein. In some cases, two amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, three amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, four amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, five amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, six amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, seven amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein. In some cases, eight amino acid residues in the polypeptide are each conjugated to a pair of moieties through a branched linker as described herein.

[0222] The one or more amino acid residues of the polypeptide that are conjugated to the one or more moieties may be naturally occurring amino acids, unnatural amino acids, or combinations thereof. For instance, the conjugate may include one or more drugs or active agents conjugated to one more naturally occurring amino acid residues (e.g., one or more native amino acid residues) of the polypeptide. In other instances, the conjugate may include one or more drugs or active agents conjugated to one or more unnatural amino acid residues of the polypeptide. One or more drugs or active agents may be conjugated to the polypeptide at a single natural (e.g., native) or unnatural amino acid residue as described above. One or more natural (e.g., native) or unnatural amino acid residues in the polypeptide may be conjugated to the moiety or moieties as described herein. For example, two (or more) amino acid residues (e.g., natural (e.g., native) or unnatural amino acid residues) in the polypeptide may each be conjugated to one or two moieties, such that multiple sites in the polypeptide are conjugated to the moieties of interest.

[0223] In certain embodiments, the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) are conjugated through a conjugation moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bonded) to the conjugation moiety, thus indirectly binding the polypeptide and the moiety of interest together through the conjugation moiety.Atty. Dkt: RDWD-053WO

[0224] In some cases, the conjugation moiety includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may be conjugated to a cysteine residue of the polypeptide (e.g., antibody), such as a native cysteine residue of the polypeptide (e.g., antibody), thus indirectly binding the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) together through the maleimide conjugation moiety. In some instances, the cysteine is produced via reduction of an interchain disulfide bond of the antibody. For example, reduction of one interchain disulfide bond would produce two cysteine residues, each of which may be conjugated to one or more drugs or active agents as described herein. In some cases, cysteine residues produced by reduction of interchain disulfide bonds are native cysteine residues in the antibody.

[0225] In some cases, the conjugation moiety includes an N-hydroxysuccinimide ester (NHS ester) or derivative thereof. For instance, an NHS-ester conjugation moiety may be conjugated to a lysine residue of the polypeptide (e.g., antibody), such as a native lysine residue of the polypeptide (e.g., antibody), thus indirectly binding the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) together through an NHS-ester conjugation moiety.

[0226] In some instances, a drug or active agent can be modified to include a conjugation moiety, which is then conjugated to a polypeptide (e.g., antibody) to produce a polypeptide (e.g., antibody) conjugate, thus attaching the drug or active agent to the polypeptide (e.g., antibody) through the conjugation moiety.

[0227] In certain embodiments, the conjugation moiety may be attached (e.g., covalently attached) to a linker. As such, embodiments of the present disclosure include a conjugation moiety attached to a drug or active agent through a linker. Various embodiments of the linkers that may be used to couple the conjugation moiety to the drug or active agent are described in detail herein. For example, in some instances, the linker is a cleavable linker, such as a cleavable linker as described herein.

[0228] In certain embodiments, the linker is a branched linker, such as a branched linker as described herein. In some instances, the conjugation moieties may be attached (e.g., covalently attached) to two or more linkers. As such, embodiments of the present disclosure include a conjugation moiety attached to two or more drugs or active agents each through a corresponding linker. Thus, conjugates of the present disclosure may include two or moreAtty. Dkt: RDWD-053WO linkers, where each linker attaches a corresponding drug or active agent to the conjugation moiety. Accordingly, the conjugation moiety and two or more linkers may be viewed overall as a “branched linker”, where the conjugation moiety is attached to two or more “branches”, where each branch includes a linker attached to a drug or active agent.

[0229] In certain embodiments, the polypeptide may be conjugated to one or more moieties of interest, where one or more amino acid residues of the polypeptide are native amino acid residues of the polypeptide. For example, the polypeptide may be conjugated to one or more moieties of interest, where one or more amino acid residues of the polypeptide are not modified before conjugation to the moiety of interest. Examples of native amino acid residues that may be conjugated to the one or more moieties of interest include, but are not limited to, cysteine, lysine, and the like. In some cases, the amino acid residue that is conjugated to the one or more moieties of interest is a native amino acid residue that does not have an aldehyde group, and the conjugation moiety is not a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0230] In some cases, to produce the conjugate, the polypeptide may be conjugated to the moiety of interest by reaction of an amino acid residue of the polypeptide with a compound (e.g., a compound containing a conjugation moiety, as described herein). For example, a polypeptide may be contacted with a reactive partner-containing drug under conditions suitable to provide for conjugation of the drug to the polypeptide. In some instances, the reactive partner-containing drug may include a conjugation moiety as described above. For example, a drug or active agent may be modified to include a conjugation moiety. In some cases, the drug or active agent is attached to a conjugation moiety, such as covalently attached to conjugation moiety through a linker, such as a linker as described in detail herein.

[0231] In certain embodiments, a conjugate of the present disclosure includes a polypeptide (e.g., an antibody) having at least one amino acid residue that has been attached to one or more moieties of interest (e.g., drugs or active agents). In order to make the conjugate, an amino acid residue of the polypeptide (e.g., a native amino acid residue) may be coupled to one or more drugs or active agents attached to a conjugation moiety as described above. In certain embodiments, the native amino acid residue of the polypeptide (e.g., antibody) is a cysteine or lysine residue. In certain embodiments, the amino acid residue is conjugated to a drug or active agent containing a conjugation moiety as described above to provide a conjugate of the presentAtty. Dkt: RDWD-053WO disclosure where the one or more drugs or active agents are conjugated to the polypeptide through the conjugation moiety.

[0232] In certain embodiments, the conjugate includes a polypeptide (e.g., an antibody) having at least one native amino acid residue attached to a linker as described herein, which in turn is attached to one or more drugs or active agents. For instance, the conjugate may include a polypeptide (e.g., an antibody) having at least one native amino acid residue that is conjugated to the one or more moieties of interest (e.g., one or more drugs or active agents) as described above.

[0233] In certain embodiments, the conjugation moiety includes a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl compound. For instance, a general scheme for coupling a moiety of interest to a polypeptide through a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the general reaction scheme below. Hydrazinyl-indolyl and hydrazinyl-pyrrolo- pyridinyl conjugation moiety are also referred to herein as a hydrazino-iso-Pictet-Spengler (HIPS) conjugation moiety and an aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugation moiety, respectively. R'' R'' polypeptide N

[0234] a drug or active agent) that is conjugated to the polypeptide (e.g., conjugated to the polypeptide through a cleavable linker as described herein). As shown in the reaction scheme above, a polypeptide (e.g., antibody) that includes a 2-formylglycine residue (fGly) is reacted with a drug or active agent that has been modified to include a conjugation moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety) to produce a polypeptide conjugate attached to the conjugation moiety, thus attaching the drug or active agent to the polypeptide through the conjugation moiety.

[0235] As described herein, the moiety can be any of a variety of moieties such as, but not limited to, chemical entities, such as detectable labels, or drugs or active agents. R’ and R” may each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedAtty. Dkt: RDWD-053WO alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z may be CR21, NR22, N, O or S, where R21and R22are each independently selected from any of the substituents described for R’ and R” above.

[0236] Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties may be attached (e.g., covalently attached) to a linker. As such, embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety attached to a drug or active agent through a linker. Various embodiments of the linker that may couple the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the drug or active agent are described in detail herein. For example, in some instances, the linker is a cleavable linker, such as a cleavable linker as described herein.

[0237] In some instances, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties may be attached (e.g., covalently attached) to two or more linkers. As such, embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety attached to two or more drugs or active agents each through a corresponding linker. Thus, conjugates of the present disclosure may include two or more linkers, where each linker attaches a corresponding drug or active agent to the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. Accordingly, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety and two or more linkers may be viewed overall as a “branched linker”, where the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is attached to two of more “branches”, where each branch includes a linker attached to a drug or active agent.

[0238] In certain embodiments, the polypeptide may be conjugated to one or more moieties of interest, where one or more amino acid residues of the polypeptide are modified before conjugation to the moiety of interest. Modification of one or more amino acid residues of the polypeptide may produce a polypeptide that contains one or more reactive groups suitable for conjugation to the moiety of interest. In some cases, the polypeptide may include one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation toAtty. Dkt: RDWD-053WO the moiety of interest (e.g., one or more moieties that includes a conjugation moiety, such as a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above). For example, an amino acid of the polypeptide may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be included in an “aldehyde tag” or “ald-tag”, which as used herein refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR [SEQ ID NO:2]) that has been converted by action of a formylglycine generating enzyme (FGE) to contain a 2-formylglycine residue (referred to herein as “fGly”). The fGly residue generated by an FGE may also be referred to as a “formylglycine”. Stated differently, the term “aldehyde tag” is used herein to refer to an amino acid sequence that includes a “converted” sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to fGly by action of an FGE, e.g., L(fGly)TPSR [SEQ ID NO:3]). A converted sulfatase motif may be produced from an amino acid sequence that includes an “unconverted” sulfatase motif (i.e., a sulfatase motif in which the cysteine or serine residue has not been converted to fGly by an FGE, but is capable of being converted, e.g., an unconverted sulfatase motif with the sequence: L(C / S)TPSR [SEQ ID NO:2]). By “conversion” as used in the context of action of a formylglycine generating enzyme (FGE) on a sulfatase motif refers to biochemical modification of a cysteine or serine residue in a sulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly, or Ser to fGly).

[0239] In certain embodiments, the polypeptide (e.g., antibody) may be modified to include the fGly or aldehyde tag at any desired site of the polypeptide (e.g., antibody). Thus, the present disclosure provides, for example, a polypeptide (e.g., antibody) that has been site- specifically modified to include an fGly or aldehyde tag at a site at or near the C-terminus of the polypeptide. Other examples include a polypeptide (e.g., antibody) that has been site- specifically modified to include an fGly or aldehyde tag at a position at or near the N-terminus of the polypeptide. Examples also include a polypeptide (e.g., antibody) that has been site- specifically modified to include an fGly or aldehyde tag at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above are also possible where the polypeptide (e.g., antibody) has been site-specifically modified to include an fGly or aldehyde tag at two or more specific positions. Accordingly, embodiments of the polypeptide (e.g., antibody) include those where the polypeptide (e.g., antibody) includes one or more site-specifically engineered conjugation sites, such as an fGly orAtty. Dkt: RDWD-053WO aldehyde tag as described herein. Each site-specifically engineered conjugation site may be conjugated to one or more drugs or active agents through a linker as described herein.

[0240] Additional aspects of aldehyde tags and uses thereof in site-specific protein modification are described in U.S. Patent No. 7,985,783 and U.S. Patent No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

[0241] In some cases, to produce the conjugate, the polypeptide containing the fGly residue may be conjugated to the moiety of interest by reaction of the fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above). For example, an fGly-containing polypeptide may be contacted with a reactive partner-containing drug under conditions suitable to provide for conjugation of the drug to the polypeptide. In some instances, the reactive partner-containing drug may include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above. For example, a drug or active agent may be modified to include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. In some cases, the drug or active agent is attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl, such as covalently attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl through a linker, such as a linker as described in detail herein.

[0242] In certain embodiments, a conjugate of the present disclosure includes a polypeptide (e.g., an antibody) having at least one amino acid residue that has been attached to one or more moieties of interest (e.g., drugs or active agents). In order to make the conjugate, an amino acid residue of the polypeptide may be modified and then coupled to one or more drugs or active agents attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above. In certain embodiments, an amino acid residue of the polypeptide (e.g., antibody) is a cysteine or serine residue that is modified to an fGly residue, as described above. In certain embodiments, the modified amino acid residue (e.g. fGly residue) is conjugated to a drug or active agent containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety as described above to provide a conjugate of the present disclosure where the one or more drugs or active agents are conjugated to the polypeptide through the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As used herein, the term fGly’ refers to the modified amino acid residue of the polypeptide (e.g., antibody) that is coupled to the moiety of interest (e.g., a drug or active agent).Atty. Dkt: RDWD-053WO

[0243] In certain embodiments, the conjugate includes a polypeptide (e.g. an antibody) having at least one amino acid residue attached to a linker as described herein, which in turn is attached to one or more drugs or active agents. For instance, the conjugate may include a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly’) that is conjugated to the one or more moieties of interest (e.g., one or more drugs or active agents) as described above.

[0244] For example, in some instances, the conjugate includes one or more of a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker, and one or more of a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker.

[0245] Embodiments of the present disclosure include conjugates where an antibody is conjugated to one or more drug moieties, such as two or more drug moieties, such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties. The drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugates have an average DAR from 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). ). In certain embodiments, the conjugates have an average DAR of 8 to 12 (e.g., 10). In certain embodiments, the conjugates have an average DAR of 10 to 15 (e.g., 12). In certain embodiments, the conjugates have an average DAR of 15 to 20 (e.g., 16). By average is meant the arithmetic mean. In certain embodiments, the conjugates have an average DAR ranging from 2 to 20, such as 4 to 16.Atty. Dkt: RDWD-053WO

[0246] As indicated above, embodiments of the conjugates of the present disclosure include conjugates attached to one or more of a first drug and one or more of a second drug. In some cases, DAR refers to the total DAR of the conjugate (e.g., the average number of the first drug plus the second drug linked to each antibody). In some cases, DAR refers to the DAR of one of the drugs attached to the antibody (e.g., the average number of the first drug linked to each antibody, or the average number of the second drug linked to each antibody). In some instances, the total DAR of the conjugate is the DAR of the first drug plus the DAR of the second drug.

[0247] As indicated above, embodiments of the conjugates of the present disclosure include conjugates where the first drug is attached to the antibody at a native amino acid residue of the antibody through a first linker. In certain embodiments, the antibody has a DAR of 2 or more for the first drug. In these embodiments, two or more of the first drug can be attached to the antibody through two or more of the first linker. In some instances, the first linker is not a branched linker. For example, two of the first drug can be attached to the antibody through two of the first linker (e.g., DAR of 2 for the first drug), where each of the first linker is attached to the antibody at different native amino acid residues of the antibody. In other embodiments, four of the first drug can be attached to the antibody through four of the first linker (e.g., DAR of 4 for the first drug), where each of the first linker is attached to the antibody at different native amino acid residues of the antibody. In other embodiments, six of the first drug can be attached to the antibody through six of the first linker (e.g., DAR of 6 for the first drug), where each of the first linker is attached to the antibody at different native amino acid residues of the antibody. In other embodiments, eight of the first drug can be attached to the antibody through eight of the first linker (e.g., DAR of 8 for the first drug), where each of the first linker is attached to the antibody at different native amino acid residues of the antibody.

[0248] In other embodiments, the antibody can have a DAR of 2 or more (e.g., 4 or more) for the first drug, where the first linker is a branched linker. As such, in some instances, four of the first drug can be attached to the antibody through two branched first linkers (e.g., DAR of 4 for the first drug), where each branched first linker is attached to the antibody at different native amino acid residues of the antibody. In other embodiments, eight of the first drug can be attached to the antibody through four of the branched first linker (e.g., DAR of 8 for the first drug), where each of the branched first linker is attached to the antibody at differentAtty. Dkt: RDWD-053WO native amino acid residues of the antibody. In other embodiments, twelve of the first drug can be attached to the antibody through six of the branched first linker (e.g., DAR of 12 for the first drug), where each of the branched first linker is attached to the antibody at different native amino acid residues of the antibody. In other embodiments, sixteen of the first drug can be attached to the antibody through eight of the branched first linker (e.g., DAR of 16 for the first drug), where each of the branched first linker is attached to the antibody at different native amino acid residues of the antibody.

[0249] As indicated above, embodiments of the conjugates of the present disclosure include conjugates where the second drug is attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker. In certain embodiments, the antibody has a DAR of 2 or more for the second drug. In these embodiments, two or more of the second drug can be attached to the antibody through two or more of the second linker. In some instances, the second linker is not a branched linker. For example, two of the second drug can be attached to the antibody through two of the second linker (e.g., DAR of 2 for the second drug), where each of the second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody. In other embodiments, four of the second drug can be attached to the antibody through four of the second linker (e.g., DAR of 4 for the second drug), where each of the second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody. In other embodiments, six of the second drug can be attached to the antibody through six of the second linker (e.g., DAR of 6 for the second drug), where each of the second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody. In other embodiments, eight of the second drug can be attached to the antibody through eight of the second linker (e.g., DAR of 8 for the second drug), where each of the second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody.

