CN116783208A - Glycoside double cleavage linker for antibody-drug conjugates - Google Patents

Abstract

The present disclosure provides antibody-drug conjugate structures comprising a cleavable linker connecting the antibody to the drug and having a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc. The disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using the conjugates.

Description

Glycosidic double cleavable linkers for antibody-drug conjugates

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/116,632, filed on November 20, 2020, and U.S. Provisional Application No. 63/139,685, filed on January 20, 2021, the disclosures of which are incorporated herein by reference.

Background Art

The field of protein-small molecule therapeutic conjugates has made great progress, providing many clinically beneficial drugs, and hopefully more in the coming years. Protein conjugate therapeutics can offer several advantages due to, for example, specificity, functional diversity, and relatively low off-target activity, thus fewer side effects. Chemical modification of proteins can make proteins more potent, stable, or multimodal to extend these advantages.

Summary of the invention

The present disclosure provides antibody-drug conjugate structures, which include a cleavable linker, which connects the antibody to the drug and has a first enzymatically cleavable portion and a second enzymatically cleavable portion, the second enzymatically cleavable portion including a glycoside selected from galactosides, glucosides, mannosides, fucosides, O-GlcNAc and O-GalNAc. The present disclosure also encompasses compounds and methods for producing such conjugates, and methods of using the conjugates.

Aspects of the present disclosure include a conjugate comprising an antibody, a drug, and a cleavable linker connecting the antibody to the drug and having a first enzymatically cleavable portion and a second enzymatically cleavable portion, the second enzymatically cleavable portion comprising a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In some embodiments, the conjugate has Formula (I):

in

Z is CR ⁴ or N;

X is O or NR ⁴ ;

^R1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

^R2 and ^R3 are 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or ^R2 and ^R3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl;

each ^R is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

Each R ⁵ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl;

Each ^R6 is 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;

k is an integer from 1 to 10;

^R7 comprises a second enzymatically cleavable moiety;

L ¹ is the first connector;

L ² is the second connector;

^W1 is a drug; and

^W2 is the antibody.

In some embodiments, k is 2; and the conjugate has Formula (Ia):

In some embodiments, the second enzymatically cleavable moiety comprises a galactoside. In some embodiments, the second enzymatically cleavable moiety comprises a glucoside. In some embodiments, the second enzymatically cleavable moiety comprises a mannoside. In some embodiments, the second enzymatically cleavable moiety comprises a fucoside. In some embodiments, the second enzymatically cleavable moiety comprises an O-GlcNAc. In some embodiments, the second enzymatically cleavable moiety comprises an O-GalNAc.

In some embodiments, the conjugate has Formula (Ib):

In some embodiments, the conjugate has Formula (Ic):

In some embodiments, the conjugate has Formula (Id):

In some embodiments, the conjugate has Formula (Ie):

In some embodiments, the conjugate has formula (If):

In some embodiments, the conjugate has formula (Ig):

In some embodiments, L1 comprises:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -,

in

a, b, c and d are each independently 0 or 1;

T ¹ , T ² , T ³ and T ⁴ are each independently selected from a covalent bond, a (C ₁ -C ₁₂ ) alkyl group, a substituted (C ₁ -C ₁₂ ) alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclyl group and a substituted heterocyclyl group, (EDA) _w , (PEG) _n , (AA) _p , (CR ¹³ OH) _m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide and an ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino 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;

V ¹ , V ² , V ³ and V ⁴ are each independently selected from the group consisting of a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-, wherein each q is an integer from 1 to 6;

Each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

Each R ¹⁵ is 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 some embodiments, ^L2 comprises:

-(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -,

in

e, f, g and h are each independently 0 or 1;

T ⁵ , T ⁶ , T ⁷ and T ⁸ are each independently selected from a covalent bond, a (C ₁ -C ₁₂ ) alkyl group, a substituted (C ₁ -C ₁₂ ) alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclyl group and a substituted heterocyclyl group, (EDA) _w , (PEG) _n , (AA) _p , (CR ¹³ OH) _m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide and an ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino 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;

V ⁵ , V ⁶ , V ⁷ and V ⁸ are each independently selected from the group consisting of a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-, wherein each q is an integer from 1 to 6;

Each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

Each R ¹⁵ is 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 some embodiments, ^L1 is as described herein, wherein:

T ¹ is selected from (C ₁ -C ₁₂ )alkyl and substituted (C ₁ -C ₁₂ )alkyl;

T ² , T ³ and T ⁴ are each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w , (PEG) _n , (C ₁ -C ₁₂ )alkyl, substituted (C ₁ -C ₁₂ )alkyl, (AA) _p , -(CR ¹³ OH) _m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine and an ester; and

V ¹ , V ² , V ³ , and V ⁴ are each independently selected from the group consisting of a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-;

in:

(PEG) _n is wherein n is an integer from 1 to 30;

EDA is the ethylenediamine moiety with the following structure: wherein y is an integer from 1 to 6, and r is 0 or 1;

4-Amino-piperidine (4AP) is

Each R ¹² and R ¹⁵ is independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl and substituted aryl, wherein any two adjacent R ¹² groups may be cyclically linked to form a piperazinyl ring; and

^R13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl and substituted aryl.

In some embodiments, ^L1 is as described herein, wherein:

T ¹ is (C ₁ -C ₁₂ )alkyl, and V ¹ is -CO-;

T ² is an amino acid analog, and V ² is -NH-;

T ³ is (PEG) _n , and V ³ is -CO-; and

d is 0.

In some embodiments, ^L2 is as described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is -CO-; and

f, g and h are 0.

In some embodiments, ^L2 is as described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is -CONR ¹⁵ -;

T ⁶ is (C ₁ -C ₁₂ )alkyl, and V ⁶ is -CO-; and

g and h are 0.

In some embodiments, the drug is selected from cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids and peptides. In some embodiments, the drug is selected from auristatin, maytansine and duocarmycin. In some embodiments, the drug is selected from Tubulysin M, Calichemicin, SN-38, Exatecan, STAT3 inhibitors, α-amanitin, Aurora kinase inhibitors, belotecan, 9-aminocamptothecin (9-AC) and anthracycline antibiotics.

Aspects of the present disclosure include a compound comprising a cleavable linker for attaching an antibody to a drug, wherein the cleavable linker comprises a first enzymatically cleavable portion and a second enzymatically cleavable portion, the second enzymatically cleavable portion having a glycoside selected from galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

In some embodiments, the compound has Formula (II):

in

Z is CR ⁴ or N;

X is O or NR ⁴ ;

^R2 and ^R3 are 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or ^R2 and ^R3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl;

each ^R is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

each R ⁵ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

Each ^R6 is 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;

k is an integer from 1 to 10;

^R7 comprises a second enzymatically cleavable moiety;

L ¹ is the first connector;

^L2 is the second linker; and

W ¹ is medicine.

In some embodiments, k is 2; and the compound has Formula (IIa):

In some embodiments, the second cleavable moiety comprises a galactoside. In some embodiments, the second cleavable moiety comprises a glucoside. In some embodiments, the second cleavable moiety comprises a mannoside. In some embodiments, the second enzymatically cleavable moiety comprises a fucoside. In some embodiments, the second enzymatically cleavable moiety comprises an O-GlcNAc. In some embodiments, the second enzymatically cleavable moiety comprises an O-GalNAc.

In some embodiments, the compound has Formula (IIb):

In some embodiments, the compound has Formula (IIc):

In some embodiments, the compound has Formula (IId):

In some embodiments, the compound has Formula (IIe):

In some embodiments, the compound has Formula (IIf):

In some embodiments, the compound has Formula (IIg):

In some embodiments, ^L1 comprises:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -,

in

a, b, c and d are each independently 0 or 1;

T ¹ , T ² , T ³ and T ⁴ are each independently selected from a covalent bond, a (C ₁ -C ₁₂ ) alkyl group, a substituted (C ₁ -C ₁₂ ) alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclyl group and a substituted heterocyclyl group, (EDA) _w , (PEG) _n , (AA) _p , (CR ¹³ OH) _m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide and an ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino 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;

V ¹ , V ² , V ³ and V ⁴ are each independently selected from the group consisting of a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-, wherein each q is an integer from 1 to 6;

Each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

Each R ¹⁵ is 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 some embodiments, ^L2 comprises:

-(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -,

in

e, f, g and h are each independently 0 or 1;

T ⁵ , T ⁶ , T ⁷ and T ⁸ are each independently selected from a covalent bond, a (C ₁ -C ₁₂ ) alkyl group, a substituted (C ₁ -C ₁₂ ) alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclyl group and a substituted heterocyclyl group, (EDA) _w , (PEG) _n , (AA) _p , (CR ¹³ OH) _m -, 4-amino-piperidine (4AP), an acetal group, a hydrazine, a disulfide and an ester, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino 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;

V ⁵ , V ⁶ , V ⁷ and V ⁸ are each independently selected from the group consisting of a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-, wherein each q is an integer from 1 to 6;

Each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

Each R ¹⁵ is 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 some embodiments, ^L1 is as described herein, wherein:

T ¹ is selected from (C ₁ -C ₁₂ )alkyl and substituted (C ₁ -C ₁₂ )alkyl;

^T2 , ^T3 and ^T4 are each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w , (PEG) _n , ( _C1 - _C12 ) alkyl, substituted ( _C1 - _C12 ) alkyl, (AA) _p , -( ^CR13OH ) _m- , 4-amino-piperidine (4AP), acetal group, hydrazine and ester; and

V ¹ , V ² , V ³ , and V ⁴ are each independently selected from the group consisting of a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-;

in:

(PEG) _n is wherein n is an integer from 1 to 30;

EDA is the ethylenediamine moiety with the following structure: wherein y is an integer from 1 to 6, and r is 0 or 1;

4-Amino-piperidine (4AP) is

Each R ¹² and R ¹⁵ is independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl and substituted aryl, wherein any two adjacent R ¹² groups may be cyclically linked to form a piperazinyl ring; and

^R13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl and substituted aryl.

In some embodiments, ^L1 is as described herein, wherein:

T ¹ is (C ₁ -C ₁₂ )alkyl, and V ¹ is -CO-;

T ² is an amino acid analog, and V ² is -NH-;

T ³ is (PEG) _n , and V ³ is -CO-; and

d is 0.

In some embodiments, ^L2 is as described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is -CO-; and

f, g and h are 0.

In some embodiments, ^L2 is as described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is -CONR ¹⁵ -;

T ⁶ is (C ₁ -C ₁₂ )alkyl, and V ⁶ is -CO-; and

g and h are 0.

In some embodiments, the drug is selected from cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids and peptides. In some embodiments, the drug is selected from auristatin, maytansine and duocarmycin. In some embodiments, the drug is selected from terpilesin M, calicheamicin, SN-38, exitecan, STAT3 inhibitors, α-amanitin, Aurora kinase inhibitors, belotecan, 9-aminocamptothecin (9-AC) and anthracycline antibiotics.

Aspects of the present disclosure include a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable excipient.

Aspects of the disclosure include a method for administering a conjugate to a subject, wherein the method comprises administering to the subject a conjugate as described herein.

Aspects of the disclosure include a method for treating cancer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate as described herein, wherein the administration is effective to treat the cancer in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows cytotoxins commonly used to generate therapeutic antibody-drug conjugates. Arrows indicate base-labile groups.

Figure 2 shows HIPS linkage for the synthesis of antibody-drug conjugates (ADCs). Antibodies carrying aldehyde moieties are reacted with a hydrazine-iso-Pictet-Spengler (HIPS) linker and a payload to generate site-specifically conjugated ADCs with stable azacarboline bonds.

Figure 3 shows the hydrophobic interaction column (HIC) trace of Construct 30 trastuzumab conjugate, which yielded a DAR of 1.7 as determined by HIC.

Figure 4 shows the analytical size exclusion chromatography (SEC) trace of the Construct 30 trastuzumab conjugate, which shows that the conjugate is 97.1% monomeric as determined by analytical SEC.

Figure 5 shows the HIC trace of Construct 33 trastuzumab conjugate, which yielded a DAR of 1.66 as determined by HIC.

Figure 6 shows the analytical SEC trace of the Construct 33 trastuzumab conjugate, which demonstrates that the conjugate is 97.6% monomeric as determined by analytical SEC.

Figure 7 shows the HIC trace of Construct 30 polatuzumab conjugate, which yielded a DAR of 1.78 as determined by HIC.

Figure 8 shows the analytical SEC trace of Construct 30 polotuzumab conjugate, which demonstrates that the conjugate is 95.4% monomeric as determined by analytical SEC.

Figure 9 shows the HIC trace of Construct 33 polotuzumab conjugate, which yielded a DAR of 1.6 as determined by HIC.

Figure 10 shows the analytical SEC trace of Construct 33 polotuzumab conjugate, which shows that the conjugate is 96.8% monomeric as determined by analytical SEC.

Figure 11 shows the structures of comparative molecules 35 (single-cleavage maytansine construct) and 34 (glucuronide double-cleavage MMAE construct).

FIG. 12 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying either a single-cleavage (35) or a galactoside-modified double-cleavage linker (30) against Granta-519 cells.

FIG. 13 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying either a single-cleavage (35) or a galactoside-modified double-cleavage linker (30) against Ramos-RA cells.

FIG14 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying either a single-cleavage (35) or a galactoside-modified double-cleavage linker (30) against NCI-N87 cells.

Figure 15 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying either a single-cleavage (35) or a galactoside-modified double-cleavage linker (30) against Sk-Br-3 cells.

FIG. 16 shows a graph of the in vitro potency of MMAE-conjugated ADCs carrying a glucuronide-modified dual-cleavage linker ( 34 ) or a galactoside-modified dual-cleavage linker ( 33 ) against Ramos-RA cells.

FIG. 17 shows a graph of the in vitro potency of MMAE-conjugated ADCs carrying a glucuronide-modified dual-cleavage linker ( 34 ) or a galactoside-modified dual-cleavage linker ( 33 ) against NCI-N87 cells.

FIG. 18 shows a graph of the in vitro potency of MMAE-conjugated ADCs carrying a glucuronide-modified dual-cleavage linker ( 34 ) or a galactoside-modified dual-cleavage linker ( 33 ) against Sk-Br-3 cells.

Figure 19 shows the PLRP trace of construct 46 anti-FITC conjugate, which yielded a DAR of 1.70 as determined by PLRP.

Figure 20 shows the analytical SEC trace of Construct 46 anti-FITC conjugate, which was 95.9% monomeric as determined by analytical SEC.

Figure 21 shows the PLRP trace of Construct 46 Trastuzumab conjugate, which yielded a DAR of 1.79 as determined by PLRP.

Figure 22 shows the analytical SEC trace of Construct 46 trastuzumab conjugate, which was 96.2% monomeric as determined by analytical SEC.