[0250] In other embodiments, the antibody can have a DAR of 2 or more (e.g., 4 or more) for the second drug, where the second linker is a branched linker. As such, in some instances, four of the second drug can be attached to the antibody through two branched second linkers (e.g., DAR of 4 for the second drug), where each branched second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody. In other embodiments, eight of the second drug can be attached to the antibody through four of theAtty. Dkt: RDWD-053WO branched second linker (e.g., DAR of 8 for the second drug), where each of the branched second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody. In other embodiments, twelve of the second drug can be attached to the antibody through six of the branched second linker (e.g., DAR of 12 for the second drug), where each of the branched second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody. In other embodiments, sixteen of the second drug can be attached to the antibody through eight of the branched second linker (e.g., DAR of 16 for the second drug), where each of the branched second linker is attached to the antibody at different site-specifically engineered conjugation sites of the antibody.

[0251] In certain embodiments, the conjugate has a ratio of the first drug to the second drug (e.g., a ratio of the amount of the first drug linked to the antibody to the amount of the second drug linked to the antibody; or DAR(first drug) : DAR(second drug)) ranging from 4:1 to 1:1. For example, in some cases, the ratio of the first drug to the second drug is 8:2, e.g., 4:1. As such, in these embodiments, the conjugate may include eight of the first drug attached to the antibody through eight of the first linker (e.g., non-branched first linker) and two of the second drug attached to the antibody through two of the second linker (e.g., non-branched second linker). In some cases, the ratio of the first drug to the second drug is 8:4, e.g., 2:1. As such, in these embodiments, the conjugate may include eight of the first drug attached to the antibody through eight of the first linker (e.g., non-branched first linker) and four of the second drug attached to the antibody through four of the second linker (e.g., non-branched second linker), or may include eight of the first drug attached to the antibody through eight of the first linker (e.g., non-branched first linker) and four of the second drug attached to the antibody through two of the second linker (e.g., branched second linker). In some cases, the ratio of the first drug to the second drug is 8:8, e.g., 1:1. As such, in these embodiments, the conjugate may include eight of the first drug attached to the antibody through eight of the first linker (e.g., non-branched first linker) and eight of the second drug attached to the antibody through four of the second linker (e.g., branched second linker).

[0252] Combinations of the same or different payloads (e.g., drugs or active agents) may be conjugated to the antibody through the first linker and the second linker. In certain embodiments, the first drug attached to the first linker and the second drug attached to the secondAtty. Dkt: RDWD-053WO linker are the same. In other embodiments, the first drug attached to the first linker and the second drug attached to the second linker are different drugs or active agents.

[0253] In some embodiments, where two different drugs or active agents are attached to the first and second linkers, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. By “synergistic”, “synergism” or “synergy” is meant a therapeutic effect that is greater than the sum of the effects of the drugs or active agents taken separately. For example, in some instances, the use of two different drugs or active agents attached to the first and second linkers may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC.

[0254] In some embodiments, where two different drugs or active agents are attached to the first and second linkers, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately. For example, the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).

[0255] In some embodiments, where two different drugs or active agents are attached to the first and second linkers, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways. Examples of payload combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like.

[0256] In some embodiments, where two different payloads are attached to the first and second linkers, the payloads may be selected from combinations of drugs or active agents and detectable labels. For example, a first payload may be a detectable label that is used as an imaging agent or tracer to detect the location of the ADC in vivo, while a second payload may be a drug or active agent that provides a therapeutic activity.

[0257] Various embodiments of the linkers that may couple the conjugation moiety to the drugs or active agents are described in detail herein. For example, in some instances, the linker is a cleavable linker, such as a cleavable linker as described herein.

[0258] In certain embodiments, the conjugate is a conjugate of formula (I):Atty. Dkt: RDWD-053WO wherein AL is a first linker; LBis a second linker; W1is a first drug; W2is a second drug; W3is an antibody; XAis a first conjugation moiety attached to the antibody at a native amino acid residue of the antibody; and XBis a second conjugation moiety attached to the antibody at a site-specifically engineered conjugation site of the antibody.

[0259] The substituents related to conjugates of formula (I) are described in more detail below.

[0260] In certain embodiments, W1is a first drug (or active agent). Further description of drugs (or active agents) that find use in the subject conjugates is found in the disclosure herein.

[0261] In certain embodiments, W2is a second drug (or active agent). Further description of drugs (or active agents) that find use in the subject conjugates is found in the disclosure herein.

[0262] In certain embodiments, W3is a polypeptide. For example, W3can be an antibody. In certain embodiments, W3comprises one or more native amino acid residues that are attached to the first drug, W1, through the first linker, LA, as described herein. In certain embodiments, W3comprises one or more modified amino acid residues that are attached to the second drug, W2, through the second linker, LB, as described herein. In some instances, the modified amino acid residue (e.g., fGly) is included in a site-specifically engineered conjugation site (e.g., aldehyde tag) of the antibody as described herein. Further description of polypeptides and antibodies that find use in the subject conjugates is found in the disclosure herein.Atty. Dkt: RDWD-053WO

[0263] In certain embodiments, LAis a linker (e.g., a first linker as described herein). The first linker, LA, may be attached on one end of the linker to the first drug, W1, and attached at another end of the linker to the antibody, W3. Linkers suitable for LAare described in more detail below.

[0264] In certain embodiments, LBis a linker (e.g., a second linker as described herein). The second linker, LB, may be attached on one end of the linker to the second drug, W2, and attached at another end of the linker to the antibody, W3. Linkers suitable for LBare described in more detail below.

[0265] In certain embodiments, XAis a first conjugation moiety attached to the antibody at a native amino acid residue of the antibody. In certain embodiments, the antibody and the first drug are conjugated through the first conjugation moiety, XA. For example, the antibody and the first drug may each be bound (e.g., covalently bonded) to the first conjugation moiety, XA, thus indirectly binding the antibody and the first drug together through the conjugation moiety, XA. As described herein, one or more (e.g., 2, 4, 6, 8, etc.) of the first drug can be conjugated to the antibody. For example, each of the one or more first drug can be conjugated to the antibody each through their own first conjugation moiety, XA, or one or more pairs of the first drug can be conjugated to the antibody, each pair through their own first conjugation moiety, XA(e.g., where the first linker is a branched linker).

[0266] In some cases, the first conjugation moiety, XA, includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may react with and thus be attached (e.g., covalently attached) to a cysteine residue of the antibody, thus indirectly binding the antibody and the drug together through the maleimide conjugation moiety. In some instances, the cysteine residue is a native cysteine residue of the antibody.

[0267] Other conjugation groups that may be used for attachment to a cysteine residue of the antibody include P5 phosphoramidate or thiobridge conjugation moieties.

[0268] In some cases, the first conjugation moiety, XA, includes an N- hydroxysuccinimide (NHS) or derivative thereof. For instance, an NHS-ester conjugation moiety may react with and thus be attached (e.g., covalently attached) to a lysine residue of the antibody, thus indirectly binding the antibody and the drug together through an NHS-ester conjugation moiety. In some instances, the lysine residue is a native lysine residue of the antibody.Atty. Dkt: RDWD-053WO

[0269] Other conjugation groups that may be used for attachment include conjugation moieties for attachment to an N-terminal amine of the antibody. For example, an N-terminal amine can be oxidized with N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport’s salt, RS) to afford an aldehyde or ketone tagged antibody, followed by conjugation using an oxime or hydrazone linkage.

[0270] In certain embodiments, XBis a second conjugation moiety attached to the antibody at a site-specifically engineered conjugation site of the antibody. In certain embodiments, the antibody and the second drug are conjugated through the second conjugation moiety, XB. For example, the antibody and the second drug may each be bound (e.g., covalently bonded) to the second conjugation moiety, XB, thus indirectly binding the antibody and the second drug together through the second conjugation moiety, XB. As described herein, one or more (e.g., 2, 4, 6, 8, etc.) of the second drug can be conjugated to the antibody. For example, each of the one or more second drug can be conjugated to the antibody each through their own second conjugation moiety, XB, or one or more pairs of the second drug can be conjugated to the antibody, each pair through their own second conjugation moiety, XB(e.g., where the second linker is a branched linker).

[0271] As described above, the conjugate of formula (I) includes a first linker, LA. The first linker may be utilized to bind one or more moieties of interest (e.g., drug or active agent) to one or more polypeptides (e.g., antibody) through the first conjugation moiety, XA. The first linker may be bound (e.g., covalently bonded) to the first conjugation moiety (e.g., as described herein) at any convenient position. For example, the first linker may attach the first conjugation moiety to a first drug. The first conjugation moiety may be used to conjugate the first linker (and thus the first drug) to a polypeptide (e.g., antibody). For example, the first conjugation moiety may be used to conjugate the first linker (and thus the first drug) to a native amino acid residue of the antibody, as described herein.

[0272] In some instances, as shown in formula (I) above, LAis attached to W3through the first conjugation moiety, XA, and thus W3is indirectly bonded to the first linker LAthrough the first conjugation moiety. As described above, W3is an antibody, and thus LAis attached through the first conjugation moiety, XA, to the antibody, e.g., the linker LAis indirectly bonded to the antibody through the first conjugation moiety, XA.Atty. Dkt: RDWD-053WO

[0273] As described above, the conjugate of formula (I) includes a second linker, LB. The second linker may be utilized to bind one or more moieties of interest (e.g., drug or active agent) to one or more polypeptides (e.g., antibody) through the second conjugation moiety, XB. The second linker may be bound (e.g., covalently bonded) to second conjugation moiety (e.g., as described herein) at any convenient position. For example, the second linker may attach the second conjugation moiety to a second drug. The second conjugation moiety may be used to conjugate the second linker (and thus the second drug) to a polypeptide (e.g., antibody). For example, the second conjugation moiety may be used to conjugate the second linker (and thus the second drug) to a modified amino acid residue in a site-specifically engineered conjugation site of the antibody, as described herein.

[0274] In some instances, as shown in formula (I) above, LBis attached to W3through the second conjugation moiety, XB, and thus W3is indirectly bonded to the second linker LBthrough the second conjugation moiety. As described above, W3is an antibody, and thus LBis attached through the second conjugation moiety, XB, to the antibody, e.g., the linker LBis indirectly bonded to the antibody through the second conjugation moiety, XB.

[0275] In one embodiment, of conjugates having a branched linker, the conjugate has formula (II): whereinLAis a first linker; LBis a second linker; LB1is a third linker; W1is the first drug; W2is the second drug; W2Ais a third drug; W3is an antibody; XAis a first conjugation moiety attached to the antibody at a native amino acid residue of the antibody; andAtty. Dkt: RDWD-053WO XBis a second conjugation moiety attached to the antibody at a site-specifically engineered conjugation site of the antibody.

[0276] With reference to formula (II), second and third linker can be the same or different. For example, in one embodiment of conjugates according to formula (II), LBand LB1are the same.

[0277] With continued reference to formula (II), the second drug and the third drug can be the same or different. For example, in one embodiment, of conjugates according to formula (II), W2and W2Aare the same.

[0278] Any convenient linker may be utilized for the linker LAor LBor LB1in the subject conjugates and compounds. Further description of the linker (e.g., LAor LBor LB1) is provided herein. References to the linker below are intended to describe LAor LBor LB1.

[0279] In certain embodiments, the linker may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the linker may include an alkyl or substituted alkyl group. In certain embodiments, the linker may include an alkenyl or substituted alkenyl group. In certain embodiments, the linker may include an alkynyl or substituted alkynyl group. In certain embodiments, the linker may include an alkoxy or substituted alkoxy group. In certain embodiments, the linker may include an amino or substituted amino group. In certain embodiments, the linker may include a carboxyl or carboxyl ester group. In certain embodiments, the linker may include an acyl amino group. In certain embodiments, the linker may include an alkylamide or substituted alkylamide group. In certain embodiments, the linker may include an aryl or substituted aryl group. In certain embodiments, the linker may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the linker may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the linker may include a heterocyclyl or substituted heterocyclyl group.

[0280] In certain embodiments, the linker may include a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where theAtty. Dkt: RDWD-053WO homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is a polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below.

[0281] In some embodiments, the linker (e.g., a first linker or a second linker as described herein) is described by the formula: -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-(L6)f-, wherein L1, L2, L3, L4, L5and L6are each independently a linker subunit, and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.

[0282] In certain embodiments, the sum of a, b, c, d, e and f is 1. In certain embodiments, the sum of a, b, c, d, e and f is 2. In certain embodiments, the sum of a, b, c, d, e and f is 3. In certain embodiments, the sum of a, b, c, d, e and f is 4. In certain embodiments, the sum of a, b, c, d, e and f is 5. In certain embodiments, the sum of a, b, c, d, e and f is 6. In certain embodiments, a, b, c, d, e and f are each 1. In certain embodiments, a, b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e and f are each 0. In certain embodiments, a is 1 and b, c, d, e and f are each 0.

[0283] In certain embodiments, the linker subunit L1is attached to the conjugation moiety (e.g., XAor XB, as shown in formula (I) above). In certain embodiments, the linker subunit L2, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, the linker subunit L3, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, the linker subunit L4, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, the linker subunit L5, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, the linker subunit L6, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above).

[0284] Any convenient linker subunits may be utilized in the linker. Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, amino acid residue(s), carbohydrate-based polymers orAtty. Dkt: RDWD-053WO carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, each of L1, L2, L3, L4, L5and L6(if present) comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine).

[0285] In some embodiments, L1(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L1comprises a polyethylene glycol. In some embodiments, L1comprises a modified polyethylene glycol. In some embodiments, L1comprises an amino acid residue. In some embodiments, L1comprises an alkyl group or a substituted alkyl. In some embodiments, L1comprises an aryl group or a substituted aryl group. In some embodiments, L1comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0286] In some embodiments, L2(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L2comprises a polyethylene glycol. In some embodiments, L2comprises a modified polyethylene glycol. In some embodiments, L2comprises an amino acid residue. In some embodiments, L2comprises an alkyl group or a substituted alkyl. In some embodiments, L2comprises an aryl group or a substituted aryl group. In some embodiments, L2comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0287] In some embodiments, L3(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L3comprises a polyethylene glycol. In some embodiments, L3comprises a modified polyethylene glycol. In some embodiments, L3comprises an amino acid residue. In some embodiments, L3comprises an alkyl group or a substituted alkyl. In some embodiments, L3comprises an aryl group or a substituted aryl group. In some embodiments, L3comprises a diamine (e.g., a linking group comprising an alkylene diamine).Atty. Dkt: RDWD-053WO

[0288] In some embodiments, L4(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L4comprises a polyethylene glycol. In some embodiments, L4comprises a modified polyethylene glycol. In some embodiments, L4comprises an amino acid residue. In some embodiments, L4comprises an alkyl group or a substituted alkyl. In some embodiments, L4comprises an aryl group or a substituted aryl group. In some embodiments, L4comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0289] In some embodiments, L5(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L5comprises a polyethylene glycol. In some embodiments, L5comprises a modified polyethylene glycol. In some embodiments, L5comprises an amino acid residue. In some embodiments, L5comprises an alkyl group or a substituted alkyl. In some embodiments, L5comprises an aryl group or a substituted aryl group. In some embodiments, L5comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0290] In some embodiments, L6(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L6comprises a polyethylene glycol. In some embodiments, L6comprises a modified polyethylene glycol. In some embodiments, L6comprises an amino acid residue. In some embodiments, L6comprises an alkyl group or a substituted alkyl. In some embodiments, L6comprises an aryl group or a substituted aryl group. In some embodiments, L6comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0291] In some embodiments, the linker comprises -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-(L6)f-, where: -(L1)a- is -(T1-V1)a-; -(L2)b- is -(T2-V2)b-; -(L3)c- is -(T3-V3)c-; -(L4)d- is -(T4-V4)d-; -(L5)e- is -(T5-V5)e-; andAtty. Dkt: RDWD-053WO -(L6)f- is -(T6-V6)f-, wherein T1, T2, T3, T4, T5and T6, if present, are tether groups; V1, V2, V3, V4, V5and V6, if present, are covalent bonds or linking functional groups; and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.

[0292] As described above, in certain embodiments, L1is attached to the conjugation moiety (e.g., XAor XB, as shown in formula (I) above). As such, in certain embodiments, T1is attached to the conjugation moiety (e.g., XAor XB, as shown in formula (I) above). In certain embodiments, V1is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, L2, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). As such, in certain embodiments, T2, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above), or V2, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, L3, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). As such, in certain embodiments, T3, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above), or V3, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, L4, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). As such, in certain embodiments, T4, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above), or V4, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, L5, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). As such, in certain embodiments, T5, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above), or V5, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). In certain embodiments, L6, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above). As such, in certain embodiments, T6, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above), or V6, if present, is attached to the drug (e.g., W1or W2or W2A, as shown in formula (I) above).

[0293] Regarding the tether groups, T1, T2, T3, T4, T5and T6, any convenient tether groups may be utilized in the subject linkers. In some embodiments, T1, T2, T3, T4, T5and T6each comprise one or more groups independently selected from a covalent bond, a (C1-C12)alkyl,Atty. Dkt: RDWD-053WO a substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, - (CR13OH)m-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino- benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, where each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12.

[0294] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes a (C1-C12)alkyl or a substituted (C1-C12)alkyl. In certain embodiments, (C1-C12)alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, (C1-C12)alkyl may be an alkyl or substituted alkyl, such as C1-C12alkyl, or C1-C10alkyl, or C1-C6alkyl, or C1-C3alkyl. In some instances, (C1-C12)alkyl is a C2-alkyl. For example, (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C1-C12 alkylene, or C1-C10 alkylene, or C1-C6 alkylene, or C1-C3 alkylene. In some instances, (C1-C12)alkyl is a C2-alkylene (e.g., CH2CH2). In some instances, (C1-C12)alkyl is a C3-alkylene (e.g., CH2CH2CH2).

[0295] In certain embodiments, substituted (C1-C12)alkyl is a straight chain or branched substituted alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, substituted (C1-C12)alkyl may be a substituted alkyl, such as substituted C1-C12 alkyl, or substituted C1-C10 alkyl, or substituted C1-C6 alkyl, or substituted C1-C3alkyl. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkyl. For example, substituted (C1-C12)alkyl may be a substituted alkylene, such as substituted C1-C12 alkylene, or substituted C1-C10 alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkylene. In some instances, substituted (C1-C12)alkyl is a substituted C3-alkylene. For example, substituted (C1-C12)alkyl may include C1-C12 alkylene (e.g., C3-alkylene or C5-alkylene) substituted with a (PEG)n group as described herein (e.g.,-CONH(PEG)3or -NHCO(PEG)7), or may include C1-C12alkylene (e.g., C3-Atty. Dkt: RDWD-053WO alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12alkylene (e.g., C5-alkylene) substituted with a -NHCOCH2SO3H group.

[0296] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5and T6) includes an aryl or substituted aryl. For example, the aryl can be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl can be substituted with one or more substituents selected from (C1-C12)alkyl, a substituted (C1- C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some instances, the substituted aryl is a substituted phenyl, where the substituent includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).

[0297] In some instances, the tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes a heteroaryl or substituted heteroaryl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5and T6) includes a cycloalkyl or substituted cycloalkyl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5and T6) includes a heterocyclyl or substituted heterocyclyl. In some instances, the substituent on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclyl includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).

[0298] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, where the aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl is produced from a reaction, such as a click-chemistry reaction. For example, click- chemistry reactions may be used to connect two portions of a linker together during synthesis of the linker. Examples of click-chemistry reactions that can be used to produce an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl tether group include, but are not limited to, azide-alkyne reactions, alkyne-DBCO (dibenzocyclooctyne) reactions, TCO-Tz (trans-cycloctene tetrazine) reactions, and the like (see, e.g., FIG. 16).Atty. Dkt: RDWD-053WO

[0299] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether group. In certain embodiments, (EDA)w includes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6). The linked ethylene diamine (EDA) moieties may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the EDA moiety is described by the structure: , 11 to 6, or is 0 or 1, and each R2is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5 or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl. In certain embodiments, any two adjacent R12groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R12groups are an alkyl group, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R12groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

[0300] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidin-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the 4AP moiety is described by the structure:Atty. Dkt: RDWD-053WO where R12is selected from hydrogen, alkyl, a polyethylene glycol moiety (e.g.,a polyethylene glycol or a modified , alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R12is a polyethylene glycol moiety. In certain embodiments, R12is a carboxy modified polyethylene glycol.

[0301] In certain embodiments, R12includes a polyethylene glycol moiety described by the formula: (PEG)k, which may be represented by the structure: , where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, k is 2. In certain embodiments, R17is selected from OH, OR, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R17is COOH. In certain embodiments, R17is OH. In certain embodiments, R17is OR, such as OCH3.

[0302] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes (PEG)n, where (PEG)n is a polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG)nis described by the structure: ,where n an 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, n is 2. In some instances, n is 3. In some instances, n is 6. In some instances, n is 12.Atty. Dkt: RDWD-053WO

[0303] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes (AA)p, where AA is an amino acid residue. Any convenient amino acids may be utilized. Amino acids of interest include but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary alpha-amino acids and beta-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as a non-naturally occurring alpha-amino acid or a non-naturally occurring beta-amino acid, etc. In certain embodiments, p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[0304] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes an amino acid analog. Amino acid analogs include compounds that are similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and / or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α- hydroxy acids, and α-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.

[0305] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes a moiety described by the formula -(CR13OH)m-, where m is 0 or n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, R13is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino,Atty. Dkt: RDWD-053WO carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R13is hydrogen. In certain embodiments, R13is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R13is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R13is alkynyl or substituted alkynyl. In certain embodiments, R13is alkoxy or substituted alkoxy. In certain embodiments, R13is amino or substituted amino. In certain embodiments, R13is carboxyl or carboxyl ester. In certain embodiments, R13is acyl or acyloxy. In certain embodiments, R13is acyl amino or amino acyl. In certain embodiments, R13is alkylamide or substituted alkylamide. In certain embodiments, R13is sulfonyl. In certain embodiments, R13is thioalkoxy or substituted thioalkoxy. In certain embodiments, R13is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R13is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6 substituted heteroaryl. In certain embodiments, R13is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R13is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.

[0306] In certain embodiments, R13is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13.

[0307] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5and / or T6) includes a meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino- benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para- amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxy-phenyl (PHP).Atty. Dkt: RDWD-053WO

[0308] In some embodiments, a tether includes a MABO group described by the following structure: . embodiments, a tether includes a MABC group described by thefollowing structure: .a tether includes a PABO group described by the following structure:structure: .a tether includes a PABA group described by the following structure:Atty. Dkt: RDWD-053WO . a tether includes a PAP group described by the followingstructure: .embodiments, a tether includes a PHP group described by the following structure: .embodiments, each R14is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0317] In certain embodiments, R14is hydrogen. In certain embodiments, each R14is hydrogen. In certain embodiments, R14is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R14is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R14is alkynyl or substituted alkynyl. In certain embodiments, R14is alkoxy or substituted alkoxy. In certain embodiments, R14is amino or substituted amino. In certain embodiments, R14is carboxyl or carboxyl ester. In certain embodiments, R14is acyl or acyloxy. In certain embodiments, R14is acyl amino or amino acyl. In certain embodiments, R14is alkylamide or substituted alkylamide. In certain embodiments, R14is sulfonyl. In certain embodiments, R14is thioalkoxy or substituted thioalkoxy. In certain embodiments, R14is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl,Atty. Dkt: RDWD-053WO or a C6aryl or C6substituted aryl. In certain embodiments, R14is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R14is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R14is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.

[0318] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0319] In certain embodiments of the linker L, one or more of the tether groups T1, T2, T3, T4, T5or T6is each optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0320] In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O- GalNAc.

[0321] For example, in some embodiments, the glycoside or glycoside derivative can be selected from the following structures:Atty. Dkt: RDWD-053WO ,

[0322] Regarding the linking functional groups, and any convenient linking functional groups may be utilized in the linker. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like. In some embodiments, V1, V2, V3, V4, V5and V6are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, - CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and - P(O)OH-, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0323] In some embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0324] In certain embodiments, R15is hydrogen. In certain embodiments, each R15is hydrogen. In certain embodiments, R15is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R15is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substitutedAtty. Dkt: RDWD-053WO alkenyl. In certain embodiments, R15is alkynyl or substituted alkynyl. In certain embodiments, R15is alkoxy or substituted alkoxy. In certain embodiments, R15is amino or substituted amino. In certain embodiments, R15is carboxyl or carboxyl ester. In certain embodiments, R15is acyl or acyloxy. In certain embodiments, R15is acyl amino or amino acyl. In certain embodiments, R15is alkylamide or substituted alkylamide. In certain embodiments, R15is sulfonyl. In certain embodiments, R15is thioalkoxy or substituted thioalkoxy. In certain embodiments, R15is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R15is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R15is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R15is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.

[0325] In certain embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15.

[0326] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.

[0327] As described above, in some embodiments, the linker comprises -(T1-V1)a-(T2- V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, where a, b, c, d, e and f are each independently 0 or 1, where the sum of a, b, c, d, e and f is 1 to 6.

[0328] In some embodiments, the linker comprises the following: T1is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl;Atty. Dkt: RDWD-053WO T2, T3, T4, T5and T6are each independently selected from (C1-C12)alkyl, substituted (C1- C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)m-, 4- amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine, and an ester; and V1, V2, V3, V4,V5and V6are each independently selected from a covalent bond, -CO-, - NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, - SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; wherein:integer from 1 to 6 and r is 0 or 1;4-amino- ; AA is an aminofrom 1 to 20; and each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; each R13is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0329] In certain embodiments, the left-hand side of the linker structure is attached to the conjugation moiety, and the right-hand side of the linker structure is attached to the drug, e.g., as shown in formula (I).Atty. Dkt: RDWD-053WO

[0330] In certain embodiments of the conjugate of formula (I), the antibody can be linked to one drug or active agent through the conjugation moiety and linker. In some instances, the antibody can be linked to more than one drug or active agent through the conjugation moiety and linker. For example, the conjugation moiety can be linked to two or more drugs or active agents. Each drug or active agent can be linked via a corresponding linker to the same conjugation moiety, which in turn can be attached to the antibody as described herein, thus linking the antibody to two or more drugs or active agents.

[0331] For example, in certain embodiments of the conjugate of formula (I), the linker (e.g., LAor LB) is a branched linker. In embodiments having a branched linker, one of T1, T2, T3, T4, T5, T6, V1, V2, V3, V4,V5or V6is a branched group. In some embodiments, the branched group is selected from -CONR15- and 4AP. In some embodiments, the branched group is - CONR15-. In some embodiments, the branched group is 4AP. In certain embodiments, the branched group is attached to a third linker, LC. As such, the branched group of the linker, LAor LB, can be attached to a two drugs, one on each branch of the branched linker.

[0332] In certain embodiments, the third linker LCis a linker described by the formula: -(L7)g-(L8)h-(L9)i-(L10)j-(L11)k-(L12)l-, wherein L7, L8, L9, L10, L11and L12are each independently a linker subunit, and g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and l is 1 to 6.

[0333] In certain embodiments, the sum of g, h, i, j, k and l is 1. In certain embodiments, the sum of g, h, i, j, k and l is 2. In certain embodiments, the sum of g, h, i, j, k and l is 3. In certain embodiments, the sum of g, h, i, j, k and l is 4. In certain embodiments, the sum of g, h, i, j, k and l is 5. In certain embodiments, the sum of g, h, i, j, k and l is 6. In certain embodiments, g, h, i, j, k and l are each 1. In certain embodiments, g, h, i, j and k are each 1 and l is 0. In certain embodiments, g, h, i and j are each 1 and k and l are each 0. In certain embodiments, g, h, and i are each 1 and j, k and l are each 0. In certain embodiments, g and h are each 1 and i, j, k and l are each 0. In certain embodiments, g is 1 and h, i, j, k and l are each 0.

[0334] In certain embodiments, the linker subunit L7is attached to the branched group. In certain embodiments, the linker subunit L8, if present, is attached to the drug. In certain embodiments, the linker subunit L9, if present, is attached to the drug. In certain embodiments, the linker subunit L10, if present, is attached to the drug. In certain embodiments, the linkerAtty. Dkt: RDWD-053WO subunit L11, if present, is attached to the drug. In certain embodiments, the linker subunit L12, if present, is attached to the drug.

[0335] Any convenient linker subunits may be utilized in the third linker LC. For example, any of the linker subunits described above in relation to L1, L2, L3, L4, L5and L6may be used for the linker subunits L7, L8, L9, L10, L11and L12.

[0336] In certain embodiments, the third linker LCis a linker comprising: -(L7)g-(L8)h-(L9)i-(L10)j-(L11)k-(L12)l-, where: -(L7)g- is -(T7-V7)g-; -(L8)h- is -(T8-V8)h-; -(L9)i- is -(T9-V9)i-; -(L10)j- is -(T10-V10)j-; -(L11)k- is -(T11-V11)k-; and -(L12)l- is -(T12-V12)l-, wherein T7, T8, T9, T10, T11and T12, if present, are tether groups; V7, V8, V9, V10, V11and V12, if present, are covalent bonds or linking functional groups; and g, h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and l is 1 to 6.

[0337] Accordingly, in certain embodiments, the third linker LCcomprises: -(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-, wherein g, h, i, j, k and 1 are each independently 0 or 1; T7, T8, T9, T10, T11and T12are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)m-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino- benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an aminoAtty. Dkt: RDWD-053WO acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12; V7, V8, V9, V10,V11and V12are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0338] Any convenient tether groups may be utilized for T7, T8, T9, T10, T11and T12. For example, any of the tether groups described above in relation to T1, T2, T3, T4, T5and T6may be used for the tether groups T7, T8, T9, T10, T11and T12.

[0339] Any convenient linking functional groups may be utilized for V7, V8, V9, V10,V11and V12. For example, any of the linking functional groups described above in relation to V1, V2, V3, V4, V5and V6may be used for the linking functional groups V7, V8, V9, V10,V11and V12.

[0340] In certain embodiments, each R13is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13.

[0341] In certain embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15. In these embodiments, various possible substituents are as described above for R15.

[0342] In certain embodiments of the third linker LC, one or more of the tether groups T7, T8, T9, T10, T11and T12is each optionally substituted with a glycoside or glycoside derivative. InAtty. Dkt: RDWD-053WO certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0343] In certain embodiments of the third linker LC, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0344] In certain embodiments of the third linker LC, the left-hand side of the linker structure is attached to the branched group of the linker (e.g., LAor LB) and the right-hand side of the linker structure is attached to the drug.

[0345] In certain embodiments, the conjugate is an antibody-drug conjugate where the antibody and the drug are linked together by a linker (e.g., LAand / or LBand / or LC), as described above. In some instances, the linker (e.g., LAand / or LBand / or LC) is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moiety includes one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separatable portions. For example, the cleavable moiety may include one or more covalent bonds, which under certain conditions, can dissociate or break apart to separate the cleavable linker into two or more portions. As such a cleavable linker can be included in an antibody-drug conjugate, such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at a desired target site of action for the drug.

[0346] In some instances, the cleavable linker is a chemically-cleavable linker, where one or more covalent bonds in the linker can be dissociated under certain chemical conditions (e.g., acid hydrolysis, etc.). In some instances, the cleavable linker is an enzymatically-cleavable linker, where one or more covalent bonds in the linker can be dissociated due to the enzymatic action of an enzyme.

[0347] In some instances, the cleavable linker includes two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configuredAtty. Dkt: RDWD-053WO such that cleavage of both cleavable moieties is needed in order to separate or release the drug from the antibody at a desired target site of action for the drug. For example, cleavage of the cleavable linker can be achieved by initially cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker includes a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety. By “hinders cleavage” is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood or substantially inhibits the cleavage of the first cleavable moiety, thus substantially reducing the amount or preventing the cleavage of the cleavable linker. For instance, the presence of uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety. The hinderance of cleavage of the first cleavable moiety by the presence of the second cleavable moiety, in turn, substantially reduces the amount or prevents the release of the drug from the antibody. For example, the premature release of the drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired target site of action for the drug.

[0348] In some cases, since the second cleavable moiety hinders cleavage of the first cleavable moiety, cleavage of the cleavable linker can be achieved by initially cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the hinderance on the cleavage of the first cleavable moiety, thus allowing the first cleavable moiety to be cleaved. Cleavage of the first cleavable moiety can result in the cleavable linker dissociating or separating into two or more portions as described above to release the drug from the antibody-drug conjugate. In some instances, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. By substantially is meant that about 10% or less cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety, such as about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety.

[0349] Stated another way, the second cleavable moiety can protect the first cleavable moiety from cleavage. For instance, the presence of uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, and thus substantially reduce or preventAtty. Dkt: RDWD-053WO premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. As such, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thus allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, which, in turn, separates or releases the drug from the antibody at a desired target site of action for the drug as described above. In certain instances, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not in and of itself result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still needed in order to cleave the cleavable linker).