Figure 23 shows the PLRP trace of Construct 46 sacituzumab conjugate, which yielded a DAR of 1.15 as determined by PLRP.

Figure 24 shows the analytical SEC trace of Construct 46 thacizumab conjugate, which was 94.4% monomeric as determined by analytical SEC.

Figure 25 shows the PLRP trace of construct 44 anti-FITC conjugate, which yielded a DAR of 1.68 as determined by PLRP.

Figure 26 shows the analytical SEC trace of Construct 44 anti-FITC conjugate, which was 96.0% monomeric as determined by analytical SEC.

Figure 27 shows the PLRP trace of Construct 44 trastuzumab conjugate, which yielded a DAR of 1.78 as determined by PLRP.

Figure 28 shows the analytical SEC trace of Construct 44 trastuzumab conjugate, which was 96.0% monomeric as determined by analytical SEC.

Figure 29 shows the PLRP trace of Construct 44 sacituzumab conjugate, which yielded a DAR of 1.16 as determined by PLRP.

Figure 30 shows the analytical SEC trace of Construct 44 thacizumab conjugate, which was 94.5% monomeric as determined by analytical SEC.

FIG31 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying a glucoside-modified (44) or galactoside-modified dual-cleavage linker (30) against MDA-MB-468 cells.

FIG32 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying a glucoside-modified (44) or galactoside-modified dual-cleavage linker (30) against BxPC3 cells.

FIG33 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying a glucoside-modified (44) or galactoside-modified dual-cleavage linker (30) against SKBR3 cells.

FIG34 shows a graph of the in vitro potency of maytansine-conjugated ADCs carrying a glucoside-modified (44) or galactoside-modified dual-cleavage linker (30) against NCI-N87 cells.

FIG35 shows a graph of the in vitro potency of MMAE-conjugated ADCs carrying a glucuronide-modified (34) or glucosidic-modified dual-cleavage linker (46) against MDA-MB-468 cells.

FIG36 shows a graph of the in vitro potency of MMAE-conjugated ADCs carrying a glucuronide-modified (34) or glucosidic-modified dual-cleavage linker (46) against BxPC3 cells.

definition

Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

"Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, e.g., 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. The term includes, for _example , straight and branched chain hydrocarbon groups _such as methyl ( _CH3- ), ethyl ( _CH3CH2- ) _, n- _propyl (CH3CH2CH2-), _isopropyl (( _{CH3 )} 2CH-), _n -butyl ₍ CH3CH2CH2CH2-), _isobutyl (( _CH3 )2CHCH2-), sec-butyl (( _CH3 )( _{CH3CH2 ) CH-} ), tert _- butyl (( _{CH3 ) 3C-} ), _n -pentyl ( _{CH3CH2CH2CH2CH2-} ) _{, and neopentyl} (( _CH3 ) _3CCH2- ).

The term "substituted alkyl" refers to an alkyl group as defined herein in which one or more carbon atoms in the alkyl chain (excluding the _C1 carbon atom) have been optionally replaced by a heteroatom such as -O-, -N-, -S-, -S(O)-, or -S(O) _-. -(wherein n is 0 to 2), -NR-(wherein R is hydrogen or alkyl), and has 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, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, 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 selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclyl.

"Alkylene" refers to a divalent aliphatic hydrocarbon group preferably having 1 to 6 and more preferably 1 to 3 carbon atoms, which is linear or branched, and which is optionally interrupted by one or more groups selected from -O-, ^-NR10- , ^-NR10C (O)-, -C ₍ O) ^NR10- , etc. The term includes, for example, methylene ( _-CH2- ), ethylene ( _-CH2CH2- ), n-propylene ( _-CH2CH2CH2- ), _{isopropylene (} -CH2CH _{( CH3} )-), (-C ₍ CH3) _{2CH2CH2- ) , ( -C} (CH3) _2CH2C (O) _- ) _, (-C( _CH3 )2CH2C(O)NH-), (-CH( _CH3 ) _CH2- ), and the like.

"Substituted alkylene" refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described below for a carbon in the definition of "substituted".

The term "alkane" refers to alkyl and alkylene groups as defined herein.

The terms "alkylaminoalkyl," "alkylaminoalkenyl," and "alkylaminoalkynyl" refer to the group R'NHR"-, wherein R' is an alkyl group as defined herein, and R" is an alkylene, alkenylene or alkynylene group as defined herein.

The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and -substituted alkylene-aryl wherein alkylene, substituted alkylene and aryl are as defined herein.

"Alkoxy" refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-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-, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

The term "alkoxyamino" refers to the group -NH-alkoxy wherein alkoxy is as defined herein.

The term "haloalkoxy" refers to the group alkyl-O- wherein one or more hydrogen atoms on the alkyl group has been replaced with a halo group and includes groups such as trifluoromethoxy.

The term "haloalkyl" refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been replaced by a halogen group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

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.

The term "alkylthioalkoxy" refers to the groups -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.

"Alkenyl" refers to a straight or branched chain hydrocarbon radical having 2 to 6 carbon atoms, and preferably 2 to 4 carbon atoms, and having at least 1, and preferably 1 to 2, sites of double bond unsaturation. The term includes, for example, divinyl, allyl, and but-3-en-1-yl. The term includes cis and trans isomers or mixtures of these isomers.

[0063] 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, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, _-SO2 -alkyl, _-SO2 -substituted alkyl, _-SO2 -aryl and _-SO2 -heteroaryl.

"Alkynyl" refers to a straight or branched monovalent hydrocarbon radical having 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl radicals include ethynyl (-C≡CH) and propargyl ( _-CH2C≡CH ).

[0063] 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, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, _-SO2 -alkyl, _-SO2 -substituted alkyl, _-SO2 -aryl and _-SO2 -heteroaryl.

"Alkynyloxy" refers to the group -O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, for example, ethynyloxy, propynyloxy, and the like.

"Acyl" refers to the radicals HC(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)-. 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, heterocyclyl, and substituted heterocyclyl are as defined herein. For example, acyl includes the "acetyl" group CH ₃ C(O)-.

"Acylamino" refers to the groups ^-NR20C (O)alkyl, ^-NR20C (O)substituted alkyl, ^-NR20C (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)heterocycle, and ^-NR20C (O)substituted heterocycle, wherein R ²⁰ is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein.

"Aminocarbonyl" or the term "aminoacyl" refers to the group ^-C (O) ^NR21R22 , wherein ^R21 and ^R22 are independently 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, heterocyclyl, and substituted heterocyclyl, and wherein ^R21 and ^R22 are optionally joined together to form a heterocyclyl or substituted heterocyclyl group to the nitrogen to which they are bound, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"Aminocarbonylamino" refers to ^the group ^-NR21C (O) ^NR22R23 , wherein ^R21 , ^R22 and ^R23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or wherein two R groups are linked to form a heterocyclyl group.

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

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.

"Aminosulfonyl" refers to the group _-SO2NR21R22 , wherein ^R21 and ^{R22 are} independently 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, heterocyclyl, substituted heterocyclyl, and wherein ^R21 and ^R22 are optionally joined together with the nitrogen to which they are bound to form a heterocyclyl or substituted heterocyclyl group, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"_{Sulfonylamino" refers to the group -NR21SO2R22} ^, wherein ^R21 and ^{R22 are} independently 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, heterocyclyl, and substituted heterocyclyl, and wherein ^R21 and ^R22 are optionally joined together with the atoms to which they are bound to form a heterocyclyl or substituted heterocyclyl group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as found in phenyl) or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthracenyl and indanyl groups) which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring. The term includes, for example, phenyl and naphthyl. Unless otherwise limited by the definition of aryl substituents, such aryl groups may be optionally substituted with 1 to 5 substituents, or 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, amido, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, 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.

"Aryloxy" refers to the group -O-aryl wherein aryl is as defined herein including, for example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups also as defined herein.

"Amino" refers to the group _-NH2 .

The term "substituted amino" refers to the group -NRR, wherein 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, with the proviso that at least one R is not hydrogen.

The term "azido" refers to the group _-N3 .

"Carboxyl,""carboxy," or "carboxylate" refers to -CO ₂ H or a salt thereof.

"Carboxyl ester" or "carboxy ester" or the term "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 The invention also includes cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclyl, and -C(O)O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"(Carboxyester)oxy" or "carbonate" refers to the group -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-heterocyclyl, -O-C(O)O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein.

"Cyano" or "nitrile" refers to the group -CN.

"Cycloalkyl" refers to a cycloalkyl group of 3 to 10 carbon atoms having a single or multiple rings (including fused, bridged and spiro ring systems). Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc. Such cycloalkyl groups include, for example, monocyclic structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures, such as adamantyl, etc.

The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 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, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, _-SO2 -alkyl, _-SO2 -substituted alkyl, _-SO2 -aryl and _-SO2 -heteroaryl.

"Cycloalkenyl" refers to a non-aromatic cycloalkyl group of 3 to 10 carbon atoms having a single or multiple rings and having at least one double bond and preferably 1 to 2 double bonds.

The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxaaminoacyl, azido, cyano, halogen, hydroxy, keto, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, _-SO2 -alkyl, _-SO2 -substituted alkyl, _-SO2 -aryl and _-SO2 -heteroaryl.

"Cycloalkynyl" refers to a non-aromatic cycloalkyl group of 5 to 10 carbon atoms having a single or multiple rings and having at least one triple bond.

"Cycloalkoxy" refers to an -O-cycloalkyl group.

"Cycloalkenyloxy" refers to an -O-cycloalkenyl group.

"Halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

"Hydroxy" or "hydroxyl" refers to the group -OH.

"Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms such as 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups can have a monocycle (e.g., pyridyl, imidazolyl or furyl) or multiple condensed rings (e.g., in groups such as indolizinyl, quinolyl, benzofuran, benzimidazolyl or benzothienyl) in a ring system, wherein at least one ring in the ring system is aromatic. In order to meet the valence requirements, any heteroatom in such heteroaryl rings may or may not be bonded to H or a substituent group (e.g., an alkyl group or other substituents as described herein). In certain embodiments, the nitrogen and/or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide N-oxides (N→O), sulfinyl or sulfonyl moieties. The term includes, for example, pyridyl, pyrrolyl, indolyl, thienyl and furyl. Unless otherwise limited by the definition of heteroaryl substituents, such heteroaryl groups may be optionally substituted with 1 to 5 substituents, or 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, amido, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, 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.

The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, wherein alkylene and heteroaryl are as defined herein. The term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

"Heteroaryloxy" refers to an -O-heteroaryl group.

"Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple fused rings (including fused bridged rings and spiro ring systems) and having from 3 to 20 ring atoms (including from 1 to 10 heteroatoms). The ring atoms are selected from nitrogen, sulfur or oxygen, wherein in a fused ring system, one or more rings may be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)- or _-SO2- moiety. To meet valence requirements, any heteroatom in such a heterocycle may or may not be bonded to one or more H or one or more substituent groups, such as an alkyl group or other substituents as described herein.

Examples of heterocyclic and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, 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 known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl, and the like.

Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, _-SO2 -alkyl, _-SO2 -substituted alkyl, _-SO2 -aryl, _-SO2 -heteroaryl and fused heterocycle.

"Heterocyclyloxy" refers to the group -O-heterocyclyl.

The term "heterocyclylthio" refers to the group heterocyclyl-S-.

The term "heterocyclylene" refers to a diradical formed from a heterocycle as defined herein.

The term "hydroxyamino" refers to the group -NHOH.

"Nitro" refers to the radical _-NO2 .

"Oxo" refers to the (=O) atom.

"Sulfonyl" refers to the radicals _-SO2 -alkyl, _-SO2 -substituted alkyl, _-SO2 -alkenyl, _-SO2 -substituted alkenyl, _-SO2 -cycloalkyl, _-SO2 -substituted cycloalkyl, -SO2-cycloalkenyl, _-SO2 -substituted cycloalkenyl, _-SO2 -aryl, _-SO2 -substituted aryl, _-SO2 -heteroaryl, _-SO2 -substituted heteroaryl, _-SO2 -heterocyclyl, and _-SO2 -substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein. _Sulfonyl radicals include, for example, methyl- _SO2- , phenyl- _SO2- , and 4-methylphenyl- _SO2- .

"Sulfonyloxy" refers to the radical _-OSO2 -alkyl, _-OSO2 -substituted alkyl, _-OSO2 -alkenyl, _-OSO2 -substituted alkenyl, _-OSO2 -cycloalkyl, _-OSO2 -substituted cycloalkyl, -OSO2-cycloalkenyl, _-OSO2 -substituted cycloalkenyl, _-OSO2 -aryl, _-OSO2 -substituted aryl, _-OSO2 -heteroaryl, _-OSO2 -substituted heteroaryl, _-OSO2 -heterocyclyl, and _{-OSO2 -} substituted heterocyclyl wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"Sulfate" or "sulfate ester" refers to the groups -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 cycloalkyl, -O-SO2 _- O-cycloalkenyl, -O-SO2-O-substituted cycloalkenyl, -O- _{SO2 -} O-aryl, -O- _SO2 -O-substituted aryl, -O-SO2 _- O-heteroaryl, -O- _SO2 -O-substituted heteroaryl, -O- _SO2 -O-heterocyclyl, and -O-SO2-O-cycloalkyl _. -O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein.

The term "aminocarbonyloxy" refers to the group -OC(O)NRR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

"Thiol" refers to the group -SH.

"Thio" or the term "thioketo" refers to the atom (=S).

"Alkylthio" or the term "thioalkoxy" refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur can be oxidized to -S(O)-. Sulfoxides can exist as one or more stereoisomers.

The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.

The term "thioaryloxy" refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also as defined herein.

The term "thioheteroaryloxy" refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups also as defined herein.

The term "thioheterocyclyl" refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including an optionally substituted heterocyclyl group also as defined herein.