[0350] The cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety and the second cleavable moiety can be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more portions as described above through the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved through the enzymatic action of an enzyme, such as, but not limited to, a peptide, a glycoside, and the like. In some instances, the enzyme that cleaves the enzymatically cleavable moiety is present at a desired target site of action, such as the desired target site of action of the drug that is to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in a significant amount in other areas, such as in whole blood, plasma or serum. As such, the cleavage of an enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, whereas cleavage does not significantly occur in other areas or before the antibody-drug conjugate reaches the desired site of action.

[0351] For example, as described herein, antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, such as for the delivery of a cancer therapeutic drug to a desired site of action where the cancer cells are present. In some cases, enzymes, such as the protease enzyme cathepsin B, can be a biomarker for cancer that is overexpressed in cancer cells. The overexpression, and thus localization, of certain enzymes in cancer can be used in the context of the enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desiredAtty. Dkt: RDWD-053WO site of action (i.e., the site of the cancer (and overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme that is overexpressed in cancer cells. For instance, the enzyme can be the protease enzyme cathepsin B. As such, in some instances, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by a protease enzyme, such as cathepsin B.

[0352] In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide can be any peptide suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as an enzymatically cleavable moiety include, for example, Val-Ala, Phe-Lys, and the like. For example, the first cleavable moiety described above (i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can include a peptide. The presence of uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from cleavage by a protease enzyme (e.g., cathepsin B), and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. In some instances, one of the amino acid residues of the peptide that comprises the first cleavable moiety is linked to or includes a substituent, where the substituent comprises the second cleavable moiety. In some instances, the second cleavable moiety includes a glycoside or glycoside derivative as described herein.

[0353] In some embodiments, the enzymatically cleavable moiety is sugar moiety, such as a glycoside (or glyosyl) or glycoside derivative. In some cases, the glycoside or glycoside derivative can facilitate an increase in the hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside or glycoside derivative. The glycoside can be any glycoside or glycoside derivative suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative. For instance, in some embodiments, the first cleavable moiety includes a peptide and the second cleavable moiety includes a glycoside or glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O- GlcNAc, and O-GalNAc. In some instances, the second cleavable moiety is a glucuronide orAtty. Dkt: RDWD-053WO derivative thereof. In some instances, the second cleavable moiety is a galactoside or derivative thereof. In some instances, the second cleavable moiety is a glucoside or derivative thereof. In some instances, the second cleavable moiety is a mannoside or derivative thereof. In some instances, the second cleavable moiety is a fucoside or derivative thereof. In some instances, the second cleavable moiety is O-GlcNAc or derivative thereof. In some instances, the second cleavable moiety is O-GalNAc or derivative thereof.

[0354] The glycoside or glycoside derivative can be attached (covalently bonded) to the cleavable linker through a glycosidic bond. The glycosidic bond can link the glycoside or glycoside derivative to the cleavable linker through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O-glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). In some instances, the glycosidic bond is an O-glycosidic bond (an O-glycoside). In some cases, the glycoside or glycoside derivative can be cleaved from the cleavable linker it is attached to by an enzyme (e.g., through enzymatically-mediated hydrolysis of the glycosidic bond). A glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme that is able to carry out the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. An example of an enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is a glucuronidase, a glycosidase, such as a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like. Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate. For instance, the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, found in cells at or near the desired site of action for the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the target site where the enzyme that mediates cleavage of the first cleavable moiety is found.

[0355] Embodiments that include a linker with the two cleavable moieties as described above can also be referred to as having a “tandem-cleavage” linker.Atty. Dkt: RDWD-053WO

[0356] In some instances, T1, T2, T3, T4, T5and T6and V1, V2, V3, V4, V5and V6are selected from the following: wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is an amino acid analog and V2is -NH-; T3is (PEG)n and V3is -CO-; T4is (AA)pand V4is absent; T5is PABC and V5is absent; and f is 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)p and V3is absent; T4is PABC and V4is absent; and e and f are each 0.

[0357] As described above, in some cases, the conjugation moiety, X, includes maleimide or derivative thereof. For instance, a maleimide conjugation moiety may be attached to a cysteine residue of the antibody, such as a native cysteine residue of the antibody. In these instances and with reference to formula (I), XAis attached to W3via a cysteine residue of W3. In one embodiment of such compounds, XAis attached to W3via maleimide-based conjugation.

[0358] In one embodiment of conjugates of formula (I), wherein XAis attached to W3via maleimide based conjugation, the conjugate has formula (Ia): , wherein LA, W1, XB, LB, W2and W3are as set forth above with

[0359] In one embodiment of conjugates according to formulas (I) and (Ia), the conjugate has formula (Ib):Atty. Dkt: RDWD-053WO ,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

[0360] In one embodiment conjugates of formulas (I), (Ia) and (Ib) have a structure selected from the group consisting of: ,Atty. Dkt: RDWD-053WO , , , ,Atty. Dkt: RDWD-053WOAtty. Dkt: RDWD-053WO .

[0361] With continued reference to formula (I), in one embodiment, the conjugation moiety, X, includes an acetamide or derivative thereof. For instance, an acetamide conjugation moiety may be attached to a cysteine residue of the antibody, such as via alkylation of a native cysteine residue of the antibody with an alpha-haloacetamide, such as alpha-bromoacetamide. In these instances and with reference to formula (I), XAis attached to W3via a cysteine residue of W3. In one embodiment of such compounds, XAis attached to W3via acetamide-based conjugation.

[0362] In one embodiment, compounds of formula (I) attached to W3via acetamide- based conjugation have formula (Ic): ,R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl.

[0363] In one embodiment of conjugates according to formulas (I) and (Ic), the conjugate has formula (Id):Atty. Dkt: RDWD-053WO ,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

[0364] In certain embodiments, conjugates of formulas (I), (Ic) and (Id) are selected from the group consisting of:Atty. Dkt: RDWD-053WO .

[0365] As mentioned above, in one embodiment, conjugates of formula (I) are attached W3via a formyl glycine residue of W3. Thus in one embodiment, XBis attached to W3via a formyl glycine residue of W3. In one such embodiment, XBis attached to W3via HIPS or azaHIPS-based conjugation. In another embodiment of conjugates according to formula (I), XBis attached to W3via thiazolidine conjugation.

[0366] In one embodiment of conjugates linked according to HIPs or azaHIPS-based conjugation, conjugates of formulas (I), (Ia), (Ib), (Ic), and (Id) have formula (Ie): ,R’ and R” are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; Z represents CR21, NR22, N, O or S; andAtty. Dkt: RDWD-053WO R21and R22are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0367] In a further embodiment of conjugates of formulas (I), (Ia), (Ib), (Ic), (Id) and (Ie), the conjugate has formula (If): . of conjugates of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie)and (If), the conjugate has formula (Ig): , wherein:each R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; R3comprises a cleavable moiety.Atty. Dkt: RDWD-053WO

[0369] In certain embodiments, conjugates according to formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If) and (Ig), the conjugate is selected from the group consisting of: , .to W3via a formyl glycine residue of W3using thiazolidine conjugation. In one embodiment, conjugates of formulas (I) through (Id) have formula (Ih): .of conjugates according to formulas (I), (Ia), (Ib), (Ic), (Id) and (Ih), the conjugate has formula (Ii)..

[0372] By way of example, a conjugate of formulas (I), (Ia), (Ib), (Ic), (Id), (Ih) and (Ii) may have the structure:Atty. Dkt: RDWD-053WO

[0373] Embodiments of branched conjugates disclosed herein, such as those of formula (II), can be conjugated to W3by similar methods as with respect to unbranched conjugates, such as those illustrated above. For example, one embodiment of formula (II) uses maleimide-based conjugation to a native cysteine of W3. In one embodiment, such conjugates have formula (IIa): .formulas (II) and (Iia) may have formula (IIb): (IIb),each R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; andAtty. Dkt: RDWD-053WO R3comprises a cleavable moiety.

[0375] Further embodiments of branched conjugates may rely on acetamide-based conjugation. Accordingly, certain embodiments of conjugates have formula (IIc): (IIc),R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl.

[0376] Further conjugates of formulas (II) and (IIc) may have formula (IId): (IId),each R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

[0377] In one embodiment, a branched conjugate according to formulas (II), (IIa), (IIb), (IIc) and (IId) has formula (IIe):Atty. Dkt: RDWD-053WO W3LAXAW1N .

[0378] a branched conjugate according to formulas (II), (IIa), (IIb), (IIc), (IId) and (IIe) has the structure: .Atty. Dkt: RDWD-053WO .

[0380] Any of the chemical entities, linkers and coupling moieties set forth in the structures above may be adapted for use in the subject compounds and conjugates.

[0381] Additional disclosure related to hydrazinyl-indolyl and hydrazinyl-pyrrolo- pyridinyl compounds and methods for producing a conjugate is found in U.S. Patent No. 9,310,374 and U.S. Patent No. 9,493,413, the disclosures of each of which are incorporated herein by reference. Additional disclosure related to cleavable linkers is found in U.S. Application No. 17 / 423,796, filed January 22, 2020, and U.S. Application No. 17 / 531,343, filed November 19, 2021, the disclosures of each of which are incorporated herein by reference. COMPOUNDSUSEFUL FORPRODUCINGCONJUGATES

[0382] The present disclosure provides compounds useful for producing the conjugates described herein. In certain embodiments, the compound may include a conjugation moiety useful for conjugation of a polypeptide (e.g., an antibody) and a drug or active agent. For example, the compound may be bound to the polypeptide (antibody) and also bound to the drug or active agent, thus indirectly attaching the polypeptide (antibody) and the drug together.

[0383] Compounds of the present disclosure can include a conjugation moiety, e.g., XAor XB, as described herein. As described herein, the conjugation moiety can be a conjugation moiety that can attach to a native amino acid residue of an antibody (e.g., XA), or attach to a site- specifically engineered conjugation site of the antibody (e.g., XB).

[0384] In certain embodiments, the compound is a compound of formula (III) or (IV):Atty. Dkt: RDWD-053WO (III), or wherein AL is a first linker; LBis a second linker; W1is a first drug; W2is a second drug; XAis a first conjugation moiety configured to attach to the antibody at a native amino acid residue of the antibody; and XBis a second conjugation moiety configured to attach to the antibody at a site- specifically engineered conjugation site of the antibody.

[0385] Regarding compounds of formulae (II) and (III), the substituents LA, LB, LC, W1, W2, XA, and XBare as described above in relation to the conjugates of formula (I). Similarly, regarding the first linker LAand the second linker LBof formulae (II) and (III), the T1, T2, T3, T4, T5, T6, V1, V2, V3, V4, V5and V6, and T7, T8, T9, T10, T11, T12, V7, V8, V9, V10, V11and V12substituents are as described above in relation to the conjugates of formula (I).

[0386] For example, in some instances, T1, T2, T3, T4, T5and T6and V1, V2, V3, V4, V5and V6are selected from the following: wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is an amino acid analog and V2is -NH-; T3is (PEG)nand V3is -CO-; T4is (AA)p and V4is absent; T5is PABC and V5is absent; and f is 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)p and V3is absent;Atty. Dkt: RDWD-053WO T4is PABC and V4is absent; and e and f are each 0.

[0387] Compounds of formulae (II) and (III) can be used in conjugation reactions described herein, where a drug or active agent attached to a conjugation moiety is conjugated to a polypeptide (e.g., antibody) to form an antibody-drug conjugate.

[0388] In certain embodiments, the compound of formulae (II) or (III) has the following structure:

[0389] Any of the chemical entities, linkers and conjugation moieties set forth in the structures above may be adapted for use in the subject compounds and conjugates. POLYPEPTIDES ANDANTIBODIES

[0390] As noted above, a subject conjugate can comprise as substituent W3a polypeptide (e.g., an antibody). As used herein, amino acids may be referred to by their standard name, their standard three letter abbreviation and / or their standard one letter abbreviation, such as: AlanineAtty. Dkt: RDWD-053WO or Ala or A; Cysteine or Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E; Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H; Isoleucine or Ile or I; Lysine or Lys or K; Leucine or Leu or L; Methionine or Met or M; Asparagine or Asn or N; Proline or Pro or P; Glutamine or Gln or Q; Arginine or Arg or R; Serine or Ser or S; Threonine or Thr or T; Valine or Val or V; Tryptophan or Trp or W; and Tyrosine or Tyr or Y.

[0391] In certain embodiments, the amino acid sequence of the polypeptide (antibody) includes one or more native amino acid residues that can be conjugated to a linker-drug as described herein. In these embodiments, the conjugation moiety (e.g., XA) can be attached to the antibody at a native amino acid residue of the antibody. For example, maleimide-based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native cysteine residues of the polypeptide (e.g., antibody). The native cysteine residues of the antibody can be derived from cysteine residues that are involved in disulfide bonds, such as the 8 hinge cysteine residues that typically form intrachain disulfide bonds of an antibody. In some instances, the disulfide bonds can be reduced to provide cysteine residues where the sulfhydryl groups of the cysteine residues are available to react with and be attached (e.g., covalently attached) to a conjugation moiety (e.g., maleimide conjugation moiety) as described herein.

[0392] In other instances, N-hydroxysuccinimide ester (NHS ester) based conjugation can be used to attach the one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native lysine residues of the polypeptide (e.g., antibody). The native lysine residues of the antibody can include an amine group, which is available to react with and be attached (e.g., covalently attached) to a conjugation moiety (e.g., NHS-ester conjugation moiety) as described herein.

[0393] In certain embodiments, the amino acid sequence of the polypeptide (antibody) has been modified to include one or more 2-formylglycine (fGly) residues. In certain embodiments, the amino acid sequence of the polypeptide (antibody) is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to a 2-formylglycine (fGly) residue by action of a formylglycine generating enzyme (FGE) either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as an FGE-modification site.Atty. Dkt: RDWD-053WO Sulfatase motifs

[0394] A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 amino acid residues in length, usually no more than 6 amino acid residues in length. Sulfatase motifs provided in an Ig polypeptide are at least 5 or 6 amino acid residues, and can be, for example, from 5 to 16, 6- 16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acid residues in length.

[0395] In certain embodiments, polypeptides of interest include those where one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide for a sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide includes a modification (insertion, addition, deletion, and / or substitution / replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide. Where an amino acid sequence native to the polypeptide (e.g., antibody) contains one or more residues of the desired sulfatase motif, the total number of modifications of residues can be reduced, e.g., by site-specification modification (insertion, addition, deletion, substitution / replacement) of amino acid residues flanking the native amino acid residues to provide a sequence of the desired sulfatase motif. In certain embodiments, the extent of modification of the native amino acid sequence of the target antibody is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target antibody may minimize the impact such modifications may have upon antibody function and / or structure.

[0396] It should be noted that while aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to a “consensus sulfatase motif”), it will be readily appreciated that longer aldehyde tags are both contemplated and encompassed by the present disclosure and can find use in the compositions and methods of the present disclosure. Aldehyde tags can thus comprise a minimal sulfatase motif of 5 or 6 residues, or canAtty. Dkt: RDWD-053WO be longer and comprise a minimal sulfatase motif which can be flanked at the N- and / or C- terminal sides of the motif by additional amino acid residues. Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.

[0397] An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C- terminus of a native, wild-type Ig heavy chain. An aldehyde tag can be present within a CH1 domain of an Ig heavy chain. An aldehyde tag can be present within a CH2 domain of an Ig heavy chain. An aldehyde tag can be present within a CH3 domain of an Ig heavy chain. An aldehyde tag can be present in an Ig light chain constant region, e.g., in a kappa light chain constant region or a lambda light chain constant region.

[0398] In certain embodiments, the sulfatase motif used may be described by the formula: X1Z10X2Z20X3Z30(I’) where Z10is cysteine or serine (which can also be represented by (C / S)); Z20is either a proline or alanine residue (which can also be represented by (P / A)); Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X1is present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; and X2and X3independently can be any amino acid, though usually an aliphatic amino acid, a polar, uncharged amino acid, or a sulfur containing amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[0399] The amino acid sequence of an antibody heavy and / or light chain can be modified to provide a sequence of at least 5 amino acids of the formula X1Z10X2Z20X3Z30, where Z10is cysteine or serine;Atty. Dkt: RDWD-053WO Z20is a proline or alanine residue; Z30is an aliphatic amino acid or a basic amino acid; X1is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the polypeptide, X1is present; X2and X3are each independently any amino acid.

[0400] The sulfatase motif is generally selected so as to be capable of conversion by a selected FGE, e.g., an FGE present in a host cell in which the aldehyde tagged polypeptide is expressed or an FGE which is to be contacted with the aldehyde tagged polypeptide in a cell-free in vitro method.

[0401] For example, where the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including a human FGE), the sulfatase motif can be of the formula: X1CX2PX3Z30(I”) where X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V or G; and Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I.