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

In addition to the groups disclosed with respect to the various terms herein, unless otherwise specified, substituent groups used to replace one or more hydrogens on a saturated carbon atom in a specified group or radical (any two hydrogens on a single carbon may be replaced by =0, =NR ⁷⁰ , =N-OR ⁷⁰ , =N ₂ , or =S) are -R ⁶⁰ , halogen, =0, -OR ⁷⁰ , -SR ⁷⁰ , -NR ⁸⁰ R ⁸⁰ , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO ₂ , =N ₂ , -N ₃ , -SO ₂ R ⁷⁰ , -SO ₂ O ^- M ⁺ , -SO ₂ OR ⁷⁰ , -OSO ₂ R ⁷⁰ , -OSO ₂ O ^- M ⁺ , -OSO ₂ OR ⁷⁰ , -P(O)(O ^- ) ₂ (M ⁺ ) ₂ , -P(O)(OR ⁷⁰ )O ^- M ⁺ , -P(O)(OR ⁷⁰ ) ₂ , -C(O)R ⁷⁰ , -C(S)R ⁷⁰ , -C(NR ⁷⁰ )R ⁷⁰ , -C(O)O ^- M ⁺ , -C(O)OR ⁷⁰ , -C(S)OR ⁷⁰ , -C(O)NR ⁸⁰ R ⁸⁰ , -C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , -OC(O )R ⁷⁰ , -OC(S)R ⁷⁰ , -OC(O)O ^- M ⁺ , -OC(O)OR ⁷⁰ , -OC(S)OR ⁷⁰ , -NR ⁷⁰ C(O)R ⁷⁰ , -NR ⁷⁰ C(S)R ⁷⁰ , -NR ⁷⁰ CO ₂ ^- M ⁺ , -NR ⁷⁰ CO ₂ R ⁷⁰ , -NR ⁷⁰ C(S)OR ⁷⁰ , -NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ , -NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and -NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , wherein R ⁶⁰ is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R ⁷⁰ is independently hydrogen or R ⁶⁰ ; each R ⁸⁰ is independently R ⁷⁰ , or alternatively, two R ⁸⁰ together with the nitrogen atom to which they are bound form a 5-membered, 6-membered or 7-membered heterocycloalkyl group, which may optionally include 1 to 4 identical or different additional heteroatoms selected from the group consisting of O, N and S, wherein N may have a -H or C ₁ -C ₃ alkyl substituent; and each M ⁺ is a counterion having a net single positive charge. Each M ⁺ can independently be, for example, a basic ion, such as K ⁺ , Na ⁺ , Li ⁺ ; an ammonium ion, such as ⁺ N( ^R60 ) ₄ ; or an alkaline earth metal ion, such as [Ca2 ⁺ ] _0.5 , [Mg2 ⁺ ] _0.5 , or [Ba2 ⁺ ] _0.5 ("subscript 0.5" means that one of the counterions of such divalent alkaline earth metal ions can be the ionized form of the compound of the present invention, and the other is a typical counterion, such as chloride, or the two ionic compounds disclosed herein can serve as the counterions of such divalent alkaline earth metal ions, or the diionic compounds of the present invention can serve as the counterions of such divalent alkaline earth metal ions). As specific examples, ^-NR80R80 is intended to include _-NH2 , -NH-alkyl, ^N -pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl, and N-morpholinyl.

In addition to the disclosure herein, and unless otherwise indicated, substituent groups for hydrogen on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl and heteroaryl groups are -R ⁶⁰ , halogen, -O ^- M ⁺ , -OR ⁷⁰ , -SR ⁷⁰ , -SM ⁺ , -NR ⁸⁰ R ⁸⁰ , trihalomethyl, -CF ₃ , -CN, -OCN, -SCN, -NO, -NO ₂ , -N ₃ , -SO ₂ R ⁷⁰ , -SO ₃ ^- M ⁺ , -SO ₃ R ⁷⁰ , -OSO ₂ R ⁷⁰ , -OSO ₃ -M ⁺ , -OSO ₃ R ⁷⁰ , -PO ₃ ^-2 (M ⁺ ) ₂ , -P(O)(OR ⁷⁰ )O ^- M ⁺ , -P(O)(OR ⁷⁰ ) ₂ , -C(O)R ^{70 , -} , -C(S)R ⁷⁰ , -C(NR ⁷⁰ )R ⁷⁰ , -CO ₂ ^- M ⁺ , -CO ₂ R ⁷⁰ , -C(S)OR ⁷⁰ , -C(O)NR ⁸⁰ R ⁸⁰ , -C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , -OC(O)R ⁷⁰ , -OC(S)R ⁷⁰ , -OCO ₂ ^- M ⁺ , - OCO ₂ R ⁷⁰ , -OC(S)OR ⁷⁰ , -NR ⁷⁰ C(O)R ⁷⁰ , -NR ⁷⁰ C(S)R ⁷⁰ , -NR ⁷⁰ CO ₂ ^- M ⁺ , -NR ⁷⁰ CO ₂ R ⁷⁰ , -NR ⁷⁰ C(S)OR ⁷⁰ , -NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ , -NR ⁷⁰ C(NR ^{-NR 70} )R ⁷⁰ and -NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , wherein R ⁶⁰ , R ⁷⁰ , R ⁸⁰ and M ⁺ are as previously defined, with the proviso that in the case of a substituted alkene or alkyne, the substituent is not ^-O- M ⁺ , -OR ⁷⁰ , -SR ⁷⁰ or ^-S- M ⁺ .

In addition to the groups disclosed for each term herein, and unless otherwise specified, substituent groups for hydrogen on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are ^-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) _2O ^- M ⁺ , -OS(O)2OR70, -P(O)(O-)2(M+) ₂ , -P(O)( ^OR70 )O-M ⁺ , -P(O)(OR70)( ^OR70 ), -C(O) ^R70 , -S(O) _2O ^- M+, -S(O) _2OR70 , -OS(O)2R70, -OS( _O )2O-M+, ^-OS (O) _2OR70 , -P(O)(O-)2(M ⁺ ) ₂ , -P(O)( ^OR70 )O ^- M ⁺ , -P(O)( ^OR70 )( ^OR70 ), -C(O)R70, -S(O) ² , -C(S)R ⁷⁰ , -C(NR ⁷⁰ )R ⁷⁰ , -C(O)OR ⁷⁰ , -C(S)OR ⁷⁰ , -C(O)NR ⁸⁰ R ⁸⁰ , -C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , -OC(O)R ⁷⁰ , -OC(S)R ⁷⁰ , -OC(O)OR ⁷⁰ , -OC(S)OR ^{7 0} , -NR ⁷⁰ C(O)R ⁷⁰ , -NR ⁷⁰ C(S)R ⁷⁰ , -NR ⁷⁰ C(O)OR ⁷⁰ , -NR ⁷⁰ C(S)OR ⁷⁰ , -NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ , -NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and -NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ , wherein R ⁶⁰ , R ⁷⁰ , R ⁸⁰ and M ⁺ are as previously defined.

In addition to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

It is understood that, in all substituted groups defined above, polymers obtained by defining a substituent with itself further substituents (e.g., a substituted aryl having a substituted aryl group as a substituent, itself substituted with a substituted aryl group, a substituted aryl group further substituted with a substituted aryl group, etc.) are not intended to be included herein. In this case, the maximum number of such substitutions is 3. For example, continuous substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

Unless otherwise indicated, substituents not expressly defined herein are named by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl)-(alkyl)-O-C(O)-.

With respect to any group disclosed herein containing one or more substituents, it is of course understood that such groups do not contain any substitution or substitution pattern that is sterically impractical and/or synthetically unfeasible. In addition, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

The term "pharmaceutically acceptable salt" means an acceptable salt for administration to a patient (e.g., a mammal) (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of a compound, which are derived from a variety of organic and inorganic counterions well known in the art, and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, and the like.

The term "salt thereof" refers to a compound formed when the proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not intended to be administered to a patient. For example, salts of the compounds of the invention include those in which the compound is protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid serves as the anionic component of the salt.

"Solvate" refers to a complex formed by the combination of solvent molecules and molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of the two. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

"Stereoisomers" refers to compounds with the same atomic connectivity but different arrangements of the atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers and diastereomers.

"Tautomers" refers to alternative forms of a molecule that differ only in the position of electron bonding and/or protons of atoms, such as enol-keto and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing an -N=C(H)-NH- ring atom arrangement, such as pyrazole, imidazole, benzimidazole, triazole, and tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom arrangements are possible.

It should be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include all permutations of salts, solvates and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compound.

"Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a particular condition or disease or one or more symptoms thereof and/or to prevent the occurrence of a disease or condition. With respect to neoplastic proliferative disorders, a pharmaceutically or therapeutically effective amount includes an amount sufficient to cause tumor shrinkage or reduce the rate of tumor growth.

"Patient" refers to human and non-human subjects, particularly mammalian subjects.

As used herein, the terms "treating" or "treatment" refer to treating or treating a disease or medical condition in a patient, such as a mammal (particularly a human), including: (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) inhibiting the disease or medical condition, such as by slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating the symptoms of the disease or medical condition in a patient.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length. Unless otherwise specifically indicated, "polypeptide", "peptide" and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells); immunolabeled proteins; and the like.

"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 being modified to include modified amino acid residues.

The terms "amino acid analogs", "non-natural amino acids", etc. are used interchangeably and include amino acid-like compounds (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) that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins. Amino acid analogs also include natural amino acids with modified side chains or main chains. Amino acid analogs also include amino acid analogs that have the same stereochemistry as naturally occurring D- and L-type amino acid analogs. In some cases, amino acid analogs share the main chain structure and/or side chain structure of one or more natural amino acids, with the difference being one or more modifying groups in the molecule. Such modifications may include, but are not limited to, substitution of related atoms (e.g., S) with atoms (e.g., N), addition of groups (e.g., methyl or hydroxyl, etc.) or atoms (e.g., Cl or Br, etc.), deletion of groups, substitution of covalent bonds (single bonds for double bonds, etc.), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids, etc.

The term "amino acid side chain" or "amino acid side chain" and the like can be used to refer to the substituent attached to the α-carbon of an amino acid residue (including natural amino acids, non-natural amino acids and amino acid analogs). The amino acid side chain can also include the amino acid side chains described in the context of the modified amino acids and/or conjugates described herein.

The term "carbohydrate" and the like can be used to refer to monomeric units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar can be used to refer to smaller carbohydrates, such as monosaccharides, disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical method), or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and are suitable for use in the subject compounds and conjugates.

The term "glycoside" or "glycosyl" refers to a sugar molecule or group that is bound to a moiety by a glycosidic bond. For example, the moiety to which the glycoside is bound can be a cleavable linker as described herein. Glycosidic bonds can connect glycosides to other moieties by various types of bonds, such as, but not limited to, O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyls). In some cases, glycosides can be cleaved from the moieties to which they are attached, such as by chemically mediated hydrolysis or enzyme-mediated hydrolysis.

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), etc. Antibodies are capable of binding to a target antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th edition, Garland Publishing, New York). The target antigen may have one or more binding sites, also called epitopes, recognized by the complementarity determining regions (CDRs) formed by one or more variable regions of the antibody.

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

The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody containing one or more amino acids (e.g., in the framework region, constant region, or CDR) that have been substituted with correspondingly positioned amino acids from a human antibody. In general, humanized antibodies produce a reduced immune response in a human host compared to a non-humanized form 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 the interactions of CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Other 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 a specific embodiment, the subject rabbit antibody can be humanized according to the methods described in US20040086979 and US20050033031. Therefore, the above antibodies can be humanized using methods well known in the art.

The term "chimeric antibody" refers to an antibody whose light and heavy chain genes are usually constructed by genetic engineering from antibody variable and constant region genes belonging to different species. For example, the variable fragments of the genes from mouse monoclonal antibodies can be connected to human constant fragments, such as γ1 and γ3. An example of a therapeutic chimeric antibody is a hybrid protein composed of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody, although domains from other mammalian species can be used.

Immunoglobulin polypeptides The immunoglobulin light or heavy chain variable region consists of a framework region (FR) interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs"). The extent of the framework regions and CDRs has 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 an antibody, i.e., the combined framework regions that make up the light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to the epitope of an antigen.

A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence of a constant region lacking an aldehyde tag as described herein. A 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).

As used herein, the term "isolated" is intended to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. "Isolated" means a compound included within a sample that is substantially enriched for the compound of interest and/or wherein the compound of interest is partially or substantially purified.

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 greater than 98% free from other components with which it is naturally associated.

The term "physiological conditions" is intended to encompass those conditions that are compatible with living cells, such as predominantly aqueous conditions of temperature, pH, salinity, etc., that are compatible with living cells.

" reactive partner " refers to the molecule or molecular part that reacts with another reactive partner specificity to produce reaction product.Exemplary reactive partner includes cysteine or serine and formylglycine generating enzyme (FGE) of sulfatase motif, and its reaction forms the reaction product of the aldehyde label of the conversion of formylglycine (FGly) containing cysteine or serine in replacement motif.Other exemplary reactive partners include aldehyde (for example, reactive aldehyde group) and " aldehyde reactive reactive partner " of the fGly residue of the aldehyde label of conversion, it comprises aldehyde reactive group and part of interest, and its reaction is to form the reaction product of the polypeptide of modified aldehyde label, and the polypeptide of described modified aldehyde label has the part of interest that is conjugated to modified polypeptide by modified fGly residue.

"N-terminus" refers to the terminal amino acid residue of a polypeptide having a free amine group, the amine groups in non-N-terminal amino acid residues typically forming part of the covalent backbone of the polypeptide.

"C-terminus" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, the carboxyl groups in non-C-terminal amino acid residues typically forming part of the covalent backbone of the polypeptide.

An "internal site" as used with reference to a polypeptide or amino acid sequence of a polypeptide refers to a region of a polypeptide that is not at the N-terminus or the C-terminus.

Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as such may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be restrictive, as the scope of the present invention will be limited only by the appended claims.

Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly indicates otherwise) to one tenth of the lower limit, as well as any other specified or intermediate values within the specified range, is included in the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included in the present invention, subject to any specifically excluded limitations within the range. Where the range includes one or two limits, ranges excluding one or both of those included limits are also included in the present invention.

It should be understood that, for the sake of clarity, certain features of the present invention described in the context of a separate embodiment may also be provided in combination in a single embodiment. On the contrary, for the sake of brevity, the various features of the present invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments of the present invention are specifically included by the present invention and disclosed herein, just as each combination is individually and clearly disclosed, to the extent that such combinations include, for example, compounds that are stable compounds (i.e., compounds that can be prepared, separated, characterized and tested for biological activity). In addition, all sub-combinations of various embodiments and their elements (e.g., elements of chemical groups listed in the embodiments describing such variables) are also specifically encompassed by the present invention and disclosed herein, just as each such sub-combination is individually and clearly disclosed herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the 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, 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 associated with the cited publications.

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 should also be noted that the claims may be drafted to exclude any optional elements. Thus, 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 for use of a "negative" limitation.

It should be understood that, for the sake of clarity, certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, the various features of the present invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination.

The publications discussed herein are provided only for their disclosures prior to the filing date of the present application. Anything herein should not be construed as admitting that the present invention has no right to precede such disclosure due to prior invention. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

DETAILED DESCRIPTION

The present disclosure provides antibody-drug conjugate structures, which include a cleavable linker connecting the antibody to the drug. The cleavable linker includes a first enzymatically cleavable portion and a second enzymatically cleavable portion, and the second enzymatically cleavable portion includes a glycoside selected from galactosides, glucosides, mannosides, fucosides, O-GlcNAc and O-GalNAc. The present disclosure also covers methods for preparing such conjugates and methods for using the conjugates.

Antibody-drug conjugates

The present disclosure provides a conjugate, such as an antibody-drug conjugate (ADC). "Conjugate" refers to a first part (e.g., an antibody) stably bound to a second part (e.g., a drug or an active agent). For example, an antibody-drug conjugate includes a drug or an active agent stably bound to another part (e.g., an antibody). "Stably bound" refers to a part that is bound to another part or structure under standard conditions. In certain embodiments, the first part and the second part are bound to each other through one or more functional groups and covalent bonds. For example, one or more functional groups and covalent bonds may include a cleavable linker as described herein.

In certain embodiments, the conjugate is a polypeptide conjugate, which includes a polypeptide (e.g., an antibody) conjugated to a second portion. In certain embodiments, the portion conjugated to the polypeptide can 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 portion for fixing the polypeptide to a membrane or surface. In certain embodiments, the conjugate is a drug conjugate, wherein the polypeptide is an antibody, thereby providing an antibody-drug conjugate. For example, the conjugate can be a drug conjugate, wherein the polypeptide is conjugated to a drug or an active agent. Various types of drugs or active agents can be used in the conjugate, as described in more detail below.

In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and the drug are connected together by a joint. In some cases, the joint is a cleavable joint. A cleavable joint is a joint comprising one or more cleavable parts, wherein the cleavable part comprises one or more keys that can be dissociated under certain conditions, thereby separating the cleavable joint into two or more separable parts. For example, the cleavable part may include one or more covalent bonds, which may dissociate or break under certain conditions to separate the cleavable joint into two or more parts. Therefore, such a cleavable joint may be included in the antibody-drug conjugate, so that under appropriate conditions, the cleavable joint is cleaved to separate or release the drug from the antibody at the desired target site of the drug's action.

In some cases, the cleavable linker includes two cleavable parts, such as a first cleavable part and a second cleavable part. The cleavable part can be configured so that the two cleavable parts need to be cleaved to separate or release the drug from the antibody at the desired target site of the drug. For example, the cleavage of the cleavable linker can be achieved by first cleaving one of the two cleavable parts and then cleaving the other of the two cleavable parts. In certain embodiments, the cleavable linker includes a first cleavable part and a second cleavable part that hinders the cleavage of the first cleavable part. "Hindering cleavage" means that the presence of the uncleaved second cleavable part reduces the possibility of cleavage of the first cleavable part or substantially inhibits the cleavage of the first cleavable part, thereby substantially reducing the amount of the cleavable linker or preventing the cleavage of the cleavable linker. For example, the presence of the uncleaved second cleavable part can hinder the cleavage of the first cleavable part. The cleavage of the first cleavable part is hindered by the presence of the second cleavable part, thereby significantly reducing the amount of the drug or preventing the drug from being released 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 the drug.

In some cases, since the second cleavable part hinders the cleavage of the first cleavable part, the cleavage of the cleavable linker can be achieved by first cleaving the second cleavable part and then cleaving the first cleavable part. The cleavage of the second cleavable part can reduce or eliminate the hindrance to the cleavage of the first cleavable part, thereby allowing the first cleavable part to be cleaved. The cleavage of the first cleavable part can cause the cleavable linker to dissociate or separate into two or more parts as described above to release the drug from the antibody-drug conjugate. In some cases, the cleavage of the first cleavable part does not occur substantially in the presence of the second cleavable part that is not cleaved. "Substantially" means that the cleavage of about 10% or less of the first cleavable part occurs when there is an uncleaved second cleavable part, for example, 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 of the cleavage of the first cleavable part occurs when there is an uncleaved second cleavable part.

In other words, the second cleavable part can protect the first cleavable part from cleavage. For example, the presence of the second cleavable part that is not cleaved can protect the first cleavable part from cleavage, and therefore substantially reduce or prevent the premature release of the drug from the antibody, until the antibody-drug conjugate is in or close to the desired target site of action of the drug. Therefore, the cleavage of the second cleavable part exposes the first cleavable part (for example, the first cleavable part is deprotected), thereby allowing the first cleavable part to be cleaved, which leads to the cleavage of the cleavable joint, and then separates or releases the drug from the antibody at the desired target site of action of the drug as described above. In some cases, the cleavage of the second cleavable part exposes the first cleavable part to subsequent cleavage, but the cleavage of the second cleavable part itself does not lead to the cleavage of the cleavable joint (that is, the cleavage of the first cleavable part is still required to cleave the cleavable joint).

The cleavable part contained in the cleavable linker can each be an enzymatic cleavable part.For example, the first cleavable part can be a first enzymatic cleavable part, and the second cleavable part can be a second enzymatic cleavable part.The enzymatic cleavable part is a cleavable part that can be separated into two or more parts as described above by the enzymatic action of an enzyme.The enzymatic cleavable part can be any cleavable part that can be cleaved by the enzymatic action of an enzyme, such as, but not limited to, peptides, glycosides, etc. In some cases, the enzyme that cleaves the enzymatic cleavable part is present at the desired target site, such as the desired target site of the drug to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatic cleavable part is not present in other regions, such as whole blood, plasma or serum, in significant amounts. Therefore, the cleavage of the enzymatic cleavable part can be controlled so that substantial cleavage occurs at the desired site of action, and cleavage does not occur significantly in other regions or before the antibody-drug conjugate reaches the desired site of action.

For example, as described herein, the antibody-drug conjugates of the present disclosure can be used to treat cancer, for example, for delivering cancer therapeutic drugs to the desired site of action where cancer cells are present. In some cases, enzymes, such as protease cathepsin B, can be biomarkers of cancers overexpressed in cancer cells. Overexpression of certain enzymes in cancer and the positioning thereof can be used in the context of enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release drugs at the desired site of action (i.e., the site of cancer (and overexpressed enzymes)). Therefore, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be a protease cathepsin B. Therefore, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by a protease such as cathepsin B.

In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide can be any peptide that is suitable for a cleavable joint and can be cleaved by the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as enzymatically cleavable moieties include, for example, Val-Ala, Phe-Lys, etc. For example, the above-mentioned first cleavable moiety (i.e., the cleavable moiety that is protected from premature cleavage by the second cleavable moiety) can include a peptide. The presence of the uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from being cleaved by a protease (e.g., cathepsin B), and therefore substantially reduce or prevent the premature release of the drug from the antibody until the antibody-drug conjugate is in or near the desired target site of action of the drug. In some cases, one of the amino acid residues of the peptide comprising the first cleavable moiety is connected to a substituent or includes a substituent, wherein the substituent includes the second cleavable moiety. In some cases, the second cleavable moiety includes a glycoside.

In some embodiments, the enzymatically cleavable part is a sugar moiety, such as a glycoside (or glycosyl). In some cases, compared with a cleavable joint that does not include a glycoside, a glycoside can promote the hydrophilicity of the cleavable joint to increase. A glycoside can be any glycoside that is suitable for a cleavable joint and can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable part (i.e., the cleavable part that protects the first cleavable part from premature cleavage) can be a glycoside. For example, in some embodiments, the first cleavable part includes a peptide, and the second cleavable part includes a glycoside. In certain embodiments, the second cleavable part is a glycoside selected from galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc. In some cases, the second cleavable part is a galactoside. In some cases, the second cleavable part is a glucoside. In some cases, the second cleavable part is a mannoside. In some cases, the second cleavable part is a fucoside. In some cases, the second cleavable part is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

Glycoside can be connected (covalently bonded) to cleavable linker by glycosidic bond.Glycosidic bond can connect glycoside to cleavable linker by various types of bond, such as but not limited to O-glycosidic bond (O-glycoside), N-glycosidic bond (glucosamine), S-glycosidic bond (thioglycoside) or C-glycosidic bond (C-glycoside or C-glycosyl).In some cases, glycosidic bond is O-glycosidic bond (O-glycoside).In some cases, glycoside can be cleaved from the cleavable linker to which it is connected by enzyme (for example, by enzyme-mediated hydrolysis of glycosidic bond).Glycoside can be removed or cleaved from cleavable linker by any convenient enzyme that can cleave (hydrolyze) the glycosidic bond connecting glycoside to cleavable linker.The example of the enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond connecting glycoside to cleavable linker is glycosidase, such as galactosidase, glucosidase, mannosidase, fucosidase etc. Other suitable enzymes can also be used to mediate the cracking (hydrolysis) of the glycosidic bond that glycoside is connected to the cleavable linker. In some cases, the enzyme used to mediate the cracking (hydrolysis) of the glycosidic bond that glycoside is connected to the cleavable linker is found near the desired action site of the drug of the antibody-drug conjugate. For example, the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, which is present in the desired action site of the drug of the antibody-drug conjugate in the cell or near it. In some cases, the enzyme is an enzyme found at or near the enzyme target site of the cracking of the first cleavable part found to mediate.

The moiety of interest (e.g., a drug or an active agent) can be conjugated to a polypeptide (e.g., an antibody) at any desired site of the polypeptide. Therefore, the present disclosure provides, for example, a modified polypeptide having a moiety conjugated at a site near or at the C-terminus of the polypeptide. Other examples include modified polypeptides having a moiety conjugated at a position near or at the N-terminus of the polypeptide. Examples also include modified polypeptides having a moiety conjugated at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). When the modified polypeptide is conjugated to two or more moieties, the above combinations are also possible.

In certain embodiments, the conjugates of the present disclosure include a drug or active agent conjugated to an amino acid residue of a polypeptide at the α-carbon of the amino acid residue. In other words, the conjugate includes a polypeptide wherein the side chain of one or more amino acid residues in the polypeptide has been modified to be connected to the drug or active agent (e.g., connected to the drug or active agent via a linker as described herein). For example, the conjugate includes a polypeptide wherein the α-carbon of one or more amino acid residues in the polypeptide has been modified to be connected to the drug or active agent (e.g., connected to the drug or active agent via a linker as described herein).

Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more parts, such as 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, or 10 or more parts. The parts can be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more parts can be conjugated to a single amino acid residue of the polypeptide. In some cases, one part is conjugated to an amino acid residue of the polypeptide. In other embodiments, the two parts can be conjugated to the same amino acid residue of the polypeptide. In other embodiments, the first part is conjugated to the first amino acid residue of the polypeptide, and the second part is conjugated to the second amino acid residue of the polypeptide. The above combination is also possible, for example, when the polypeptide is conjugated to the first part at the first amino acid residue and to two other parts at the second amino acid residue. Other combinations are also possible, such as, but not limited to, polypeptides conjugated to the first part and the second part at the first amino acid residue and to the third and fourth parts at the second amino acid residue, etc.

One or more amino acid residues of the polypeptide conjugated to one or more parts can be naturally occurring amino acids, non-natural amino acids or combinations thereof. For example, the conjugate may include a part conjugated to a naturally occurring amino acid residue of the polypeptide. In other cases, the conjugate may include a part conjugated to a non-natural amino acid residue of the polypeptide. One or more parts can be conjugated to the polypeptide at a single natural or non-natural amino acid residue. One or more natural or non-natural amino acid residues in the polypeptide can be conjugated to one or more parts described herein. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in the polypeptide can each be conjugated to one or two parts so that multiple sites in the polypeptide are modified.

As described herein, a polypeptide can be conjugated to one or more moieties. In certain embodiments, the moiety of interest is a chemical entity, such as a drug, an active agent, or a detectable marker. For example, a drug (or active agent) can be conjugated to a polypeptide, or in other embodiments, a detectable marker can be conjugated to a polypeptide. Thus, for example, embodiments of the present disclosure include, but are not limited to, the following: a conjugate of a polypeptide and a drug; a conjugate of a polypeptide and an active agent; a conjugate of a polypeptide and a detectable marker; a conjugate of two or more drugs and a polypeptide; a conjugate of two or more detectable markers and a polypeptide; and the like.

In certain embodiments, polypeptide (e.g., antibody) and part of interest (e.g., drug or active agent) are conjugated by coupling moiety. For example, polypeptide and part of interest can be each combined (e.g., covalently bound) to coupling moiety, so that polypeptide and part of interest are indirectly combined together by coupling moiety. In some cases, coupling moiety includes hydrazino-indolyl or hydrazino-pyrrolo-pyridyl compound, or derivative of hydrazino-indolyl or hydrazino-pyrrolo-pyridyl compound. For example, the general scheme of coupling part of interest with polypeptide by hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moiety is shown in the general reaction scheme below. Hydrazino-indolyl and hydrazino-pyrrolo-pyridyl coupling moieties are also referred to as hydrazino-iso-Pickett-Spengler (HIPS) coupling moieties and aza-hydrazino-iso-Pickett-Spengler (aza-HIPS) coupling moieties, respectively, in this article.

In the above reaction scheme, R comprises a moiety of interest (e.g., a drug or an active agent) conjugated to a polypeptide (e.g., conjugated to a polypeptide via a cleavable linker as described herein). As shown in the above reaction scheme, a polypeptide comprising a 2-formylglycine residue (fGly) is reacted with a drug or an active agent that has been modified to include a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety) to produce a polypeptide conjugate linked to the coupling moiety, thereby linking the drug or active agent to the polypeptide via the coupling moiety.

As described herein, the moiety can be any of a variety of moieties, such as, but not limited to, a chemical entity, such as a detectable label, or a drug or active agent. R′ and R″ can each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z can be CR ²¹ , NR ²² , N, O, or S, wherein R ²¹ and R ²² are each independently selected from any of the substituents described above for R′ and R″.

Other hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties are also possible, as shown in the conjugates and compounds described herein. For example, hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties can be modified to be connected (e.g., covalently linked) to a joint. Therefore, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties connected to a drug or an activating agent by a joint. Various embodiments of the joints of hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties that can be coupled to a drug or an activating agent are described in detail herein. For example, in some cases, the joint is a cleavable joint, such as a cleavable joint as described herein.

In certain embodiments, the polypeptide can be conjugated to a part of interest, wherein the polypeptide is modified before being conjugated to the part of interest. The modification of the polypeptide can produce a modified polypeptide containing one or more reactive groups suitable for being conjugated to the part of interest. In some cases, the polypeptide can be modified at one or more amino acid residues to provide one or more reactive groups suitable for being conjugated to the part of interest (e.g., including a coupling moiety, such as the part of the hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moiety described above). For example, the polypeptide can be modified to include a reactive aldehyde group (e.g., reactive aldehyde). Reactive aldehydes can be included in "aldehyde tags" or "ald-tags", as used herein, refer to amino acid sequences derived from sulfatase motifs (e.g., L(C/S)TPSR), which have been transformed by the action of formylglycine generating enzyme (FGE) to contain 2-formylglycine residues (referred to herein as "FGly"). The FGly residue generated by FGE may also be referred to as "formylglycine". In other words, the term "aldehyde tag" is used herein to refer to an amino acid sequence comprising a "converted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to FGly by the action of FGE, e.g., L(FGly)TPSR). The converted sulfatase motif may be derived from an amino acid sequence comprising an "unconverted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has not been converted to FGly by FGE, but is capable of being converted, e.g., an unconverted sulfatase motif having the following sequence: L(C/S)TPSR). "Conversion" used in the context of the action of a formylglycine generating enzyme (FGE) on a sulfatase motif refers to the 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). Other aspects of aldehyde tags and their use in site-specific protein modification are described in US Pat. No. 7,985,783 and US Pat. No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

In some cases, the modified polypeptide containing FGly residues can be conjugated to the part of interest by the reaction of FGly and a compound (e.g., a compound containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moiety, as described above). For example, a polypeptide containing FGly can be contacted with a drug containing a reactive partner under conditions suitable for conjugating the drug to the polypeptide. In some cases, the drug containing a reactive partner may include a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moiety as described above. For example, a drug or an activating agent may be modified to include a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moiety. In some cases, a drug or an activating agent is connected to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl, for example, by a joint covalently connected to a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl, and the joint is, for example, a cleavable joint described in detail herein.