[0402] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO:1), MCTPSR (SEQ ID NO:4), VCTPSR (SEQ ID NO:5), LCSPSR (SEQ ID NO:6), LCAPSR (SEQ ID NO:7), LCVPSR (SEQ ID NO:8), LCGPSR (SEQ ID NO:9), ICTPAR (SEQ ID NO:10), LCTPSK (SEQ ID NO:11), MCTPSK (SEQ ID NO:12), VCTPSK (SEQ ID NO:13), LCSPSK (SEQ ID NO:14), LCAPSK (SEQ ID NO:15), LCVPSK (SEQ ID NO:16), LCGPSK (SEQ ID NO:17), LCTPSA (SEQ ID NO:18), ICTPAA (SEQ ID NO:19), MCTPSA (SEQ ID NO:20), VCTPSA (SEQ ID NO:21), LCSPSA (SEQ ID NO:22), LCAPSA (SEQ ID NO:23), LCVPSA (SEQ ID NO:24), and LCGPSA (SEQ ID NO:25).Atty. Dkt: RDWD-053WO fGly-containing sequences

[0403] Upon action of FGE on the antibody heavy and / or light chain, the serine or the cysteine in the sulfatase motif is modified to fGly. Thus, the fGly-containing sulfatase motif can be of the formula: X1(fGly)X2Z20X3Z30(I’’’) where fGly is the formylglycine residue; Z20is either a proline or alanine residue (which can also be represented by (P / A)); Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; and X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[0404] As described above, to produce the conjugate, the polypeptide containing the fGly residue may be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety (e.g., a conjugation moiety, as described herein) of a linker attached to the drug or active agent to produce an fGly’-containing sulfatase motif. As used herein, the term fGly’ refers to the amino acid residue of the sulfatase motif that is coupled to the drug or active agent through a linker, as described herein. Thus, the fGly’-containing sulfatase motif can be of the formula: X1(fGly’)X2Z20X3Z30(II) where fGly’ is the amino acid residue coupled to the drug or active agent through a linker as described herein; Z20is either a proline or alanine residue (which can also be represented by (P / A));Atty. Dkt: RDWD-053WO Z30is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X1may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X1is present; and X2and X3independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. Site of modification

[0405] As noted above, in certain embodiments, the amino acid sequence of the polypeptide (antibody) is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an fGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system). The antibody used to generate a conjugate of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region. Such Ig polypeptides are referred to herein as “target Ig polypeptides” or “target antibodies”.

[0406] The site in an antibody into which a sulfatase motif is introduced can be any convenient site. As noted above, in some instances, the extent of modification of the native amino acid sequence of the target polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and / or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target antibody may minimize the impact such modifications may have upon antibody function and / or structure.

[0407] An antibody heavy chain constant region can include Ig constant regions of any heavy chain isotype, non-naturally occurring Ig heavy chain constant regions (includingAtty. Dkt: RDWD-053WO consensus Ig heavy chain constant regions). An Ig constant region amino acid sequence can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent a solvent- accessible loop region of the Ig constant region. An Ig constant region amino acid sequence can be modified by insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above.

[0408] In some cases, an aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain). The aldehyde- tagged Ig heavy chain constant region can include heavy chain constant region sequences of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant of same, e.g., human heavy chain constant region sequences or mouse heavy chain constant region sequences, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly-modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4.

[0409] In some cases, an aldehyde-tagged antibody comprises an aldehyde-tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region can include constant region sequences of a kappa light chain, a lambda light chain, e.g., human kappa or lambda light chain constant regions, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, etc., that includes at least one sulfatase motif that can be modified by an FGE to generate an fGly-modified antibody. Exemplary constant regions include human gamma 1 and gamma 3 regions. With the exception of the sulfatase motif, a modified constant region may have a wild-type amino acid sequence, or it may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to a wild type amino acid sequence.

[0410] In some embodiments the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain. As noted above, an isolated aldehyde- tagged antibody can comprise a heavy chain constant region amino acid sequence modified to include a sulfatase motif as described above, where the sulfatase motif is in or adjacent a surface- accessible loop region of the antibody heavy chain constant region.Atty. Dkt: RDWD-053WO

[0411] A sulfatase motif can be provided within or adjacent one or more of these amino acid sequences of such modification sites of an Ig heavy chain. For example, an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and / or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif adjacent and N-terminal and / or adjacent and C- terminal to these modification sites. Alternatively or in addition, an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and / or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modifications sites. In some embodiments, an Ig heavy chain polypeptide amino acid sequence may be modified to include two motifs, which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids. Alternatively or in addition, where a native amino acid sequence provides for one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues of the modification sites of an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and / or substitutions) so as to provide a sulfatase motif at the modification site.

[0412] An antibody used in an antibody-drug conjugate of the present disclosure can have any of a variety of antigen-binding specificities, including but not limited to, e.g., an antigen present on a cancer cell; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on a diseased cell; and the like. For example, an antibody conjugate can bind an antigen, where the antigen is present on the surface of the cell. An antibody conjugate of the present disclosure can bind antigen with a suitable binding affinity, e.g., from 5 x 10-6M to 10-7M, from 10-7M to 5 x 10-7M, from 5 x 10-7M to 10-8M, from 10-8M to 5 x 10-8M, from 5 x 10-8M to 10-9M, or a binding affinity greater than 10-9M.

[0413] As non-limiting examples, a subject antibody conjugate can bind an antigen present on a cancer cell (e.g., a tumor-specific antigen; an antigen that is over-expressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., aAtty. Dkt: RDWD-053WO cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, a subject antibody conjugate can be specific for an antigen on a cancer cell, where the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

[0414] As further non-limiting examples, a subject antibody conjugate can bind an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the virus; where the antigen is expressed on a cell type that is infected by a virus; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor. For example, a subject antibody conjugate can bind an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, such as a viral fusion inhibitor. DRUGS FORCONJUGATION TO APOLYPEPTIDE

[0415] The present disclosure provides drug-polypeptide conjugates (e.g., antibody-drug conjugates). Drugs suitable for use, or that can be modified to be rendered suitable for use, as a reactive partner to conjugate to a polypeptide (e.g., an antibody) are described herein.

[0416] As noted above, a conjugate or a compound of the present disclosure can include as substituents W1and W2a drug or active agent. Any of a number of drugs are suitable for use, or can be modified to be rendered suitable for use, as a reactive partner to conjugate to an antibody. Examples of drugs include small molecule drugs and peptide drugs.

[0417] “Small molecule drug” as used herein refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa. A small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.

[0418] For example, the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as a camptothecine, or an analog or derivative thereof, or a pharmaceutically active camptothecine moiety and / or a portion thereof. A topoisomerase inhibitor (e.g., camptothecine, or analog or derivative thereof) conjugated to the polypeptide can be any of a variety of topoisomerase inhibitors, for example camptothecine or camptothecine moieties such as, but not limited to, camptothecine and analogs and derivatives thereof asAtty. Dkt: RDWD-053WO described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to, a topoisomerase inhibitor, for example camptothecine or a camptothecine derivative, such as SN-38, Belotecan, Exatecan, 9-aminocamptothecin (9-AC), topotecan, des-Me-topotecan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT / US2022 / 012325, the disclosure of which is incorporated herein by reference.

[0419] In other embodiments, the drug or active agent can be a maytansine. “Maytansine”, “maytansine moiety”, “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and / or portions thereof. A maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein (e.g., deacylmaytansine).

[0420] In other instances, the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and / or a portion thereof. An auristatin conjugated to the polypeptide can be any of a variety of auristatin moieties such as, but not limited to, an auristatin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to an auristatin or an auristatin derivative, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like.

[0421] In other cases, the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and / or a portion thereof. A duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties such as, but not limited to, a duocarmycin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to a duocarmycin or a duocarmycin derivative, such as duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like. In some embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.Atty. Dkt: RDWD-053WO

[0422] In certain embodiments, the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulatory agent, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.

[0423] For example, a cytotoxin can include any compound that leads to cell death (e.g., necrosis or apoptosis) or a decrease in cell viability.

[0424] Kinase inhibitors can include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandetanib, Vemurafenib, and the like.

[0425] For example, selective estrogen receptor modulators include, but are not limited to, Endoxifen, Tamoxifen, Afimoxifene, Toremifene, and the like.

[0426] Immunostimulatory agents can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, and the like. TLR agonists include, but are not limited to, imiquimod, resiquimod, and the like.

[0427] Oligonucleotide dugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, volanesorsen, inotersen, tofersen, tominersen, and the like.

[0428] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like.

[0429] Cytokines include, but are not limited to, Albinterferon Alfa-2B, Aldesleukin, ALT-801, Anakinra, Ancestim, Avotermin, Balugrastim, Bempegaldesleukin, Binetrakin, Cintredekin Besudotox, CTCE-0214, Darbepoetin alfa, Denileukin diftitox, Dulanermin, Edodekin alfa, Emfilermin, Epoetin delta, Erythropoietin, Human interleukin-2, Interferon alfa, Interferon alfa-2c, Interferon alfa-n1, Interferon alfa-n3, Interferon alfacon-1, Interferon beta-1a, Interferon beta-1b, Interferon gamma-1b, Interferon Kappa, Interleukin-1 alpha, Interleukin-10, Interleukin-7, Lenograstim, Leridistim, Lipegfilgrastim, Lorukafusp alfa, Maxy-G34, Methoxy polyethylene glycol-epoetin beta, Molgramostim, Muplestim, Nagrestipen, Oprelvekin, Pegfilgrastim, Pegilodecakin, Peginterferon alfa-2a, Peginterferon alfa-2b, Peginterferon beta-1a, Peginterferon lambda-1a, Recombinant CD40-ligand, Regramostim, Romiplostim,Atty. Dkt: RDWD-053WO Sargramostim, Thrombopoietin, Tucotuzumab celmoleukin, Viral Macrophage-Inflammatory Protein, and the like.

[0430] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.

[0431] “Peptide drug” as used herein refers to amino-acid containing polymeric compounds, and is meant to encompass naturally-occurring and non-naturally-occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics. The peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source). Peptide drugs can range in molecular weight, and can be from 200 Da to 10 kDa or greater in molecular weight. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti-angiogenic peptides; peptides that activate B cells; peptides that activate T cells; anti-viral peptides; peptides that inhibit viral fusion; peptides that increase production of one or more lymphocyte populations; anti-microbial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti- inflammatory peptides; peptides that regulate glucose metabolism; an anti-thrombotic peptide; an anti-nociceptive peptide; a vasodilator peptide; a platelet aggregation inhibitor; an analgesic; and the like.

[0432] Additional examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to Tubulysin M, Calicheamicin, a STAT3 inhibitor, alpha-Amanitin, an aurora kinase inhibitor, belotecan, and an anthracycline.

[0433] Other examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.

[0434] Suitable cancer chemotherapeutic agents include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., MonomethylAtty. Dkt: RDWD-053WO auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96 / 33212, WO 96 / 14856, and U.S. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).

[0435] Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0436] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6- mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8- dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.

[0437] Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicAtty. Dkt: RDWD-053WO immunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.

[0438] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0439] Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0440] Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g. hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g. aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation; therefore compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation.

[0441] Other suitable chemotherapeutic agents include metal complexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, e.g. mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4- morpholinyl)propoxy)quinazoline); etc.

[0442] Taxanes are suitable for use. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to includeAtty. Dkt: RDWD-053WO analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL ^, TAXOTERE ^ (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis).

[0443] Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere ^ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0444] Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6- thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivative described in U.S. Patent No. 5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0445] Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.

[0446] Examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agent. In certain embodiments, the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid.Atty. Dkt: RDWD-053WO

[0447] As described herein, in some instances, the antibody-drug conjugate may include a branched linker, where two drugs or active agents are attached to a branched linker. In certain embodiments, the two drugs or active agents attached to a branched linker are the same drug or active agent. For example, a first branch of a branched linker may be attached to a drug or an active agent and a second branch of the branched linker may be attached to the same drug or the same active agent as the first branch. In other embodiments, the two drugs or active agents attached to the branched linker are different drugs or active agents. For example, a first branch of a branched linker may be attached to a first drug or a first active agent and a second branch of the branched linker may be attached to a second drug or a second active agent different from the first drug or the first active agent attached to the first branch.

[0448] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. For example, in some instances, the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC.

[0449] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately, For example, the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).

[0450] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways. Examples of payload combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like.

[0451] In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an auristatin (e.g., MMAE) as described herein. In someAtty. Dkt: RDWD-053WO embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an iRGD peptide as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and an iRGD peptide as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.

[0452] Drugs to be conjugated to a polypeptide may be modified to incorporate a reactive partner for reaction with the polypeptide. Where the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be positioned at an N-terminal region, the N-terminus, a C- terminal region, the C-terminus, or at a position internal to the peptide. For example, an example of a method involves synthesizing a peptide drug having an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N-Boc group. As an example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N-protecting group occurs through standard peptide coupling chemistry. Examples of peptide coupling reagents that can be used include, but not limited to, DCCAtty. Dkt: RDWD-053WO (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1- benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3- dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium tetrafluoroborate), TSTU (O-(N-succinimidyl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1- ylmethylene]- -N-methylmethanaminium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo-3- oxazolidinyl)phosphinic chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)- tris(pyrrolidino)phosphonium tetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). As a non-limiting example, HOBt and DIC can be used as peptide coupling reagents.

[0453] Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group. Deprotection of the N-oxysuccinimide group, for example, occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. The product and intermediates can be purified by conventional means, such as HPLC purification.

[0454] The ordinarily skilled artisan will appreciate that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to reaction with a reactive partner of interest) are of importance, Modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with a polypeptide present in or on a living cell, the conditions are selected so as to be physiologically compatible. For example, the pH can be dropped temporarilyAtty. Dkt: RDWD-053WO for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (e.g., from about 30 min to 1 hour). Physiological conditions for conducting modification of polypeptides on a cell surface can be similar to those used in a ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040).

[0455] Small molecule compounds containing, or modified to contain, an α-nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure. General methods are known in the art for chemical synthetic schemes and conditions useful for synthesizing a compound of interest (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). FORMULATIONS

[0456] The conjugates of the present disclosure can be formulated in a variety of different ways. In general, where the conjugate is an antibody-drug conjugate, the conjugate is formulated in a manner compatible with the drug, the antibody, the condition to be treated, and the route of administration to be used.

[0457] In some embodiments, provided is a pharmaceutical composition that includes any of the conjugates of the present disclosure and a pharmaceutically-acceptable excipient.

[0458] The conjugate (e.g., antibody-drug conjugate) can be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, e.g., oral, topical or parenteral administration. Where the conjugate is provided as a liquid injectable (such as in those embodiments where they are administered intravenously or directly into a tissue), the conjugate can be provided as a ready-to- use dosage form, or as a reconstitutable storage-stable powder or liquid composed of pharmaceutically acceptable carriers and excipients.

[0459] Methods for formulating conjugates can be adapted from those readily available. For example, conjugates can be provided in a pharmaceutical composition comprising a therapeutically effective amount of a conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers,Atty. Dkt: RDWD-053WO stabilizers, preservatives, and the like). In some embodiments, the formulations are suitable for administration to a mammal, such as those that are suitable for administration to a human. METHODS OF TREATMENT

[0460] The antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug (i.e., the drug prior to conjugation to the antibody).

[0461] In some embodiments, provided are methods that include administering to a subject an effective amount (e.g., a therapeutically effective amount) of any of the conjugates of the present disclosure.

[0462] In certain aspects, provided are methods of delivering a drug to a target site in a subject, the method including administering to the subject a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject. For example, as described herein, antibody-drug conjugates of the present disclosure can include a cleavable linker, such as an enzymatically cleavable linker that includes a first enzymatically cleavable moiety and a second enzymatically cleavable moiety. In some instances, the cleavable linker can be cleaved under appropriate conditions to separate or release the drug from the antibody at a desired target site of action for the drug. For example, the second cleavable moiety, which protects the first cleavable moiety from cleavage, may be cleaved in order to allow the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker into two or more portions, thus releasing the drug from the antibody-drug conjugate at a desired site of action.

[0463] In certain embodiments, the first cleavable moiety can be an enzymatically cleavable moiety. In some instances, the enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated). For example, a first enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein.

[0464] In certain embodiments, the second cleavable moiety can be an enzymatically cleavable moiety. In some instances, the enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to theAtty. Dkt: RDWD-053WO subject to be treated). For example, a second enzyme can be administered before, concurrently with, or after administration of an antibody-drug conjugate described herein. In certain embodiments, the first enzyme and the second enzyme are different enzymes.