In certain embodiments, the conjugate of the present disclosure includes a polypeptide (e.g., antibody) with at least one modified amino acid residue. The modified amino acid residue of the polypeptide can be coupled with a drug or an active agent containing the above-mentioned hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling portion. In certain embodiments, the modified amino acid residue of the polypeptide (e.g., antibody) can be derived from a cysteine or serine residue that has been converted into a FGly residue as described above. In certain embodiments, the FGly residue is conjugated to a drug or an active agent containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling portion as described above, to provide a conjugate of the present disclosure, wherein the drug or active agent is conjugated to the polypeptide by a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling portion. As used herein, the term FGly′ refers to a modified amino acid residue of a polypeptide (e.g., antibody) coupled to a portion of interest (e.g., a drug or an active agent).

In certain embodiments, the conjugate includes at least one modified amino acid residue as described herein, wherein the modified amino acid residue is connected to a linker as described herein (a cleavable linker), which in turn is connected to a drug or an active agent. For example, the conjugate may include at least one modified amino acid residue (FGly′) as described above. In some embodiments, the conjugate has formula (I):

in

Z is CR ⁴ or N;

X is O or NR ⁴ ;

^R1 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

^R2 and ^R3 are 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or ^R2 and ^R3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl;

each ^R is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

Each R ⁵ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl;

Each R ⁶ is 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;

k is an integer from 1 to 10;

^R7 comprises a second enzymatically cleavable moiety;

L ¹ is the first connector;

L ² is the second connector;

^W1 is a drug; and

^W2 is the antibody.

In certain embodiments of Formula (I), k is 2, and the conjugate has Formula (Ia):

In certain embodiments of Formula (I), the conjugate has Formula (Ib):

In certain embodiments of Formula (I), the conjugate has Formula (Ic):

In certain embodiments of Formula (I), the conjugate has Formula (Id):

In certain embodiments of Formula (I), the conjugate has Formula (Ie):

In certain embodiments of Formula (I), the conjugate has Formula (If):

In certain embodiments of Formula (I), the conjugate has Formula (Ig):

Substituents associated with the conjugates of formula (I) are described in more detail below. Reference to formula (I) also contemplates formulas (Ia), (Ib), (Ic), (Id), (Ie), (If) and (Ig).

In certain embodiments, Z is CR ⁴ or N. In certain embodiments, Z is CR ⁴ . In certain embodiments, Z is N.

In certain embodiments, X is O or NR ⁴ . In some cases, X is O. In some cases, X is NR ⁴ . In some cases, X is NH.

In certain embodiments, R ¹ is selected from hydrogen, 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, R ¹ is hydrogen. In certain embodiments, R ¹ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹ is methyl. In certain embodiments, R ¹ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, ^R is alkynyl or substituted alkynyl, such as _C2-6 alkenyl or _C2-6 substituted alkenyl, or _C2-4 alkenyl or _C2-4 substituted alkenyl, or _C2-3 alkenyl or _C2-3 substituted alkenyl. In certain embodiments, ^R is aryl or substituted aryl, such as _C5-8 aryl or _C5-8 substituted aryl, such as _C5 aryl or _C5 substituted aryl, or _C6 aryl or _C6 substituted aryl. In certain embodiments, ^R is heteroaryl or substituted heteroaryl, such as _C5-8 heteroaryl or _C5-8 substituted heteroaryl, such as _C5 heteroaryl or _C5 substituted heteroaryl, or _C6 heteroaryl or _C6 substituted heteroaryl. In certain embodiments, ^R is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, ^R is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, ^R and ^R are 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or ^R and ^R are optionally cyclically linked to form a 5- or 6-membered heterocyclyl.

In certain embodiments, ^R is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In certain embodiments, R is hydrogen. In certain embodiments, ^R is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, ^or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, ^R is methyl. In certain embodiments, ^R is alkenyl or substituted alkenyl, such as _C2-6 alkenyl or _C2-6 substituted alkenyl, or _C2-4 alkenyl or _C2-4 substituted alkenyl, or _C2-3 alkenyl or _C2-3 substituted alkenyl. In certain embodiments, ^R is alkynyl or substituted alkynyl. In certain embodiments, ^R is alkoxy or substituted alkoxy. In certain embodiments, ^R is amino or substituted amino. In certain embodiments, ^R is carboxyl or carboxyl ester. In certain embodiments, ^R is acyl or acyloxy. In certain embodiments, ^R is acylamino or aminoacyl. In certain embodiments, ^R is alkylamide or substituted alkylamide. In certain embodiments, ^R is sulfonyl. In certain embodiments, ^R is thioalkoxy or substituted thioalkoxy. In certain embodiments, ^R is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, ^R is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, ^R is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ² is a heterocyclyl or substituted heterocyclyl, such as a C _3-6 heterocyclyl or a C _3-6 substituted heterocyclyl, or a C _3-5 heterocyclyl or a C _3-5 substituted heterocyclyl.

In certain embodiments, ^R is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In certain embodiments, R is hydrogen. In certain embodiments, ^R is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, ^or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, ^R is methyl. In certain embodiments, ^R is alkenyl or substituted alkenyl, such as _C2-6 alkenyl or _C2-6 substituted alkenyl, or _C2-4 alkenyl or _C2-4 substituted alkenyl, or _C2-3 alkenyl or _C2-3 substituted alkenyl. In certain embodiments, ^R is alkynyl or substituted alkynyl. In certain embodiments, ^R is alkoxy or substituted alkoxy. In certain embodiments, ^R is amino or substituted amino. In certain embodiments, ^R is carboxyl or carboxyl ester. In certain embodiments, ^R is acyl or acyloxy. In certain embodiments, ^R is acylamino or aminoacyl. In certain embodiments, ^R is alkylamide or substituted alkylamide. In certain embodiments, ^R is sulfonyl. In certain embodiments, ^R is thioalkoxy or substituted thioalkoxy. In certain embodiments, ^R is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, ^R is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, ^R is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ³ is a heterocyclyl or substituted heterocyclyl, such as a C _3-8 heterocyclyl or a C _3-8 substituted heterocyclyl, such as a C _3-6 heterocyclyl or a C _3-6 substituted heterocyclyl, or a C _3-5 heterocyclyl or a C _3-5 substituted heterocyclyl.

In certain embodiments, ^R2 and ^R3 are optionally cyclically connected to form a 5- or 6-membered heterocyclic group. In certain embodiments, ^R2 and ^R3 are cyclically connected to form a 5- or 6-membered heterocyclic group. In certain embodiments, ^R2 and ^R3 are cyclically connected to form a 5-membered heterocyclic group. In certain embodiments, ^R2 and ^R3 are cyclically connected to form a 6-membered heterocyclic group.

In certain embodiments, each ^R is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

The various possibilities for each R ⁴ are described in more detail below. In certain embodiments, R ⁴ is hydrogen. In certain embodiments, each R ⁴ is hydrogen. In certain embodiments, R ⁴ is halogen, such as F, Cl, Br or I. In certain embodiments, R ⁴ is F. In certain embodiments, R ⁴ is Cl. In certain embodiments, R ⁴ is Br. In certain embodiments, R ⁴ is I. In certain embodiments, R ⁴ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ⁴ is methyl. In certain embodiments, R ⁴ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ⁴ is alkynyl or substituted alkynyl. In certain embodiments, R ⁴ is alkoxy or substituted alkoxy. In certain embodiments, R ⁴ is amino or substituted amino. In certain embodiments, R ⁴ is carboxyl or carboxyl ester. In certain embodiments, R ^{4 is acyl or acyloxy. In certain embodiments, R 4 is} acylamino or aminoacyl. In certain embodiments, R ⁴ is alkylamide or substituted alkylamide. In certain embodiments, R ⁴ is sulfonyl. In certain embodiments, R ⁴ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ⁴ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, ^R4 is heteroaryl or substituted heteroaryl, such as _C5-8 heteroaryl or _C5-8 substituted heteroaryl, such as _C5 heteroaryl or _C5 substituted heteroaryl, or _C6 heteroaryl or _C6 substituted heteroaryl. In certain embodiments, ^R4 is cycloalkyl or substituted cycloalkyl, such as _C3-8 cycloalkyl or _C3-8 substituted cycloalkyl, such as _C3-6 cycloalkyl or _C3-6 substituted cycloalkyl, or _C3-5 cycloalkyl or _C3-5 substituted cycloalkyl. In certain embodiments, ^R4 is heterocyclyl or substituted heterocyclyl, such as _C3-8 heterocyclyl or _C3-8 substituted heterocyclyl, such as _C3-6 heterocyclyl or _C3-6 substituted heterocyclyl, or _C3-5 heterocyclyl or _C3-5 substituted heterocyclyl.

In certain embodiments, each R ⁵ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl and substituted alkynyl. In certain embodiments, R ⁵ is hydrogen. In certain embodiments, R ⁵ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ⁵ is methyl. In certain embodiments, R ⁵ is ethyl. In certain embodiments, R ⁵ is propyl (e.g., n-propyl or isopropyl). In certain embodiments, R ^{5 is butyl (e.g., n-butyl, isobutyl, sec-butyl or tert-butyl). In certain embodiments, R 5 is} pentyl (e.g., n-pentyl or neopentyl, etc.). In certain embodiments, R ⁵ is neopentyl. In certain embodiments, R ⁵ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ⁵ is alkynyl or substituted alkynyl.

In certain embodiments, each R ⁶ is 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; in certain embodiments, R ⁶ is hydrogen. In certain embodiments, R ⁶ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ⁶ is methyl. In certain embodiments, R ⁶ is ethyl. In certain embodiments, R ⁶ is propyl (e.g., n-propyl or isopropyl). In certain embodiments, R ⁶ is butyl (e.g., n-butyl, isobutyl, sec-butyl or tert-butyl). In certain embodiments, R ⁶ is pentyl (e.g., n-pentyl or neopentyl, etc.). In certain embodiments, R ⁶ is neopentyl. In certain embodiments, ^R is alkenyl or substituted alkenyl, such as _C2-6 alkenyl or _C2-6 substituted alkenyl, or _C2-4 alkenyl or _C2-4 substituted alkenyl, or _C2-3 alkenyl or _C2-3 substituted alkenyl. In certain embodiments, ^R is alkynyl or substituted alkynyl.

In certain embodiments, ^R is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, ^R is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, ^R is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ⁶ is a heterocyclyl or substituted heterocyclyl, such as a C _3-8 heterocyclyl or a C _3-8 substituted heterocyclyl, such as a C _3-6 heterocyclyl or a C _3-6 substituted heterocyclyl, or a C _3-5 heterocyclyl or a C _3-5 substituted heterocyclyl.

In certain embodiments, ^R represents the side chain of an amino acid. For example, ^R may represent a substituent connected to the α-carbon of an amino acid residue (including natural amino acids, non-natural amino acids, and amino acid analogs). In some cases, ^R represents the side chain of an amino acid found in a naturally occurring protein (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). In certain embodiments, ^R represents the side chain of valine (Val); that is, ^R is an isopropyl group. In certain embodiments, ^R represents the side chain of alanine (Ala); that is, ^R is a methyl group. In certain embodiments, R ⁶ represents the side chain of phenylalanine (Phe); that is, R ⁶ is benzyl. In certain embodiments, R ⁶ represents the side chain of lysine (Lys); that is, R ⁶ is 4-amino-butyl.

In some embodiments, k is an integer from 1 to 10. In some embodiments, k is 1. In some embodiments, k is 2. In some embodiments, k is 3. In some embodiments, k is 4. In some embodiments, k is 5. In some embodiments, k is 6. In some embodiments, k is 7. In some embodiments, k is 8. In some embodiments, k is 9. In some embodiments, k is 10.

In certain embodiments, the moiety in formula (I) surrounded by brackets-subscript k represents one or more amino acids (e.g., peptides). For example, as described above, the conjugates of the present disclosure may include a first enzymatically cleavable moiety, wherein the first enzymatically cleavable moiety is a peptide. As shown in formula (I), the one or more amino acids may be a peptide comprising a first enzymatically cleavable moiety.

In certain embodiments, ^R is a second enzymatically cleavable moiety as described herein. For example, ^R can comprise a glycoside selected from galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc. In some cases, ^R comprises galactosides. In some cases, ^R comprises glucosides. In some cases, ^R comprises mannosides. In some cases, ^R comprises fucosides. In some cases, ^R comprises O-GlcNAc. In some cases, ^R comprises O-GalNAc.

In certain embodiments, ^L1 is a first linker. Suitable linkers for ^L1 are described in more detail below.

In certain embodiments, ^L2 is a second linker. Linkers suitable for ^L2 are described in more detail below.

In certain embodiments, W ¹ is a drug (or active agent). Drugs and active agents suitable for use in the conjugates and compounds described herein are further described in more detail below.

In certain embodiments, ^W2 is an antibody. Further description of antibodies that can be used in the conjugates of the present invention is provided in the disclosure herein.

In certain embodiments, the conjugate of formula (I) includes one or more joints.Joints can be used to combine coupling moieties with one or more parts of interest and/or one or more polypeptides.In certain embodiments, coupling moieties are combined with polypeptides or chemical entities such as drugs by joints.Joints can be combined (e.g., covalently bonded) to coupling moieties (e.g., as described herein) at any convenient position.For example, joints can be connected to hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties to drugs (e.g., maytansine or auristatin).Hydrazino-indolyl or hydrazino-pyrrolo-pyridyl coupling moieties can be used to conjugate joints (thereby conjugating drugs) to polypeptides, such as antibodies.For example, coupling moieties can be used to conjugate joints (and therefore conjugating drugs) to modified amino acid residues of polypeptides, such as FGly residues of antibodies.

In certain embodiments, the linker includes one or more linkers, such as a first linker ^L1 and a second linker ^L2 . In addition, the linker may include one or more cleavable moieties (e.g., a first cleavable moiety and a second cleavable moiety) as described herein. In some cases, the linker includes one or more linkers, such as a first linker ^L1 and a second linker ^L2 . For example, the linker may include a first linker ( ^L1 ) that connects the first cleavable moiety to a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein), and a second linker ( ^L2 ) that connects the first cleavable moiety to a chemical entity (e.g., a drug or active agent as described herein). Therefore, the linker may include a first linker ( ^L1 ) that connects the first cleavable moiety to an antibody (e.g., through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein), and a second linker ( ^L2 ) that connects the first cleavable moiety to a chemical entity (e.g., a drug or active agent as described herein).

For example, as shown in formula (I) above, ^L1 is connected to ^W2 through a coupling moiety, and thus ^W2 is indirectly bonded to the first linker ^L1 through a coupling moiety. As described above, ^W2 is an antibody, and thus ^L1 is connected to the antibody through a coupling moiety, for example, the first linker ^L1 is indirectly bound to the antibody through a coupling moiety (e.g., through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein). In addition, as shown in formula (I) above, ^L1 is (indirectly) connected to ^L2 , and ^L2 is connected to ^W1 . As described above, ^W1 is a drug, and thus the second linker ^L2 connects the drug to the antibody ^W2 through the first linker ^L1 and a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein).

Any convenient linker can be used for the first linker (L ¹ ) and the second linker (L ² ) in the conjugates and compounds of the present invention. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include an alkyl or substituted alkyl group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include an alkenyl or substituted alkenyl group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include an alkoxy group or a substituted alkoxy group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include an amino group or a substituted amino group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include a carboxyl group or a carboxyl ester group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include an acylamino group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include an alkylamide group or a substituted alkylamide group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include an aryl group or a substituted aryl group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) may each independently include a heteroaryl group or a substituted heteroaryl group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include a heterocyclyl or substituted heterocyclyl group.

In certain embodiments, the first linker (L ¹ ) and the second linker (L ² ) can each independently include a polymer. For example, the polymer can include polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., wherein the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinyl pyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below.

In some embodiments, L ¹ is a first linker described by the formula -(L ¹¹ ) _a -(L ¹² ) _b -(L ¹³ ) _c -(L ¹⁴ ) _d -, wherein L ¹¹ , L ¹² , L ¹³ and L ¹⁴ are each independently a first linker subunit, and a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4.

In some embodiments, the sum of a, b, c, and d is 1. In some embodiments, the sum of a, b, c, and d is 2. In some embodiments, the sum of a, b, c, and d is 3. In some embodiments, the sum of a, b, c, and d is 4. In some embodiments, a, b, c, and d are each 1. In some embodiments, a, b, and c are each 1, and d is 0. In some embodiments, a and b are each 1, and c and d are each 0. In some embodiments, a is 1, and b, c, and d are each 0.

In certain embodiments, L ¹¹ is connected to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in Formula (I) above). In certain embodiments, L ¹² (if present) is connected to a first cleavable moiety. In certain embodiments, L ¹³ (if present) is connected to a first cleavable moiety. In certain embodiments, L ¹⁴ (if present) is connected to a first cleavable moiety.

Any convenient linker subunit can be used in the first linker ^L. Interested linker subunits include, but are not limited to, polymer units (e.g., polyethylene glycol, polyethylene, and polyacrylates), amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, ^L , ^L , ^L , and ^L (if present) each comprise one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linker groups comprising alkylenediamines).

In some embodiments, L ¹¹ (if present) comprises polyethylene glycol, modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L ¹¹ comprises polyethylene glycol. In some embodiments, L ¹¹ comprises a modified polyethylene glycol. In some embodiments, L ¹¹ comprises an amino acid residue. In some embodiments, L ^{11 comprises an alkyl group or a substituted alkyl group. In some embodiments, L 11} comprises an aryl group or a substituted aryl group. In some embodiments, L ¹¹ comprises a diamine (e.g., a linker group ^comprising an alkylenediamine).

In some embodiments, L ¹² (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ¹² comprises polyethylene glycol. In some embodiments, L ¹² comprises modified polyethylene glycol. In some embodiments, L ¹² comprises amino acid residue. In some embodiments, L ¹² comprises alkyl group or substituted alkyl. In some embodiments, L ^{12 comprises aryl group or substituted aryl group. In some embodiments, L 12 comprises} diamine (e.g., a linker group comprising alkylenediamine).

In some embodiments, L ¹³ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ¹³ comprises polyethylene glycol. In some embodiments, L ¹³ comprises modified polyethylene glycol. In some embodiments, L ¹³ comprises amino acid residue. In some embodiments, L ¹³ comprises alkyl group or substituted alkyl. In some embodiments, L ^{13 comprises aryl group or substituted aryl group. In some embodiments, L 13 comprises} diamine (e.g., a linker group comprising alkylenediamine).

In some embodiments, L ¹⁴ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ¹⁴ comprises polyethylene glycol. In some embodiments, L ¹⁴ comprises modified polyethylene glycol. In some embodiments, L ¹⁴ comprises amino acid residue. In some embodiments, L ¹⁴ comprises alkyl group or substituted alkyl. In some embodiments, L ¹⁴ comprises aryl or substituted aryl group. In some embodiments, L ¹⁴ comprises diamine (e.g., a linker group comprising an alkylenediamine).

In some embodiments, L ¹ is a first linker comprising -(L ¹¹ ) _a -(L ¹² ) _b -(L ¹³ ) _c -(L ¹⁴ ) _d -, wherein:

-(L ¹¹ ) _a -is -(T ¹ -V ¹ ) _a -;

-(L ¹² ) _b -is -(T ² -V ² ) _b -;

-(L ¹³ ) _c - is -(T ³ -V ³ ) _c -; and

-(L ¹⁴ ) _d -is -(T ⁴ -V ⁴ ) _d -,

wherein T ¹ , T ² , T ³ and T ⁴ (if present) are tethering groups;

V ¹ , V ² , V ³ and V ⁴ (if present) are covalent bonds or linking functional groups; and

a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1-4.

As described above, in certain embodiments, L ¹¹ is connected to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in Formula (I) above). Therefore, in certain embodiments, T ¹ is connected to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in Formula (I) above). In certain embodiments, V ¹ is connected to a first cleavable moiety. In certain embodiments, L ¹² (if present) is connected to a first cleavable moiety. Therefore, in certain embodiments, T ² (if present) is connected to a first cleavable moiety, or V ² (if present) is connected to a first cleavable moiety. In certain embodiments, L ¹³ (if present) is connected to a first cleavable moiety. Therefore, in certain embodiments, T ³ (if present) is connected to a first cleavable moiety, or V ³ (if present) is connected to a first cleavable moiety. In certain embodiments, L ¹⁴ (if present) is connected to a first cleavable moiety. Thus, in certain embodiments, ^T4 (if present) is linked to the first cleavable moiety, or ^V4 (if present) is linked to the first cleavable moiety.

With respect to linker groups T ¹ , T ² , T ³ and T ⁴ , any convenient linker group may be used in the subject linker. In some embodiments, each of T ¹ , T ² , T ³ and T ⁴ comprises one or more groups independently selected from (C ₁ -C ₁₂ ) alkyl, substituted (C ₁ -C ₁₂ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w , (PEG) _n , (AA) _p , -(CR ¹³ OH) _h -, 4-amino-piperidine (4AP), acetal groups, disulfides, hydrazines and esters, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12.

In certain embodiments, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes a (C ₁ -C ₁₂ ) alkyl group or a substituted (C ₁ -C ₁₂ ) alkyl group. In certain embodiments, the (C ₁ -C ₁₂ ) alkyl group is a straight or branched alkyl group including 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 cases, the (C ₁ -C ₁₂ ) alkyl group can be an alkyl group or a substituted alkyl group, such as a C ₁ -C ₁₂ alkyl group, or a C ₁ -C 10 alkyl group, or a _{C 1} -C ₆ alkyl group, or a C ₁ -C ₃ alkyl group. In some cases, the (C ₁ -C ₁₂ ) alkyl group is a C ₂ -alkyl group. For example, (C ₁ -C ₁₂ )alkyl can be alkylene or substituted alkylene, such as C ₁ -C ₁₂ alkylene, or C ₁ -C ₁₀ alkylene, or C ₁ -C ₆ alkylene, or C ₁ -C ₃ alkylene. In some cases, (C ₁ -C ₁₂ )alkyl is C ₂ -alkylene (eg, CH ₂ CH ₂ ).

In certain embodiments, substituted (C ₁ -C ₁₂ ) alkyl is a linear or branched substituted alkyl group comprising 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 cases, substituted (C ₁ -C ₁₂ ) alkyl can be a substituted alkyl, such as a substituted C ₁ -C ₁₂ alkyl, or a substituted C ₁ -C ₁₀ alkyl, or a substituted C ₁ -C ₆ alkyl, or a substituted C ₁ -C ₃ alkyl. In some cases, substituted (C _l -C ₁₂ ) alkyl is a substituted C ₂ -alkyl. For example, the substituted (C ₁ -C ₁₂ ) alkyl group can be a substituted alkylene group, such as a substituted C ₁ -C ₁₂ alkylene group, or a substituted C ₁ -C ₁₀ alkylene group, or a substituted C ₁ -C ₆ alkylene group, or a substituted C ₁ -C ₃ alkylene group. In some cases, the substituted (C ₁ -C ₁₂ ) alkyl group is a substituted C ₂ -alkylene group.

In certain embodiments, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes an aryl, a substituted aryl, a heteroaryl, a substituted heteroaryl, a cycloalkyl, a substituted cycloalkyl, a heterocyclyl or a substituted heterocyclyl. In some cases, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes an aryl or a substituted aryl. For example, the aryl can be a phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl can be substituted with one or more substituents selected from (C ₁ -C ₁₂ ) alkyl, a substituted (C ₁ -C ₁₂ ) alkyl, an aryl, a substituted aryl, a heteroaryl, a substituted heteroaryl, a cycloalkyl, a substituted cycloalkyl, a heterocyclyl and a substituted heterocyclyl. In some cases, the substituted aryl is a substituted phenyl, wherein the substituent includes a second cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside).

In some cases, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes a heteroaryl or substituted heteroaryl. In some cases, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes a cycloalkyl or substituted cycloalkyl. In some cases, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes a heterocyclyl or substituted heterocyclyl. In some cases, the substituents on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclyl include a second cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside).

In certain embodiments, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes an ethylenediamine (EDA) moiety, e.g., an EDA-containing tether. In certain embodiments, (EDA) _w includes one or more EDA moieties, e.g., wherein w is an integer from 1 to 50, e.g., from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12, or from 1 to 6, e.g., from 1, 2, 3, 4, 5, or 6. The ethylenediamine (EDA) moiety of the connection may be optionally substituted at one or more convenient positions by any convenient substituent, e.g., by an alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the EDA moiety is described by the following structure:

Wherein y is 1 to 6, or is an integer of 0 or 1, and each R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, 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 R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. In certain embodiments, any two adjacent R ¹² groups of EDA can be cyclically connected, for example, to form a piperazinyl ring. In certain embodiments, y is 1, and two adjacent R ¹² groups are alkyl groups, which are cyclically connected to form a piperazinyl ring. In certain embodiments, y is 1, and adjacent R ¹² groups are selected from hydrogen, alkyl (e.g., methyl) and substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be optionally substituted at one or more convenient positions with any convenient substituent, such as an alkyl, substituted alkyl, a polyethylene glycol moiety, an acyl, substituted acyl, an aryl, or a substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure:

Wherein R ¹² is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In certain embodiments, R ¹² is a polyethylene glycol moiety. In certain embodiments, R ¹² is a carboxyl modified polyethylene glycol.

In certain embodiments, R ¹² includes a polyethylene glycol moiety described by the formula: (PEG) _k , which can be represented by the following structure:

Wherein k is an integer from 1 to 20, such as 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 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 cases, k is 2. In certain embodiments, R ¹⁷ is selected from OH, COOH or COOR, wherein 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, R ¹⁷ is COOH.

In certain embodiments, the tethering group (eg, T ¹ , T ² , T ³ and/or T ⁴ ) comprises (PEG) _n , wherein (PEG) _n is polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG) _n is described by the following structure:

wherein n is an integer from 1 to 50, such as from 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 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 cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

In certain embodiments, the tethering group (e.g., T ¹ , T ² , T ³ and/or T ⁴ ) comprises (AA) _p , wherein AA is an amino acid residue. Any convenient amino acid may be used. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids (e.g., any of the 20 primary α-amino acids and β-alanine), non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids, etc. In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 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.

In certain embodiments, the tethering group (e.g., ^T1 , ^T2 , ^T3 , and/or ^T4 ) includes a moiety described by the formula -( ^CR13OH ) _h- , wherein h is 0, or n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R ¹³ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In certain embodiments, R ¹³ is hydrogen. In certain embodiments, R ¹³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹³ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹³ is alkynyl or substituted alkynyl. In certain embodiments, R ¹³ is alkoxy or substituted alkoxy. In certain embodiments, R ¹³ is amino or substituted amino. In certain embodiments, R ¹³ is carboxyl or carboxyl ester. In certain embodiments, R ^{13 is acyl or acyloxy. In certain embodiments, R 13 is} acylamino or aminoacyl. In certain embodiments, R ¹³ is alkylamide or substituted alkylamide. In certain embodiments, R ¹³ is sulfonyl. In certain embodiments, R ¹³ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹³ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹³ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹³ is a heterocyclyl or substituted heterocyclyl, such as a C _3-8 heterocyclyl or a C _3-8 substituted heterocyclyl, such as a C _3-6 heterocyclyl or a C _3-6 substituted heterocyclyl, or a C _3-5 heterocyclyl or a C _3-5 substituted heterocyclyl.

In certain embodiments, R ¹³ is 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 R ¹⁵ .

With respect to the linking functional groups V ¹ , V ² , V ³ and V ⁴ , any convenient linking functional group may be used in the first linker L ^1. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phosphate, phosphoramidate, phosphorothioate, and the like. In some embodiments, V ¹ , V ² , V ³ , and V ⁴ are each independently selected from a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ -, and -P(O)OH-, wherein 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 some embodiments, each ^R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

The various possibilities of each R ¹⁵ are described in more detail below. In certain embodiments, R ¹⁵ is hydrogen. In certain embodiments, each R ¹⁵ is hydrogen. In certain embodiments, R ¹⁵ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹⁵ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹⁵ is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁵ is alkoxy or substituted alkoxy. In certain embodiments, R ¹⁵ is amino or substituted amino. In certain embodiments, R ¹⁵ is carboxyl or carboxyl ester. In certain embodiments, R ¹⁵ is acyl or acyloxy. In certain embodiments, R ¹⁵ is acylamino or aminoacyl. In certain embodiments, R ¹⁵ is alkylamide or substituted alkylamide. In certain embodiments, R ¹⁵ is sulfonyl. In certain embodiments, R ¹⁵ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹⁵ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹⁵ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹⁵ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹⁵ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, each ^R is 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, 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 substituents are as described above for ^R.