[0465] In other instances, the first enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated). For instance, the first enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the first enzyme. In some instances, the first enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated. For example, the first enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated. In some instances, the first enzyme is present in an overabundance at the desired site of action due to localization of the first enzyme at a particular area or location. For instance, the first enzyme may be associated with a certain structure within the desired site of action, such as lysosomes. In some cases, the first enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject. In some embodiments, the lysosomes that include the first enzyme, are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug. In certain embodiments, the first enzyme is a protease, such as a human protease enzyme (e.g., cathepsin B).

[0466] In certain embodiments, the second enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated). For instance, the second enzyme may be present at the desired site of action for the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate may be specifically targeted to a desired site of action (e.g., may specifically bind to an antigen present at a desired site of action), where the desired site of action also includes the presence of the second enzyme. In some instances, the second enzyme is present in an overabundance at the desired site of action as compared to other areas in the body of the subject to be treated. For example, the second enzyme may be overexpressed at the desired site of action as compared to other areas in the body of the subject to be treated. In some instances, the second enzyme isAtty. Dkt: RDWD-053WO present in an overabundance at the desired site of action due to localization of the second enzyme at a particular area or location. For instance, the second enzyme may be associated with a certain structure within the desired site of action, such as lysosomes. In some cases, the second enzyme is present in an overabundance in lysosomes as compared to other areas in the body of the subject. In some embodiments, the lysosomes that include the second enzyme, are found at a desired site of action for the drug of the antibody-drug conjugate, such as the site of a cancer or tumor that is to be treated with the drug. In certain embodiments, the second enzyme is a glycosidase, such as a galactosidase, a glucosidase, or a mannosidase.

[0467] Any suitable enzymes can be used for cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein. Other enzymes may also be suitable for use in cleavage of the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein, such as but not limited to, enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs, quails, goats, dogs, rabbits, etc.).

[0468] In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions. By substantially stable is meant that the cleavable linker of the antibody- drug conjugate does not undergo a significant amount of cleavage in the absence of a first enzyme and a second enzyme as described above. For example, as described above, the second cleavable moiety can protect the first cleavable moiety from being cleaved, and as such the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of a second enzyme as described above. For instance, the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate is cleaved in the absence of the first enzyme and / or second enzyme, such as 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less. In some cases, the antibody-drug conjugate is substantially stable such that the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the first enzyme and / or second enzyme, but can be cleaved when in the presence of the first enzyme and the second enzyme. For example, the antibody-drug conjugate can be substantially stable after administration to a subject. In some cases, the antibody-drug conjugate is substantially stable after administration to a subject, and then, when the antibody-drug conjugate is in the presence of the second enzyme at a desired site of action, the second cleavable moiety can be cleaved from the cleavable linker, thus exposing the firstAtty. Dkt: RDWD-053WO cleavable moiety to subsequent cleavage by the first enzyme, which in turn releases the drug at the desired site of action. In certain embodiments, after administration to a subject the antibody- drug conjugate is stable for an extended period of time in the absence of the first enzyme and / or second enzyme, such as 1 hr or more, or 2 hrs or more, or 3 hrs or more, or 4 hrs or more, or 5 hrs or more, or 6 hrs or more, or 7 hrs or more, or 8 hrs or more, or 9 hrs or more, or 10 hrs or more, or 15 hrs or more, or 20 hrs or more, or 24 hrs (1 day) or more, or 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days (1 week) or more. In certain embodiments, the antibody-drug conjugate is stable at a range pH values for an extended period of time in the absence of the first enzyme and / or second enzyme, such as at a pH ranging from 2 to 10, or from 3 to 9, or from 4 to 8, or from 5 to 8, or from 6 to 8, or from 7 to 8.

[0469] As described above, the antibody-drug conjugates of the present disclosure find use in treatment of a condition or disease in a subject that is amenable to treatment by administration of the parent drug. By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease; and / or (iii) relief, that is, causing the regression of clinical symptoms.

[0470] The subject to be treated can be one that is in need of therapy, where the subject to be treated is one amenable to treatment using the parent drug. Accordingly, a variety of subjects may be amenable to treatment using the antibody-drug conjugates disclosed herein. Generally, such subjects are “mammals”, with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees and monkeys).Atty. Dkt: RDWD-053WO

[0471] The amount of antibody-drug conjugate administered can be initially determined based on guidance of a dose and / or dosage regimen of the parent drug. In general, the antibody- drug conjugates can provide for targeted delivery and / or enhanced serum half-life of the bound drug, thus providing for at least one of reduced dose or reduced administrations in a dosage regimen. Thus, the antibody-drug conjugates can provide for reduced dose and / or reduced administration in a dosage regimen relative to the parent drug prior to being conjugated in an antibody-drug conjugate of the present disclosure.

[0472] Furthermore, as noted above, because the antibody-drug conjugates can provide for controlled stoichiometry of drug delivery, dosages of antibody-drug conjugates can be calculated based on the number of drug molecules provided on a per antibody-drug conjugate basis.

[0473] In some embodiments, multiple doses of an antibody-drug conjugate are administered. The frequency of administration of an antibody-drug conjugate can vary depending on any of a variety of factors, e.g., severity of the symptoms, condition of the subject, etc. For example, in some embodiments, an antibody-drug conjugate is administered once per month, twice per month, three times per month, every other week, once per week (qwk), twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily (qd / od), twice a day (bds / bid), or three times a day (tds / tid), etc. Methods of treating cancer

[0474] The present disclosure provides methods that include delivering a conjugate of the present disclosure to an individual having a cancer. The methods are useful for treating a wide variety of cancers, including, but not limited to breast, ovarian, colon, lung, stomach, and pancreatic cancer. In the context of cancer, the term “treating” includes one or more (e.g., each) of: reducing growth of a solid tumor, inhibiting replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with a cancer.

[0475] Carcinomas that can be treated using a subject method include, but are not limited to, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma,Atty. Dkt: RDWD-053WO choriocarcinoma, seminoma, embryonal carcinoma, cervical carcinoma, uterine carcinoma, testicular carcinoma, and epithelial carcinoma, etc.

[0476] In certain aspects, provided are methods of treating cancer in a subject, such methods including administering to the subject a therapeutically effective amount of a pharmaceutical composition including any of the conjugates of the present disclosure, where the administering is effective to treat cancer in the subject. METHODS OF PRODUCTION

[0477] The conjugates of compounds of the present disclosure can be prepared using standard reactions and methods of production. In some instances, the method of producing an antibody-drug conjugate according to the present disclosure includes contacting an antibody comprising a site-specifically engineered conjugation site with: (1) a first drug-linker compound comprising a first reactive group configured to conjugate to a native amino acid residue of the antibody; and (2) a second drug-linker compound comprising a second reactive group configured to conjugate to the site-specifically engineered conjugation site. For example, the first drug- linker compound may be a compound of formula (III) as described herein, and the second drug- linker compound may be a compound of formula (IV) as described herein. In some instances, the first reactive group of the first drug-linker compound is the first conjugation moiety, XA, and the second reactive group of the second drug-linker compound is the second conjugation moiety, XB.

[0478] In certain embodiments, the method includes combining the first drug-linker compound and the second drug-linker compound in a one-pot reaction to produce the antibody- drug conjugate. By “one-pot reaction” is meant a reaction in which all the reagents and starting materials are combined in a single step. For example, methods of producing an antibody-drug conjugate according to the present disclosure included combining the antibody, the first drug- linker compound and the second drug-linker compound together to produce the antibody-drug conjugate. In these instances, the first reactive group (e.g., the first conjugation moiety, XA) reacts with and conjugates to a native amino acid residue of the antibody and the second reactive group (e.g., the second conjugation moiety, XB) reacts with and conjugates to a site-specifically engineered conjugation site (e.g., an aldehyde tag, which can include an fGly residue) of the antibody, as described herein.Atty. Dkt: RDWD-053WO

[0479] In other embodiments of the method of producing an antibody-drug conjugate, the contacting comprises contacting the antibody with the first drug-linker compound and then contacting the antibody with the second drug-linker compound. For example, the production of the antibody-drug conjugate can be performed in a stepwise manner involving a first reaction where the antibody and the first drug-linker compound are contacted together such that the first reactive group (e.g., the first conjugation moiety, XA) reacts with and conjugates to a native amino acid residue of the antibody, followed by a second reaction, where the antibody produced by the first reaction and the second drug-linker compound are contacted together such that the second reactive group (e.g., the second conjugation moiety, XB) reacts with and conjugates to a site-specifically engineered conjugation site (e.g., an aldehyde tag, which can include an fGly residue) of the antibody. In other instances, the order of the reactions can be reversed, such that for example, the first reaction includes contacting the antibody with the second drug-linker compound and then the second rection includes contacting the antibody with the first drug-linker compound.

[0480] Methods of producing an antibody-drug conjugate according to the present disclosure also include methods where prior to contacting the antibody with the first drug-linker compound and the second drug-linker compound, the method comprises reducing one or more interchain disulfide bonds of the antibody to produce two or more cysteine residues. For example, these embodiments can be used to prepare the antibody for reaction to a first drug- linker compound where the first drug-linker compound includes a maleimide as the first reactive group (e.g., a maleimide as the first conjugation moiety, XA). In certain instances, the antibody has four interchain disulfide bonds, and thus reduction of the four interchain disulfide bonds produces eight cysteine residues that are then available for conjugation to the first drug-linker compound. For example, eight of the first drug-linker compound can be conjugated to the eight cysteine residues (e.g., one of the first drug-linker compound conjugated to each of the eight cysteine residues), thus producing an antibody-drug conjugate having a DAR of 8 for the first drug. CERTAINEMBODIMENTS

[0481] The present disclosure contemplates, among other things, the following numbered embodiments:Atty. Dkt: RDWD-053WO 1. An antibody-drug conjugate comprising: an antibody; a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker; and a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker. 2. The conjugate of Embodiment 1, wherein the native amino acid residue of the antibody is a cysteine. 3. The conjugate of Embodiment 2, wherein the cysteine is produced via reduction of an interchain disulfide bond of the antibody. 4. The conjugate of any of Embodiments 1 to 3, wherein the site-specifically engineered conjugation site of the antibody comprises a formylglycine (fGly). 5. The conjugate of any of Embodiments 1 to 4, wherein the antibody has a drug-antibody ratio (DAR) of 2 or more for the first drug. 6. The conjugate of Embodiment 5, wherein two or more of the first drug are attached to the antibody through two or more of the first linker. 7. The conjugate of Embodiment 5, wherein the antibody has a DAR of 8 for the first drug. 8. The conjugate of Embodiment 7, wherein eight of the first drug are attached to the antibody through eight of the first linker. 9. The conjugate of any of Embodiments 1 to 9, wherein the antibody has a DAR of 2 or more for the second drug.Atty. Dkt: RDWD-053WO 10. The conjugate of Embodiment 9, wherein two or more of the second drug are attached to the antibody through two or more of the second linker. 11. The conjugate of any of Embodiments 9 to 10, wherein the antibody has a DAR of 2 for the second drug. 12. The conjugate of Embodiment 11, wherein two of the second drug are attached to the antibody through two of the second linker. 13. The conjugate of any of Embodiments 9 to 10, wherein the antibody has a DAR of 4 for the second drug. 14. The conjugate of Embodiment 13, wherein four of the second drug are attached to the antibody through four of the second linker. 15. The conjugate of Embodiment 13, wherein four of the second drug are attached to the antibody through two of the second linker, wherein each of the second linker is a branched linker. 16. The conjugate of any of Embodiments 9 to 10, wherein the antibody has a DAR of 8 for the second drug. 17. The conjugate of Embodiment 16, wherein eight of the second drug are attached to the antibody through four of the second linker, wherein each of the second linker is a branched linker. 18. The conjugate of any of Embodiments 1 to 17, wherein the antibody has a total DAR for the first drug and the second drug of 4 to 16. 19. A pharmaceutical composition comprising: a conjugate of any one of Embodiments 1 to 18; andAtty. Dkt: RDWD-053WO a pharmaceutically-acceptable excipient. 20. A method comprising: administering to a subject an amount of a conjugate of any one of Embodiments 1 to 18. 21. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of any one of Embodiments 1 to 18, wherein the administering is effective to treat cancer in the subject. 22. A method of producing an antibody-drug conjugate, comprising: contacting an antibody comprising a site-specifically engineered conjugation site with: (1) a first drug-linker compound comprising a first reactive group configured to conjugate to a native amino acid residue of the antibody; and (2) a second drug-linker compound comprising a second reactive group configured to conjugate to the site-specifically engineered conjugation site. 23. The method of Embodiment 22, wherein the contacting comprises combining the antibody, the first drug-linker compound and the second drug-linker compound in a one-pot reaction. 24. The method of Embodiment 22, wherein the contacting comprises contacting the antibody with the first drug-linker compound and then contacting the antibody with the second drug-linker compound. 25. The method of Embodiment 22, wherein the contacting comprises contacting the antibody with the second drug-linker compound and then contacting the antibody with the first drug-linker compound.Atty. Dkt: RDWD-053WO 26. The method of any of Embodiments 22 to 25, wherein prior to the contacting the method comprises reducing one or more interchain disulfide bonds of the antibody to produce two or more cysteine residues. 27. The method of Embodiment 26, wherein the first reactive group comprises a maleimide. 28. The method of any of Embodiments 22-27, wherein the site-specifically engineered conjugation site of the antibody comprises an fGly. 29. The method of Embodiment 28, wherein the second reactive group comprises a hydrazinyl-indole or hydrazinyl-pyrrolo-pyridine group. EXAMPLES

[0482] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. By “average” is meant the arithmetic mean. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. General Synthetic Procedures

[0483] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, FifthAtty. Dkt: RDWD-053WO Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0484] Compounds as described herein can be purified by any purification protocol known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and / or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer- Verlag, New York, 1969.

[0485] During any of the processes for preparation of the subject compounds, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie”, Houben-Weyl, 4thedition, Vol. 15 / l, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and / or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.

[0486] The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and / or starting materials prepared by conventional synthetic methods. A variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.Atty. Dkt: RDWD-053WO EXAMPLE 1 Summary

[0487] Dual-payload branched linkers (Fig. 2A) possess high molecular complexity and as such are cumbersome to synthesize and may have manufacturability limitations. Moreover, due to the synthetic limitations, this mode is intrinsically limited to an internal payload ratio of 1:1, making it difficult to leverage combinations of payloads that differ in biological activity (potency). When two stochastic conjugation methods utilize naturally-occurring amino acid residues as chemical handles (e.g., cysteine and lysine) and corresponding single-payload linkers, the resulting ADC will likely be highly heterogeneous with poorly predictable properties, problematic manufacturing and analysis (Fig. 2B) (Wakankar et al., mAbs, 3, 161-172 (2011)). The extent of conjugation and therefore the ratio between payloads in this system would also be hard to control and manage. Viewed at this angle, the use of two site-specific orthogonal conjugation methods appears to be more attractive as it would allow for homogeneous conjugates with predictable payload ratios (Fig. 2C). However, this approach may also require extensive protein engineering and optimization which would significantly lengthen the development times and increase cost of goods (Wijdeven et al., mAbs, 14:1, (2022)).

[0488] An unique approach towards generating dual-payload ADCs that is simple, comprehensive, modular and versatile is described in the present disclosure and shown in Fig. 3A and 3B. At the core of the disclosed method is the utility of the highly efficient stochastic conjugation method (e.g. thiol-maleimide) which attaches the first payload of interest to an antibody in well-controlled manner. The maleimide conjugation reaction (which, depending on the stoichiometry employed, can yield heterogeneous conjugates with a range of drug-to- antibody ratios (DARs) in a mixture) is employed here so as to fully-occupy the available 8 hinge cysteines that typically form intrachain disulfide bonds. The result is a homogeneous maleimide conjugate with a drug to antibody ratio (DAR) of 8. The second payload of interest is conjugated to the antibody using the site-specific Hydrazino-iso-Pictet-Spengler (HIPS) reaction to an orthogonal aldehyde chemical handle as shown in Fig. 3A (Agarwal et al. Bioconjugate Chemistry 24, 846-851 (2013)). The latter is installed in a site-specific manner at a desired location(s) in the protein through the genetic encoding of a six amino acid consensus sequence (aldehyde tag) followed by the cotranslational enzymatic transformation of cysteine toAtty. Dkt: RDWD-053WO formylglycine (fGly) mediated by formylglycine generating enzyme (FGE) (Huang et al., mAbs, 10, 1182-1189 (2018); Drake et al. Bioconjugate Chemistry, 25, 1331-1341 (2014)).

[0489] Such a conjugation approach reliably led to uniform dual-payload ADCs with highly predictable and discrete payload ratios within the conjugate. As shown in Fig. 3A, the antibody’s interchain disulfide bonds were reduced to liberate eight cysteine residues which fully react with maleimide drug-linker to produce a DAR8 thioether conjugate. The two aldehyde groups, which are chemically orthogonal to the maleimide unit were left untouched during this process. The aldehyde groups were reacted via HIPS conjugation to give a conjugate which has DAR2 for the second payload, thus generating a dual-payload ADC with a payload ratio of 1:4. If needed, the payload ratio can easily be adjusted to 1:2 by using a double-tagged antibody (carrying two aldehyde tag sites) with 4 aldehyde groups installed or a single-tagged antibody conjugated to a branched HIPS linker bearing two units of a single type of payload. Furthermore, using a double- tagged antibody with branched HIPS linkers carrying two of the same payloads per one conjugation unit in combination with a fully-conjugated (DAR8) thioether (maleimide) conjugate will give 1:1 ratio between payloads with an overall DAR of 16 (Fig. 3B). Access to easily tunable payload ratios may become important when the dual-payload ADC is constructed from two payloads with significantly different potencies (e.g., auristatins and topoisomerase inhibitors).

[0490] It is further demonstrated that the disclosed molecules can be made using conjugations that are carried out in a variety of ways: two-step conjugations can be run with either the maleimide or HIPS reaction first followed by the other reaction. Surprisingly, both conjugations can also proceed at the same time in a one-pot fashion without the need to purify the intermediate conjugate. This facile outcome was due to the compatibility of HIPS and maleimide conjugation chemistries and their overlapping requirements for conjugation conditions.

[0491] HIPS conjugation chemistry, which requires an acidic environment, is not readily compatible with certain acid-sensitive payloads (such as anthracyclines). By contrast, the maleimide-functionalized linker, which can be conjugated under a wider range of conditions, including neutral or basic pH, may be used in combination with a broad variety of acid-sensitive payloads, thus widening the scope of application for preparing a defined dual-payload ADC.Atty. Dkt: RDWD-053WO

[0492] Furthermore, the dual-payload conjugates can be made with linkers containing a broad array of functionalities and design elements, including those that impart stability, hydrophilicity, and good biophysical properties to the conjugate. Such linker elements include the tandem-cleavage motif, which requires two consecutive enzymatic steps to occur in order to release the payload as shown in Fig. 4. The monosaccharide unit attached to the phenolic self- immolation moiety (PABA) serves as a protective element and has to be removed first by the corresponding lysosomal glycosidase to expose the peptide portion of the linker which is then readily cleaved by proteases to liberate the payload. Because glucuronidase is only activated in the low pH environment of the lysosomal compartment, it is generally not active when found in serum. Thus, in order for the tandem-cleavage linker to be enzymatically degraded, it first must be internalized into a cell and trafficked to the lysosome. This requirement greatly reduces release of linker during ADC circulation.

[0493] Such a method of making dual-payload ADCs, as disclosed herein, is expected to be widely applicable to various payload types and combinations, while staying manufacturable and analytically simple. This approach also opens an avenue for the rapid preparation and screening of various payload combinations in different ratios to find an optimal ADC that will help a broader patient population as compared to ADCs carrying a single payload. EXAMPLE2 Materials and Methods General

[0494] Synthetic reagents were purchased from Sigma-Aldrich, Acros, AK Scientific, or other commercial sources and were used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Compounds 1, 6, 19, 32, and 34 were obtained commercially from Shanghai Medicilon and used without purification. Payloads 2, 7, 41, and 49 were purchased from commercial sources and used as received. Resin-bound peptide 55 was obtained from New England Peptide; compounds 59 and 61 were commercially available. In all cases, solvent was removed under reduced pressure with a Buchi Rotovapor R-114 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage chromatography system. Preparative HPLC purifications were performed using Waters preparative HPLC unit equipped with Phenomenex Kinetex 5 µm EVO C18150 x 21.2 mmAtty. Dkt: RDWD-053WO column. HPLC analyses were conducted on an Agilent 1100 Series Analytical HPLC equipped with a Model G1322A Degasser, Model G1311A Quarternary Pump, Model G1329A Autosampler, Model G1314 Variable Wavelength Detector, Agilent Poroshell 120 SB C18, 4.6 mm x 50 mm column at room temperature using a 10-100% gradient of water and acetonitrile containing 0.05% trifluoroacetic acid. HPLCs were monitored at 254 or 205 nm. Low-resolution mass spectra (LRMS) were acquired on Agilent Technology 6120 Quadrupole LC / MS, equipped with Agilent 1260 Infinity HPLC system, G1314 variable wavelength detector, and Agilent Poroshell 120 SB C18, 4.6 mm x 50 mm column at room temperature using 10-100% gradient of water and acetonitrile containing 0.1% formic acid. EXAMPLE 3 Synthesis of drug-linkers Preparation of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)- 3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin- 1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)- 2-((2S,5S,18R)-22-(2-((1,2-dimethylhydrazineyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-5- isopropyl-2-methyl-4,7,17,20-tetraoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19- tetraazadocosanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (5)Atty. Dkt: RDWD-053WO

[0495] In a 20 mL glass vial were combined monomethyl auristatin A 2 (MMAE, 720 mg, 1.0 mmol), 5 mL of anhydrous DMF, and 0.35 mL of DIPEA (2.0 mmol) at room temperature. The resulting mixture was stirred and treated with PNP carbonate 1 (1014 mg, 1.0 mmol) as a solid in a few small portions, followed by the addition of HOAt (136 mg, 1.0 mmol) in one portion at room temperature. Reaction mixture was stirred for 6 h until reaction was judged complete (HPLC). Reaction mixture was poured into 30 mL of water, and the resulting white precipitate was separated by spinning and collected, washed with 5 mL of water, and dried briefly under high vacuum to give 1.87 g of crude coupling product as a yellow solid, which was taken to the next step without purification.

[0496] A solution of crude intermediate (1.87 g) in 15 mL of THF was cooled down to 0 °C in an ice bath and treated slowly with 1 M aqueous lithium hydroxide solution (3 mL). Reaction mixture was stirred at 0 °C for 3 hours, then warmed up to ambient temperature, treated with 3 mL of 1 M aqueous lithium hydroxide and diluted with 3 mL of methanol. The resulting mixture was stirred at room temperature for 3 hours until hydrolysis was found complete (HPLC), then quenched by adding 1 M aqueous HCl solution to pH 7. Reaction mixture was then concentrated under reduced pressure and washed with 10 mL of MTBE. Aqueous layer wasAtty. Dkt: RDWD-053WO purified by reversed-phase chromatography (C18 column, 0-40% acetonitrile-water with 0.05% TFA). Pure product fractions were combined, concentrated under reduced pressure, and lyophilized to give compound 3 as a white powder (735 mg, 0.60 mmol, 60% yield over 2 steps). LRMS (ESI): m / z 1229.7 [M+H]+, Calcd for C61H96N8O18m / z 1229.7.

[0497] To a stirred solution of compound 3 (735 mg, 0.60 mmol) in 3 mL of anhydrous DMA were added DIPEA (0.21 mL, 1.2 mmol) and a solution of PFP-ester 4 (575 mg, 0.60 mmol) in 2 mL of DMA at room temperature, followed by the addition of HOAt (84 mg, 0.60 mmol). The resulting mixture was stirred for 30 minutes until coupling was judged complete (HPLC analysis), then treated directly with 1.2 mL of piperidine at room temperature. After 15 minutes, reaction mixture was purified by reversed-phase chromatography (C18 column, 0-40% gradient of acetonitrile-water, no acid additive). Pure fractions were combined, concentrated under reduced pressure to ~50 mL final volume, and lyophilized to give compound 5 (808 mg, 0.45 mmol, 75% yield) as a white fluffy powder. LRMS (ESI): m / z 1783.9 [M+H]+, Calcd for C84H130N14O26S m / z 1783.9.

[0498] Synthesis of compound 4.(1.23 g, 6.68 mmol) in 6.5 mL of anhydrous DMF. This mixture was treated with EDCI-HCl (0.64 g, 3.34 mmol) in one portion at room temperature and stirred for 20 h until 6 was fully consumed as judged by HPLC analysis. Reaction mixture was loaded onto a C18 column and eluted with 0-80% gradient of acetonitrile-water with 0.05% TFA additive. Pure fractions were combined, concentrated under reduced pressure until slightly murky, and lyophilized to give PFP-ester product 4 (1.40 g, 1.46 mmol, 87% yield) as a tan powder. LRMS (ESI): m / z 961.2 [M+H]+, Calcd for C44H45F5N6O11S m / z 961.3.Atty. Dkt: RDWD-053WO EXAMPLE 4 Preparation of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23- octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo- 3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (15)

[0500] Belotecan hydrochloride 7 (2.35 g, 5.0 mmol) was suspended in a mixture of 30 mL of anhydrous DMF and 1.75 mL of DIPEA (10 mmol). The resulting mixture was stirred and treated with HOAt (0.68 g, 5 mmol), followed by PNP-carbonate 1 (5.1 g, 5 mmol) in a few small portions at room temperature. Reaction mixture was stirred at RT for 8 hours until starting materials were judged fully consumed based on HPLC analysis. The mixture was poured ontoAtty. Dkt: RDWD-053WO 300 mL of ice with vigorous stirring, the resulting yellowish precipitate was collected by filtration, washed with 30 mL of cold water twice, dried on air overnight to give 6.7 g of crude coupling product as a light-yellow powder which was taken to the next step without purification.

[0501] A solution of crude coupling intermediate (6.7 g) in 30 mL of THF was cooled down to 0 °C in an ice bath and treated slowly with 2 M aqueous lithium hydroxide solution (10 mL). Reaction mixture was stirred at 0 °C for 1 hour, then another 10 mL of 2 M LiOH solution was added and stirring continued for 15 minutes before warming the reaction mixture to room temperature and adding another 10 mL of 2 M lithium hydroxide. The resulting mixture was stirred for 1 hour at room temperature, then quenched by adding 2 M aqueous HCl solution to pH ~2-3 and let stir for 30 minutes. The mixture was transferred to a separatory funnel and washed with MTBE (2 x 50 mL). Aqueous layer was separated, then directly loaded on a C18 column, and eluted with 0-40% CH3CN-H2O with 0.05% TFA. Pure fractions were combined, concentrated under reduced pressure to ~70 mL volume, and lyophilized to give 3.4 g of product 8 (3.6 mmol, 72% yield over 2 steps) as a bright-yellow fluffy powder. LRMS (ESI): m / z 945.4 [M+H]+, Calcd for C47H56N6O15 m / z 945.4.

[0502] To a stirred mixture of compound 8 (945 mg, 1.0 mmol) and DIPEA (0.35 mL, 2 mmol) in 4 mL DMF were added Boc-Lys(Fmoc)-OPfp 9 (635 mg, 1 mmol) in one portion at room temperature. The resulting turbid mixture was stirred for 30 minutes until it became a clear yellow solution, while the reaction was judged complete by LCMS analysis. Reaction mixture was poured into 50 mL of 10% aq. citric acid solution and extracted with ethyl acetate (2x100 mL). Organic layer was washed once with brine, dried over sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography using 5-10% MeOH in DCM as an eluent. Solvents removed under vacuum to give 1.15 g (0.82 mmol, 82% yield) of product 10 as a bright yellow solid. LRMS (ESI): m / z 1395.6 [M+H]+, Calcd for C73H86N8O20m / z 1395.6.

[0503] To a stirred solution of compound 10 (1.15 g, 0.82 mmol) in 5 mL of anhydrous DCM were added 5 mL of TFA at RT. Reaction mixture was stirred for 15 minutes, monitored for completion by LCMS. Solvents were immediately removed under reduced pressure, the dark oily residue was briefly dried under high vacuum and triturated with 25 mL MTBE. The resulting light-yellow precipitate was collected by filtration, transferred to a round-bottom flask,Atty. Dkt: RDWD-053WO and dried under high vacuum overnight to give 1.2 g of crude product 11 as light-yellow powder (TFA salt). LRMS (ESI): m / z 1295.5 [M+H]+, Calcd for C68H78N8O18 m / z 1295.6.

[0504] To a solution of mPEG8 acid (340 mg, 0.82 mmol) in 5 mL of anhydrous THF were added pentafluorophenol (180 mg, 0.98 mmol), followed by DCC (170 mg, 0.82 mmol) at room temperature. The resulting mixture was stirred overnight, solids were filtered off, and washed with THF on filter. Combined filtrate was concentrated under reduced pressure, to give crude compound 12 (clear oil), which was re-dissolved in 2 mL of DMF and combined with a stirred mixture of crude compound 11 (0.82 mmol) and DIPEA (0.43 mL, 2.5 mmol) in 4 mL of DMF. Reaction mixture was stirred for 1 hour at room temperature until judged complete by HPLC analysis based on consumption of 11. Reaction mixture was directly treated with 3 mL of triethylamine and stirring continued at room temperature for 7 hours until Fmoc-deprotection was complete based on HPLC analysis. Triethylamine and partially DMF were removed under reduced pressure, and the residual mixture was purified by reversed-phase chromatography (C18 column 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure to ~70 mL final volume, and lyophilized to give 932 mg of product 13 as a bright-yellow light powder (0.64 mmol, 78% yield over 3 steps starting from 10). LRMS (ESI): m / z 1467.7 [M+H]+, Calcd for C71H102N8O25m / z 1467.7.

[0505] Compound 13 (20 mg, 14 µmol) was dissolved in 2 mL of anhydrous DMF and combined with HOAt (14 µmol, 14 µL of 1M solution in DMA) and DIPEA (6 µL). To this mixture were added compound 14 (5 mg, 14 µmol) at room temperature. Reaction mixture was stirred for 10 minutes until judged complete by LCMS analysis and purified directly by reversed- phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were lyophilized to give 20 mg of compound 15 as a yellow solid (12 µmol, 86% yield). LRMS (ESI): m / z 1660.8 [M+H]+, Calcd C81H113N9O28m / z 1660.8. Synthesis of Boc-Lys(Fmoc)-OPfp 9.

[0506] To a stirred solution of Boc-Lys(Fmoc)-OH (4.7 g, 10 mmol) in 40 mL of anhydrous THF were added pentafluorophenol (2.2 g, 12 mmol), followed by DCC (2.1 g, 10 mmol) in small portions at room temperature. The resulting mixture was stirred overnight, solids were filtered off, washed with THF on filter. Combined filtrate was concentrated under vacuum, and the white solid residue was triturated with DCM-EtOAc mixture, collected by filtration, andAtty. Dkt: RDWD-053WO dried under vacuum to give 4.9 g (7.8 mmol, 78% yield) of Boc-Lys(Fmoc)-OPfp 9 as a white powder. LRMS (ESI): m / z 657.3 [M+Na]+, Calcd for C32H31F5N2O6 m / z 657.2. Synthesis of compound 14

[0507] To a mixture of 6-maleimidohexanoic acid (2.11 g, 10 mmol) and pentafluorophenol (2.2 g, 12 mmol) in 25 mL of anhydrous THF were added DCC (2.06 g, 10 mmol) in one portion at room temperature. The resulting mixture was stirred overnight, then solids were filtered off and washed with THF. Combined filtrates were concentrated under vacuum and then purified by silica gel chromatography using (0-15% EtOAc-hexanes) to give 3.22 g of product 14 as a white solid (8.5 mmol, 85% yield). LRMS (ESI): m / z 400.1 [M+Na]+, Calcd for C16H12F5NO4m / z 400.1. EXAMPLE5 Synthesis of (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-((S)-2-amino-3-mercapto-3-methylbutanamido)- 3-methylbutanamido)propanamido)-5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2- ((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa- 4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (17)

[0508] To a solution of acetonide 16 (2 mg, 10 µmol) in anhydrous DMF (2 mL) were added HATU (4 mg, 10 µmol) and DIPEA (4 uL, 20 µmol) at RT. The mixture was stirred for 30 seconds before adding a solution of compound 3 (13 mg, 10 µmol) in DMF (0.5 mL). ReactionAtty. Dkt: RDWD-053WO mixture was stirred at RT for 1 h and then directly purified by reversed-phase prep HPLC (C18, acetonitrile-water with 0.05% TFA). Pure fractions were combined and concentrated under reduced pressure to remove acetonitrile. The resulting solution was diluted with 1 mL of 10 % (v / v) TFA in water. The mixture was then purged with N2, sealed, and kept at RT overnight, followed by direct lyophilization to obtain pure product 17 (11 mg, 8 µmol, 80% yield) as a white foam. LRMS (ESI): m / z 1360.7 [M+H]+, Calcd for C66H105N9O19S m / z 1360.7. Synthesis of (S)-2,2,5,5-tetramethylthiazolidine-4-carboxylic acid (16)(100 mL) was refluxed at 55 °C - 60 °C until the majority of solids dissolved (~ 9 h). The remaining solids were filtered, and the filtrate was allowed to cool to room temperature to start crystallization and placed in a -20 °C freezer overnight. The resulting precipitate was collected by filtration, washed with minimal cold acetone, and dried on air to give acetonide 16 as white sharp needles (0.90 g, 4.8 mmol, 72% yield) and used directly for next step. EXAMPLE6 Synthesis of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3- (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1- yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2- ((S)-2-((S)-2-(3-(2-((1,2-dimethylhydrazineyl)methyl)-5-((R)-27-oxo-29-(sulfomethyl)- 2,5,8,11,14,17,20,23,26-nonaoxa-28-azatriacontan-30-amido)-1H-indol-1-yl)propanamido)-3- methylbutanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (26)Atty. Dkt: RDWD-053WO

[0510] Nitro compound 19 (116 mg, 0.20 mmol) was dissolved in 1 mL of THF and combined with a solution of ammonium chloride (85 mg, 1.6 mmol) in 0.5 mL of water and 1 mL of methanol. The resulting mixture was vigorously stirred at room temperature and treated with zinc powder (104 mg, 1.6 mmol) in small portions over 5 minutes. Reaction mixture was stirred for 2 hours, solids were filtered off, filtrate was diluted with 20 mL of saturated aqueous ammonium chloride solution and extracted with ethyl acetate (2x25 mL). Organic extracts were dried over sodium sulfate, solvents removed under vacuum to give crude product 20 which was taken to the next step without purification. LRMS (ESI): m / z 555.3 [M+H]+, Calcd for C33H38N4O4m / z 555.3.