In certain embodiments, the tethering group comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tethering group comprises an acetal group. In some embodiments, the tethering group comprises a disulfide. In some embodiments, the tethering group comprises a hydrazine. In some embodiments, the tethering group comprises an ester.

As described above, in some embodiments, ^L1 is a first linker comprising -( ^T1 - ^V1 ) _a- ( ^T2 - ^V2 ) _b- ( ^T3 - ^V3 ) _c- ( ^T4 - ^V4 ) _d- , wherein a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4.

In some embodiments, in the first joint ^L1 :

T ¹ is selected from (C ₁ -C ₁₂ )alkyl and substituted (C ₁ -C ₁₂ )alkyl;

^T2 , ^T3 and ^T4 are 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 ) _h- , 4-amino-piperidine (4AP), acetal group, disulfide, hydrazine and ester; and

V ¹ , V ² , V ³ and V ⁴ are each independently selected from a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ - and -P(O)OH-, wherein q is an integer from 1 to 6;

in:

(PEG) _n is wherein n is an integer from 1 to 30;

EDA is the ethylenediamine moiety with the following structure:

wherein y is an integer from 1 to 6, and r is 0 or 1;

4-Amino-piperidine (4AP) is

AA is an amino acid residue, wherein p is an integer from 1 to 20; and

Each R ¹⁵ and R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl, wherein any two adjacent R ¹² groups may be cyclically linked to form a piperazinyl ring; and

^R13 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl and substituted aryl.

In certain embodiments, T ¹ , T ² , T ³ and T ⁴ and V ¹ , V ² , V ³ and V ⁴ are selected from the following:

T ¹ is (C ₁ -C ₁₂ )alkyl, and V ¹ is -CO-;

T ² is an amino acid analog, and V ² is -NH-;

T ³ is (PEG) _n , and V ³ is -CO-; and

d is 0 (ie, T ⁴ and V ⁴ are absent).

In certain embodiments, the left hand side of the linker structure is connected to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, and the right hand side of the linker structure is connected to the first cleavable moiety. For example, where the first cleavable moiety comprises a peptide, the right hand side of the linker structure can be connected to an amino acid of the peptide comprising the first cleavable moiety. In some cases, the carbonyl group on the right hand side of the linker structure can form an amide bond with an amino acid of the peptide comprising the first cleavable moiety.

In some embodiments, ^L2 is a second linker described by the formula -( ^L21 ) _e- ( ^L22 ) _f- ( ^L23 ) _g- ( ^L24 ) _h- , wherein ^L21 , ^L22 , ^L23 and ^L24 are each independently a second linker subunit, and e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4.

In certain embodiments, the sum of e, f, g, and h is 0. In these cases, the second linker ^L2 is not present. In other words, when the sum of e, f, g, and h is 0, the second linker ^L2 is a covalent bond. In certain embodiments, the sum of e, f, g, and h is 1. In certain embodiments, the sum of e, f, g, and h is 2. In certain embodiments, the sum of e, f, g, and h is 3. In certain embodiments, the sum of e, f, g, and h is 4. In certain embodiments, e, f, g, and h are each 1. In certain embodiments, e, f, and g are each 1, and h is 0. In certain embodiments, e and f are each 1, and g and h are each 0. In certain embodiments, e is 1, and f, g, and h are each 0.

In certain embodiments, L ²¹ is connected to a drug (e.g., W ¹ as shown in Formula (I) above). In certain embodiments, L ²² (if present) is connected to a drug. In certain embodiments, L ²³ (if present) is connected to a drug. In certain embodiments, L ²⁴ (if present) is connected to a drug.

Any convenient linker subunit can be used in the second linker ^L2 . Linker subunits of interest include, but are not limited to, polymer units (e.g., polyethylene glycol, polyethylene, and polyacrylates), amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, ^L21 , ^L22 , ^L23 , and ^L24 (if present) each comprise one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linker groups comprising alkylenediamines).

In some embodiments, L ²¹ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ²¹ comprises polyethylene glycol. In some embodiments, L ²¹ comprises modified polyethylene glycol. In some embodiments, L ²¹ comprises amino acid residue. In some embodiments, L ²¹ comprises alkyl group or substituted alkyl. In some embodiments, L ²¹ comprises aryl group or substituted aryl group. In some embodiments, L ²¹ comprises diamine (e.g., a linker group comprising alkylenediamine).

In some embodiments, L ²² (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ²² comprises polyethylene glycol. In some embodiments, L ²² comprises modified polyethylene glycol. In some embodiments, L ²² comprises amino acid residue. In some embodiments, L ²² comprises alkyl group or substituted alkyl. In some embodiments, L ²² comprises aryl group or substituted aryl group. In some embodiments, L ²² comprises diamine (e.g., a linker group comprising alkylenediamine).

In some embodiments, L ²³ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ²³ comprises polyethylene glycol. In some embodiments, L ²³ comprises modified polyethylene glycol. In some embodiments, L ²³ comprises amino acid residue. In some embodiments, L ²³ comprises alkyl group or substituted alkyl. In some embodiments, L ²³ comprises aryl group or substituted aryl group. In some embodiments, L ²³ comprises diamine (e.g., a linker group comprising alkylenediamine).

In some embodiments, L ²⁴ (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residue, alkyl group, substituted alkyl, aryl group, substituted aryl group or diamine. In some embodiments, L ²⁴ comprises polyethylene glycol. In some embodiments, L ²⁴ comprises modified polyethylene glycol. In some embodiments, L ²⁴ comprises amino acid residue. In some embodiments, L ²⁴ comprises alkyl group or substituted alkyl. In some embodiments, L ²⁴ comprises aryl group or substituted aryl group. In some embodiments, L ²⁴ comprises diamine (e.g., a linker group comprising an alkylenediamine).

In some embodiments, ^L2 is a second linker comprising -( ^L21 ) _e- ( ^L22 ) _f- ( ^L23 ) _g- ( ^L24 ) _h- , wherein:

-(L ²¹ ) _e - is -(T ⁵ -V ⁵ ) _e -;

-(L ²² ) _f -is -(T ⁶ -V ⁶ ) _f -;

-(L ²³ ) _g - is -(T ⁷ -V ⁷ ) _g -; and

-(L ²⁴ ) _h -is -(T ⁸ -V ⁸ ) _h -,

wherein T ⁵ , T ⁶ , T ⁷ and T ⁸ (if present) are tethering groups;

V ⁵ , V ⁶ , V ⁷ and V ⁸ (if present) are covalent bonds or linking functional groups; and

e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0-4.

In certain embodiments, L ²¹ is connected to a first cleavable moiety. Thus, in certain embodiments, T ⁵ is connected to a first cleavable moiety. In certain embodiments, V ⁵ is connected to a drug (e.g., W ¹ as shown in Formula (I) above). In certain embodiments, L ²² (if present) is connected to a drug. Thus, in certain embodiments, T 6 (if present) is connected to a drug, or V ⁶ (if present) is connected to a drug. In certain embodiments, L ²³ (if present) is connected to a drug. Thus, in certain embodiments, T ⁷ (if present) is connected to a drug, or V ⁷ (if present) is connected to a drug. In certain embodiments, L ²⁴ (if present) is connected to a drug. Thus, in certain embodiments, T ⁸ (if present) is connected to a drug, or V ⁸ (if present) is connected to a drug.

With respect to linker groups T ⁵ , T ⁶ , T ⁷ and T ⁸ , any convenient linker group may be used in the subject linker. In some embodiments, T ⁵ , T ⁶ , T ⁷ and T ⁸ each comprise one or more groups independently selected from (C ₁ -C ₁₂ ) alkyl, substituted (C ₁ -C ₁₂ ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w , (PEG) _n , (AA) _p , -(CR ¹³ OH) _h -, 4-amino-piperidine (4AP), acetal groups, disulfides, hydrazines and esters, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12.

In certain embodiments, the tethering group (e.g., T ⁵ , T ⁶ , T ⁷ , and/or T ⁸ ) includes a (C ₁ -C ₁₂ ) alkyl group or a substituted (C ₁ -C ₁₂ ) alkyl group. In certain embodiments, the (C ₁ -C ₁₂ ) alkyl group is a straight or branched alkyl group including 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 cases, the (C ₁ -C ₁₂ ) alkyl group can be an alkyl group or a substituted alkyl group, such as a C ₁ -C ₁₂ alkyl group, or a C ₁ -C ₁₀ alkyl group, or a C ₁ -C ₆ alkyl group, or a C ₁ -C ₃ alkyl group. In some cases, the (C ₁ -C ₁₂ ) alkyl group is a C ₂ -alkyl group. For example, (C ₁ -C ₁₂ )alkyl can be alkylene or substituted alkylene, such as C ₁ -C ₁₂ alkylene, or C ₁ -C ₁₀ alkylene, or C ₁ -C ₆ alkylene, or C ₁ -C ₃ alkylene. In some cases, (C ₁ -C ₁₂ )alkyl is C ₂ -alkylene (e.g., CH ₂ CH ₂ ). In some cases, (C ₁ -C ₁₂ )alkyl is C ₁ -alkylene (e.g., CH ₂ ).

In certain embodiments, substituted (C ₁ -C ₁₂ ) alkyl is a linear or branched substituted alkyl group comprising 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 cases, substituted (C ₁ -C ₁₂ ) alkyl can be a substituted alkyl, such as a substituted C ₁ -C ₁₂ alkyl, or a substituted C ₁ -C ₁₀ alkyl, or a substituted C ₁ -C ₆ alkyl, or a substituted C ₁ -C ₃ alkyl. In some cases, substituted (C ₁ -C ₁₂ ) alkyl is a substituted C ₂ -alkyl. For example, the substituted (C ₁ -C ₁₂ ) alkyl group can be a substituted alkylene group, such as a substituted C ₁ -C ₁₂ alkylene group, or a substituted C ₁ -C ₁₀ alkylene group, or a substituted C ₁ -C ₆ alkylene group, or a substituted C ₁ -C ₃ alkylene group. In some cases, the substituted (C ₁ -C ₁₂ ) alkyl group is a substituted C ₂ -alkylene group. In some cases, the substituted (C ₁ -C ₁₂ ) alkyl group is a substituted C ₁ -alkylene group.

In certain embodiments, the tethering group (e.g., T ⁵ , T ⁶ , T ⁷ and/or T ⁸ ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl. In some cases, the tethering group includes aryl or substituted aryl. For example, the aryl can be phenyl or substituted phenyl. In some cases, the tethering group (e.g., T ⁵ , T ⁶ , T ⁷ and/or T ⁸ ) includes heteroaryl or substituted heteroaryl. In some cases, the tethering group (e.g., T ⁵ , T ⁶ , T ⁷ and/or T ⁸ ) includes cycloalkyl or substituted cycloalkyl. In some cases, the tethering group (e.g., T ⁵ , T ⁶ , T ⁷ and/or T ⁸ ) includes heterocyclyl or substituted heterocyclyl.

In certain embodiments, the tethering group (e.g., ^T5 , ^T6 , ^T7 , and/or ^T8 ) includes an ethylenediamine (EDA) moiety, e.g., an EDA moiety as described above, e.g., an EDA moiety depicted by the following structure:

Wherein y is 1 to 6, or is an integer of 0 or 1, and each R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, 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 R ¹² is independently selected from hydrogen, alkyl, substituted alkyl, aryl and substituted aryl. In certain embodiments, any two adjacent R ¹² groups of EDA can be cyclically connected, for example, to form a piperazinyl ring. In certain embodiments, y is 1, and two adjacent R ¹² groups are alkyl groups, which are cyclically connected to form a piperazinyl ring. In certain embodiments, y is 1, and adjacent R ¹² groups are selected from hydrogen, alkyl (e.g., methyl) and substituted alkyl (e.g., low alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tethering group (e.g., ^T5 , ^T6 , ^T7 , and/or ^T8 ) includes a 4-amino-piperidine (4AP) moiety as described above, such as the 4AP moiety depicted by the following structure:

Wherein R ¹² is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In certain embodiments, R ¹² is a polyethylene glycol moiety. In certain embodiments, R ¹² is a carboxyl modified polyethylene glycol.

In certain embodiments, R ¹² includes a polyethylene glycol moiety described by the formula: (PEG) _k , which can be represented by the following structure:

Wherein k is an integer from 1 to 20, such as 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 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 cases, k is 2. In certain embodiments, R ¹⁷ is selected from OH, COOH or COOR, wherein 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, R ¹⁷ is COOH.

In certain embodiments, the tethering group (e.g., ^T5 , ^T6 , ^T7 , and/or ^T8 ) includes a polyethylene glycol moiety (PEG) _n as described above, such as the (PEG) _n moiety depicted by the following structure:

wherein n is an integer from 1 to 50, such as from 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 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 cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

In certain embodiments, the tethering group (e.g., T ⁵ , T ⁶ , T ⁷ and/or T ⁸ ) comprises (AA) _p , wherein AA is an amino acid residue. Any convenient amino acid may be used. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids (e.g., any of the 20 primary α-amino acids and β-alanine), non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids, etc. In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 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.

In certain embodiments, the tethering group (e.g., ^T5 , ^T6 , ^T7 , and/or ^T8 ) includes a moiety described by the formula -( ^CR13OH ) _h- , wherein h is 0, or n is an integer from 1 to 50, such as from 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R ¹³ is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl. In certain embodiments, R ¹³ is hydrogen. In certain embodiments, R ¹³ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹³ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹³ is alkynyl or substituted alkynyl. In certain embodiments, R ¹³ is alkoxy or substituted alkoxy. In certain embodiments, R ¹³ is amino or substituted amino. In certain embodiments, R ¹³ is carboxyl or carboxyl ester. In certain embodiments, R ^{13 is acyl or acyloxy. In certain embodiments, R 13 is} acylamino or aminoacyl. In certain embodiments, R ¹³ is alkylamide or substituted alkylamide. In certain embodiments, R ¹³ is sulfonyl. In certain embodiments, R ¹³ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹³ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹³ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹³ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹³ is a heterocyclyl or substituted heterocyclyl, such as a C _3-8 heterocyclyl or a C _3-8 substituted heterocyclyl, such as a C _3-6 heterocyclyl or a C _3-6 substituted heterocyclyl, or a C _3-5 heterocyclyl or a C _3-5 substituted heterocyclyl.

In certain embodiments, R ¹³ is 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 R ¹⁵ .