[0511] In an oven dried 100 mL round bottom flask were combined Boc-L-cysteic acid (100 mg, 371 µmol), HATU (155 mg, 408 µmol), and DIPEA (209 µL, 1.2 mmol) in 5 mL of DMF at RT. To this mixture were added compound 19 (206 mg, 371 µmol) in one portion. After 30 minutes, reaction was judged complete by LCMS analysis. The mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and evaporated under reduced pressure to give 250 mg of compound 21 (310 µmol, 84% yield) as a colorless oil. LRMS (ESI): m / z 806.4 [M+H]+, Calcd for C41H52N5O10S m / z 806.3.

[0512] Compound 21 (250 mg, 310 µmol) was dissolved in 3 mL of hexafluoro isopropanol (HFIP) and treated with 0.3 mL of concentrated HCl at RT. After 20 mins, reactionAtty. Dkt: RDWD-053WO mixture was diluted with 2-3 mL of water and purified by reversed-phase prep HPLC (C18, 0- 75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 170 mg of compound 22 (262 µmol, 84% yield) as a white powder. LRMS (ESI): m / z 650.2 [M+H]+, Calcd for C32H36N5O8S m / z 650.2.

[0513] In an oven dried 20 mL glass vial were combined compound 22 (17 mg, 26 µmol), HOAt (26 µmol, 26 µL of 1 M in DMA), and DIPEA (14 µL, 79 µmol) in 2 mL of DMF at RT. To this mixture were added m-PEG8 PNP carbonate 23 (15 mg, 27 µmol) in one portion. After 2 h, reaction was judged complete by LCMS analysis. Reaction mixture was directly purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and concentrated to dryness under reduced pressure to give 20 mg of compound 24 as colorless oil (19 µmol, 72% yield). LRMS (ESI): m / z 1060.4 [M+H]+, Calcd for C50H70N5O18S m / z 1060.4.

[0514] In an oven dried 20 mL glass vial were combined compound 24 (50 mg, 47 µmol), pentafluorophenol trifluoroacetate (0.5 mL), and 1 mL pyridine in 3 mL of DCM at RT. The mixture was allowed to stir at RT overnight. Reaction mixture was quenched by 10% TFA in water until the pH reached 4.5 and DCM was removed in vacuum. The residue was purified by reversed-phase prep HPLC (C18, 0-70% MeCN / H2O with 0.05% TFA). Pure fractions were collected and lyophilized to give 45 mg of compound 25 as colorless oil (37 µmol, 79% yield). LRMS (ESI): m / z 1226.4 [M+H]+, Calcd for C56H69F5N5O18S m / z 1226.4.

[0515] In an oven dried 20 mL glass vial were combined compound 3 (34 mg, 28 µmol), HOAt (36 µmol, 36 µL of 1 M in DMA), and DIPEA (19 µL, 108 µmol) in 3 mL of DMF at RT. To this mixture were added PFP-ester 25 (37 mg, 30 µmol) in one portion. After 10 minutes, 56 µL of piperidine were added directly to the reaction mixture. The resulting mixture was stirred for 30 min and then purified by reversed-phase prep HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). Pure fractions were combined and lyophilized to give 40 mg of compound 26 as a white powder (20 µmol, 67% yield). LRMS (ESI): m / z 1025.6 [M+2H]2+, Calcd for C96H155N13O33S m / z 1025.5.Atty. Dkt: RDWD-053WO Synthesis of mPEG8-PNP carbonate (23) and mP8c-Lys(Fmoc)-OH (29)anhydrous DCM were added DIPEA (0.35 mL, 2 mmol), followed by bis-(4-nitrophenyl) carbonate (335 mg, 1.1 mmol) at room temperature. Reaction mixture was stirred overnight and then directly purified by silica gel chromatography using 0-5% MeOH-DCM gradient to obtain 507 mg of compound 23 (0.92 mmol, 92% yield) as a light-yellow clear oil. LRMS (ESI): m / z 550.3 [M+H]+, Calcd for C24H39NO13m / z 550.2.

[0517] Amino acid H-Lys(Fmoc)-OH (28) (184 mg, 0.5 mmol) was suspended in 2 mL of anhydrous DMF, treated with DIPEA (0.17 mL, 1 mmol) at RT, and stirred for 5 min. To this mixture were added HOAt (0.6 mL of 1M in DMA, 0.6 mmol), followed by PNP-carbonate 23 (330 mg, 0.6 mmol) as a solution in 1 mL of DMF. The resulting mixture was stirred for 2 h at RT and then directly purified by reversed-phase chromatography (C18, 0-70% acetonitrile- water / 0.05% TFA). Pure fractions were combined and lyophilized to give 220 mg of compound 29 (0.28 mmol, 56% yield) as a clear colorless oil. LRMS (ESI): m / z 779.4 [M+H]+, Calcd for C39H58N2O14 m / z 779.4. EXAMPLE 7 Synthesis of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)-28-(((S)-1-(((S)-1-((2- (((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4-((((2-((S)-4- ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin- 11-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl- 1-oxobutan-2-yl)carbamoyl)-26,34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,33- diazaheptatriacontan-37-amido)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-1- yl)propanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa- 27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14- tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-Atty. Dkt: RDWD-053WO yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (31)

[0518] To a stirred solution of compound 13 (3.5 g, 2.4 mmol) in 16 mL of anhydrous DMA were added DIPEA (0.84 mL, 4.8 mmol) and HOAt (0.33 g, 2.4 mmol) at room temperature. The resulting mixture was treated with a separately prepared solution of bis-PFP- ester 30 (1.0 g, 1.1 mmol in 4 mL of DMA) in small portions (2 mL, 1 mL, 0.75 mL, and 0.5 mL) with 10 minutes intervals between additions. After the addition was complete, reaction mixture was stirred for 15 minutes at room temperature and treated with 2.1 mL of piperidine (22Atty. Dkt: RDWD-053WO mmol). After 20 minutes, the reaction mixture was directly purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure to ~100 mL final volume, and lyophilized to obtain 3.2 g of compound 31 as a bright-yellow fluffy powder (0.98 mmol, 89% yield for two steps based on 30). LRMS (ESI): m / z 1638.3 [M+H]2+, Calcd for C160H224N20O53 m / z 1638.8.

[0519] Synthesis of bis-PFP ester 30.combined with 6.2 g (33.4 mmol) of pentafluorophenol. The mixture was stirred and treated with DCC (2.0 g, 10 mmol) in a few small portions at room temperature. Reaction mixture was stirred for 48 hours at RT, then all solids were removed by filtration and washed with THF on filter. Combined filtrates were concentrated under vacuum and purified by silica gel chromatography (0-25-35% gradient of EtOAc-Hexanes) to give 2.4 g of bis-PFP ester 30 (2.6 mmol, 77% yield) as a white foaming solid. LRMS (ESI): m / z 931.2 [M+H]+, Calcd for C45H32F10N4O7 m / z 931.2. EXAMPLE 8 Synthesis of (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3- (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1- yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2- ((29S,32S,35S)-29-(4-(3-(2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-1- yl)propanamido)butyl)-...

Claims

Atty. Dkt: RDWD-053WO What is claimed is:

1. An antibody-drug conjugate comprising: an antibody; a first drug attached to the antibody at a native amino acid residue of the antibody through a first linker; and a second drug attached to the antibody at a site-specifically engineered conjugation site of the antibody through a second linker.

2. The conjugate of Claim 1, wherein the native amino acid residue of the antibody is a cysteine.

3. The conjugate of Claim 2, wherein the cysteine is produced via reduction of an interchain disulfide bond of the antibody.

4. The conjugate of any of Claims 1 to 3, wherein the site-specifically engineered conjugation site of the antibody comprises a formylglycine (fGly).

5. The conjugate of any of Claims 1 to 4, wherein the antibody has a drug-antibody ratio (DAR) of 2 or more for the first drug.

6. The conjugate of Claim 5, wherein two or more of the first drug are attached to the antibody through two or more of the first linker.

7. The conjugate of Claim 5, wherein the antibody has a DAR of 8 for the first drug.

8. The conjugate of Claim 7, wherein eight of the first drug are attached to the antibody through eight of the first linker.

9. The conjugate of any of Claims 1 to 8, wherein the antibody has a DAR of 2 or more for the second drug.Atty. Dkt: RDWD-053WO 10. The conjugate of Claim 9, wherein two or more of the second drug are attached to the antibody through two or more of the second linker.

11. The conjugate of any of Claims 9 to 10, wherein the antibody has a DAR of 2 for the second drug.

12. The conjugate of Claim 11, wherein two of the second drug are attached to the antibody through two of the second linker.

13. The conjugate of any of Claims 9 to 10, wherein the antibody has a DAR of 4 for the second drug.

14. The conjugate of Claim 13, wherein four of the second drug are attached to the antibody through four of the second linker.

15. The conjugate of Claim 13, wherein four of the second drug are attached to the antibody through two of the second linker, wherein each of the second linker is a branched linker.

16. The conjugate of any of Claims 9 to 10, wherein the antibody has a DAR of 8 for the second drug.

17. The conjugate of Claim 16, wherein eight of the second drug are attached to the antibody through four of the second linker, wherein each of the second linker is a branched linker.

18. The conjugate of any of Claims 1 to 17, wherein the antibody has a total DAR for the first drug and the second drug of 4 to 16.Atty. Dkt: RDWD-053WO 19. The conjugate of any of Claims 1 to 18, wherein the conjugate has formula (I): wherein AL is a first linker; LBis a second linker; W1is the first drug; W2is the second drug; W3is an antibody; XAis a first conjugation moiety attached to the antibody at a native amino acid residue of the antibody; and XBis a second conjugation moiety attached to the antibody at a site-specifically engineered conjugation site of the antibody.

20. The conjugate of Claim 19, wherein XAis attached to W3via a cysteine residue of W3.

21. The conjugate of Claim 20, wherein XAis attached to W3via maleimide-based conjugation.

22. The conjugate of Claim 20, having formula (Ia): .Atty. Dkt: RDWD-053WO 23. The conjugate of any one of Claims 20 to 22, having formula (Ib): ,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

24. The conjugate of Claim 22 or 23, wherein the conjugate is selected from: ,Atty. Dkt: RDWD-053WO , ,Atty. Dkt: RDWD-053WO , ,Atty. Dkt: RDWD-053WO .

25. The conjugate of Claim 20, having formula (Ic): ,R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl.

26. The conjugate of Claim 20 or 24, having formula (Id): , wherein:each R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,Atty. Dkt: RDWD-053WO cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

27. The conjugate of Claim 25 or 26, wherein the conjugate is selected from .

28. The conjugate of any one of Claims 19 to 27, wherein XBis attached to W3via a formyl glycine residue of W3.

29. The conjugate of Claim 28, wherein XBis attached to W3via HIPS or azaHIPS-based conjugation.Atty. Dkt: RDWD-053WO 30. The conjugate of Claim 29, having formula (Ie): ,R’ and R” are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; Z represents CR21, NR22, N, O or S; and R21and R22are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

31. The conjugate of Claim 30, wherein the conjugate has formula (If): .Atty. Dkt: RDWD-053WO 32. The conjugate of Claim 30, wherein the conjugate has formula (Ig): , wherein:each is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; R3comprises a cleavable moiety.

33. The conjugate of any one of claims Claim 30 to 32, wherein the conjugate is selected from , .Atty. Dkt: RDWD-053WO 34. The conjugate of Claim 28, wherein XBis attached to W3via thiazolidine conjugation.

35. The conjugate of Claim 34, having formula (Ih) .

36. The conjugate of Claim 35, having formula (Ii).

37. The conjugate of Claim 35 or 36, having the structure38. The conjugate of any one of Claims 1 to 18, having formula The conjugate of any of Claims 1 to 18, wherein the conjugate has formula (II):Atty. Dkt: RDWD-053WO whereinLAis a first linker; LBis a second linker; LB1is a third linker; W1is the first drug; W2is the second drug; W2Ais a third drug; W3is an antibody; XAis a first conjugation moiety attached to the antibody at a native amino acid residue of the antibody; and XBis a second conjugation moiety attached to the antibody at a site-specifically engineered conjugation site of the antibody.

39. The conjugate of Claim 38, wherein LBand LB1are the same.

40. The conjugate of Claim 38 or 39, wherein W2and W2Aare the same.

41. The conjugate of any of Claims 38 to 40, having formula (IIa): .Atty. Dkt: RDWD-053WO 42. The conjugate of any one of Claims 39 to 41, having formula (IIb): (IIb),each R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

43. The conjugate of any of Claims 38 to 40, having formula (IIc): ,R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl.Atty. Dkt: RDWD-053WO 44. The conjugate of any one of Claims 39 to 41 and 43, having formula (IId): (IId),each R1is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; each R2is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; k1 is an integer from 1 to 10; and R3comprises a cleavable moiety.

45. The conjugate of any one of Claims 38 to 44, having formula (IIe) W3LAXAW1.

46. The conjugate of claim 45, having the structure:Atty. Dkt: RDWD-053WO OH O HO OH O O .

47. A pharmaceutical composition comprising: a conjugate of any one of Claims 1 to 18; and a pharmaceutically-acceptable excipient.

48. A method comprising: administering to a subject an amount of a conjugate of any one of Claims 1 to 46.

49. A method of treating cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of any one of Claims 1 to 46, wherein the administering is effective to treat cancer in the subject.Atty. Dkt: RDWD-053WO 50. A method of producing an antibody-drug conjugate, comprising: contacting an antibody comprising a site-specifically engineered conjugation site with: (1) a first drug-linker compound comprising a first reactive group configured to conjugate to a native amino acid residue of the antibody; and (2) a second drug-linker compound comprising a second reactive group configured to conjugate to the site-specifically engineered conjugation site.

51. The method of Claim 50, wherein the contacting comprises combining the antibody, the first drug-linker compound and the second drug-linker compound in a one-pot reaction.

52. The method of Claim 50, wherein the contacting comprises contacting the antibody with the first drug-linker compound and then contacting the antibody with the second drug-linker compound.

53. The method of Claim 50, wherein the contacting comprises contacting the antibody with the second drug-linker compound and then contacting the antibody with the first drug-linker compound.

54. The method of any of Claims 50 to 53, wherein prior to the contacting the method comprises reducing one or more interchain disulfide bonds of the antibody to produce two or more cysteine residues.

55. The method of Claim 54, wherein the first reactive group comprises a maleimide.

56. The method of any of Claims 50 to 55, wherein the site-specifically engineered conjugation site of the antibody comprises an fGly.

57. The method of Claim 50, wherein the second reactive group comprises a hydrazinyl- indole or hydrazinyl-pyrrolo-pyridine group.

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