With respect to the linking functional groups V ⁵ , V ⁶ , V ⁷ and V ⁸ , any convenient linking functional group may be used in the second linker L ^2. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phosphate, phosphoramidate, phosphorothioate, and the like. In some embodiments, ^V5 , ^V6 , ^V7 , and ^V8 are 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. 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 some embodiments, each ^R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

The various possibilities of each R ¹⁵ are described in more detail below. In certain embodiments, R ¹⁵ is hydrogen. In certain embodiments, each R ¹⁵ is hydrogen. In certain embodiments, R ¹⁵ is alkyl or substituted alkyl, such as C _1-6 alkyl or C _1-6 substituted alkyl, or C _1-4 alkyl or C _1-4 substituted alkyl, or C _1-3 alkyl or C _1-3 substituted alkyl. In certain embodiments, R ¹⁵ is alkenyl or substituted alkenyl, such as C _2-6 alkenyl or C _2-6 substituted alkenyl, or C _2-4 alkenyl or C _2-4 substituted alkenyl, or C _2-3 alkenyl or C _2-3 substituted alkenyl. In certain embodiments, R ¹⁵ is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁵ is alkoxy or substituted alkoxy. In certain embodiments, R ¹⁵ is amino or substituted amino. In certain embodiments, R ¹⁵ is carboxyl or carboxyl ester. In certain embodiments, R ¹⁵ is acyl or acyloxy. In certain embodiments, R ¹⁵ is acylamino or aminoacyl. In certain embodiments, R ¹⁵ is alkylamide or substituted alkylamide. In certain embodiments, R ¹⁵ is sulfonyl. In certain embodiments, R ¹⁵ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹⁵ is aryl or substituted aryl, such as C _5-8 aryl or C _5-8 substituted aryl, such as C ₅ aryl or C ₅ substituted aryl, or C ₆ aryl or C ₆ substituted aryl. In certain embodiments, R ¹⁵ is heteroaryl or substituted heteroaryl, such as C _5-8 heteroaryl or C _5-8 substituted heteroaryl, such as C ₅ heteroaryl or C ₅ substituted heteroaryl, or C ₆ heteroaryl or C ₆ substituted heteroaryl. In certain embodiments, R ¹⁵ is cycloalkyl or substituted cycloalkyl, such as C _3-8 cycloalkyl or C _3-8 substituted cycloalkyl, such as C _3-6 cycloalkyl or C _3-6 substituted cycloalkyl, or C _3-5 cycloalkyl or C _3-5 substituted cycloalkyl. In certain embodiments, R ¹⁵ is heterocyclyl or substituted heterocyclyl, such as C _3-8 heterocyclyl or C _3-8 substituted heterocyclyl, such as C _3-6 heterocyclyl or C _3-6 substituted heterocyclyl, or C _3-5 heterocyclyl or C _3-5 substituted heterocyclyl.

In certain embodiments, each ^R is 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, 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 substituents are as described above for ^R.

In certain embodiments, the tethering group comprises an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tethering group comprises an acetal group. In some embodiments, the tethering group comprises a disulfide. In some embodiments, the tethering group comprises a hydrazine. In some embodiments, the tethering group comprises an ester.

As described above, in some embodiments, ^L2 is a second linker comprising -( ^T5 - ^V5 ) _e- ( ^T6 - ^V6 ) _f- ( ^T7 - ^V7 ) _g- ( ^T8 - ^V8 ) _h- , wherein e, f, g and h are each independently 0 or 1, and wherein the sum of e, f, g and h is 0 to 4.

In some embodiments, in the second joint ^L2 :

^T5 , ^T6 , ^T7 and ^T8 are each independently selected from ( _C1 - _C12 ) alkyl, substituted ( _C1 - _C12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl, (EDA) _w , (PEG) _n , (AA) _p , -( ^CR13OH ) _h- , 4-amino-piperidine (4AP), acetal group, disulfide, hydrazine and ester; and

V ⁵ , V ⁶ , V ⁷ and V ⁸ are each independently selected from a covalent bond, -CO-, -NR ¹⁵ -, -NR ¹⁵ (CH ₂ ) _q -, -NR ¹⁵ (C ₆ H ₄ )-, -CONR ¹⁵ -, -NR ¹⁵ CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO ₂ -, -SO ₂ NR ¹⁵ -, -NR ¹⁵ SO ₂ - and -P(O)OH-, wherein q is an integer from 1 to 6;

in:

(PEG) _n is wherein n is an integer from 1 to 30;

EDA is the ethylenediamine moiety with the following structure:

wherein y is an integer from 1 to 6, and r is 0 or 1;

4-Amino-piperidine (4AP) is

AA is an amino acid residue, wherein p is an integer from 1 to 20;

Each R ¹³ is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and

Each R ¹⁵ is 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 certain embodiments, T ⁵ , T ⁶ , T ⁷ , and T ⁸ and V ⁵ , V ⁶ , V ⁷ , and V ⁸ are absent (ie , the sum of e , f , g , and h is 0 ) .

In certain embodiments, T ⁵ , T ⁶ , T ⁷ and T ⁸ and V ⁵ , V ⁶ , V ⁷ and V ⁸ are selected from the following:

T ⁵ is a covalent bond, and V ⁵ is -C(O)-;

f is 0 (i.e., T ⁶ and V ⁶ do not exist);

g is 0 (i.e., T ⁷ and V ⁷ are absent); and

h is 0 (ie, T ⁸ and V ⁸ are absent).

In certain embodiments, T ⁵ , T ⁶ , T ⁷ and T ⁸ and V ⁵ , V ⁶ , V ⁷ and V ⁸ are selected from the following:

T ⁵ is a covalent bond, and V ⁵ is -CONR ¹⁵ -;

T ⁶ is alkyl, and V ⁶ is -CO-;

g is 0 (i.e., T ⁷ and V ⁷ are absent); and

h is 0 (ie, T ⁸ and V ⁸ are absent).

In certain embodiments, the left-hand side of the linker structure is linked to the first cleavable moiety and the right-hand side of the linker structure is linked to the drug.

Any of the chemical entities, linkers, and coupling moieties listed in the above structures may be suitable for use in the subject compounds and conjugates.

Additional disclosure related to hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds and methods of making conjugates is found in U.S. Pat. No. 9,310,374 and U.S. Pat. No. 9,493,413, the disclosures of each of which are incorporated herein by reference.

Compounds for preparing conjugates

The present disclosure provides hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds that can be used to prepare the conjugates described herein. In certain embodiments, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound can be a coupling moiety for conjugating an antibody to a drug or an active agent. For example, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound can be bound to an antibody or to a drug, thereby indirectly binding the antibody and the drug together.

In certain embodiments, the compound includes a cleavable linker for attaching the antibody to the drug, wherein the cleavable linker comprises a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of: galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

In certain embodiments, the compound is of formula (II):

in

Z is CR ⁴ or N;

X is O or NR ⁴ ;

^R2 and ^R3 are 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, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or ^R2 and ^R3 are optionally cyclically linked to form a 5- or 6-membered heterocyclyl;

each ^R is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

each R ⁵ is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

Each ^R6 is 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;

k is an integer from 1 to 10;

^R7 is a second enzymatically cleavable moiety;

L ¹ is the first connector;

^L2 is the second linker; and

W ¹ is medicine.

In some cases, k is 2, and the compound is of formula (IIa):

For example, the compound may be a compound of formula (IIb):

In some cases, the compound can be a compound of formula (IIc):

In some cases, the compound may be a compound of formula (IId):

In some cases, the compound may be a compound of formula (IIe):

In some cases, the compound can be a compound of formula (IIf):

In some cases, the compound can be a compound of formula (IIg):

Substituents associated with conjugates of formula (II) are described herein. Reference to formula (II) is also intended to encompass formulas (IIa), (IIb), (IIc), (IId), (IIe), (IIf) and (IIg).

With respect to the compound of formula (II), substituents Z, R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , L ¹ , L ² and W ¹ are as described above for the conjugate of formula (I). Similarly, with respect to the first linker L ¹ and the second linker L ² of formula (II), T ¹ , T ² , T ³ , T ⁴ , V ¹ , V ² , V ³ and V ⁴ and T ⁵ , T ⁶ , T ⁷ , T ⁸ , V ⁵ , V ⁶ , V ⁷ and V ⁸ substituents are as described above for the conjugate of formula (I).

In certain embodiments, the compound has the following structure:

Antibody

As described above, the subject conjugates may comprise an antibody as a substituent W2, wherein the antibody has been modified to include a 2-formylglycine (FGly) residue. As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and/or their standard one-letter abbreviations, e.g., Alanine 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.

In certain embodiments, the amino acid sequence of the antibody is modified to include a sulfatase motif containing a serine or cysteine residue that can be converted (oxidized) to a 2-formylglycine (FGly) residue by the action of a formylglycine generating enzyme (FGE) in vivo (e.g., when translating a protein containing an aldehyde tag in a cell) or in vitro (e.g., by contacting a protein containing an aldehyde tag with FGE in a cell-free system). Such a sulfatase motif may also be referred to herein as a FGE-modification site.

Sulfatase motif

The minimum sulfatase motif of an aldehyde tag is typically 5 or 6 amino acid residues in length, and typically does not exceed 6 amino acid residues in length. The sulfatase motif provided in an Ig polypeptide is at least 5 or 6 amino acid residues, and can be, for example, 5 to 16, 6 to 16, 5 to 15, 6 to 15, 5 to 14, 6 to 14, 5 to 13, 6 to 13, 5 to 12, 6 to 12, 5 to 11, 6 to 11, 5 to 10, 6 to 10, 5 to 9, 6 to 9, 5 to 8, or 6 to 8 amino acid residues in length 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.

In certain embodiments, the polypeptide of interest includes a polypeptide in which 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 (alternative) relative to the native amino acid sequence to provide a sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide includes modifications (insertions, additions, deletions and/or substitutions/alternatives) 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. When the native amino acid sequence of the polypeptide (e.g., antibody) contains one or more residues of the desired sulfatase motif, the total number of modifications of the residues can be reduced, for example, by providing the sequence of the desired sulfatase motif by site-specific modifications (insertions, additions, deletions, substitutions/alternatives) of the amino acid residues flanking the native amino acid residues. In certain embodiments, the degree of modification of the native amino acid sequence of the target antibody is minimized so as to minimize the number of amino acid residues inserted, deleted, substituted (alternatives) or added (e.g., to the N or C-terminus). Minimizing the degree of modification of the amino acid sequence of the target antibody can minimize the effects of such modifications on antibody function and/or structure.

It should be noted that while aldehyde tags of particular interest are those that contain at least one minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it will be readily appreciated that longer aldehyde tags are also contemplated and encompassed by the present disclosure and can be used in the compositions and methods of the present disclosure. Thus, an aldehyde tag can contain a minimal sulfatase motif of 5 or 6 residues, or can be longer and contain a minimal sulfatase motif, which can be flanked by additional amino acid residues at the N- and/or C-terminal side of the motif. 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.

The aldehyde tag may be present at or near the C-terminus of the Ig heavy chain; for example, the aldehyde tag may 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. The aldehyde tag may be present in the CH1 domain of the Ig heavy chain. The aldehyde tag may be present in the CH2 domain of the Ig heavy chain. The aldehyde tag may be present in the CH3 domain of the Ig heavy chain. The aldehyde tag may be present in the Ig light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region.

In certain embodiments, the sulfatase motif used can be described by the following formula:

X ¹ Z ¹⁰ X ² Z ²⁰ X ³ Z ³⁰ (I′)

in

Z ¹⁰ is cysteine or serine (which may also be represented by (C/S));

Z ²⁰ is a proline or alanine residue (which may also be represented by (P/A));

Z ³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), such as lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), such as A, G, L, V, or I;

^X1 is present or absent, and when present, can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, V, S, or T, such as L, M, S, or V, provided that ^X1 is present when the sulfatase motif is at the N-terminus of the target polypeptide; and

^X2 and ^X3 can independently be any amino acid, but are typically aliphatic amino acids, polar uncharged amino acids, or sulfur-containing amino acids (i.e., other than aromatic amino acids or charged amino acids), such as S, T, A, V, G, or C, e.g., S, T, A, V, or G.

^The amino ^acid sequence of the antibody ^heavy chain and/or ^light chain may be modified to provide a sequence of at least 5 amino acids of the formula ^{X1Z10X2Z20X3Z30} , ^wherein

Z ¹⁰ is cysteine or serine;

Z ²⁰ is a proline or alanine residue;

Z ³⁰ is an aliphatic amino acid or a basic amino acid;

^X1 is present or absent, and when present, is any amino acid, provided that ^X1 is present when the heterologous sulfatase motif is at the N-terminus of the polypeptide;

^X2 and ^X3 are each independently any amino acid.

The sulfatase motif is generally selected to enable transformation by a selected FGE, such as an FGE present in a host cell expressing the aldehyde-tagged polypeptide or an FGE contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method.

For example, when the FGE is eukaryotic FGE (e.g., mammalian FGE, including human FGE), the sulfatase motif can have the formula:

X ¹ CX ² pX ³ Z ³⁰ (I″)

in

X ¹ may be present or absent, and when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, S, or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X ¹ is present;

^X2 and ^X3 independently can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as S, T, A, V, G, or C, such as S, T, A, V, or G; and

Z ³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), such as lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), such as A, G, L, V, or I.

Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO: //), MCTPSR (SEQ ID NO: //), VCTPSR (SEQ ID NO: //), LCSPSR (SEQ ID NO: //), LCAPSR (SEQ ID NO: //), LCVPSR (SEQ ID NO: //), LCGPSR (SEQ ID NO: //), ICTPAR (SEQ ID NO: //), LCTPSK (SEQ ID NO: //), MCTPSK ( SEQ ID NO: //), VCTPSK (SEQ ID NO: //), LCSPSK (SEQ ID NO: //), LCAPSK (SEQ ID NO: //), LCVPSK (SEQ ID NO: //), LCGPSK (SEQ ID NO: //), LCTPSA (SEQ ID NO: //), ICTPAA (SEQ ID NO: //), MCTPSA (SEQ ID NO: //), VCTPSA (SEQ ID NO: //), LCSPSA (SEQ IDNO://), LCAPSA(SEQ ID NO://), LCVPSA(SEQ ID NO: //) and LCGPSA (SEQ ID NO: //).

Contains FGly sequence

When FGE acts on the modified antibody heavy chain and/or light chain, the serine or cysteine in the sulfatase motif is modified to FGly. Therefore, the sulfatase motif containing FGly can have the following formula:

X ¹ (FGly)X ² Z ²⁰ X ³ Z ³⁰ (I″′)

in

FGly is a formylglycine residue;

Z ²⁰ is a proline or alanine residue (which may also be represented by (P/A));

Z ³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), such as A, G, L, V, or I;

^Xi may be present or absent, and when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, V, S, or T, such as L, M, or V, provided that ^Xi is present when the sulfatase motif is at the N-terminus of the target polypeptide; and

^X2 and ^X3 independently can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G.

As described above, a modified polypeptide containing an FGly residue can be conjugated to a drug (e.g., a maytansine compound) by reaction of FGly with a drug (e.g., a drug containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, as described above) to produce a sulfatase motif containing FGly'. The term "FGly" as used herein refers to a modified amino acid residue of a sulfatase motif coupled to a drug, such as maytansine or auristatin. Thus, a sulfatase motif containing FGly' can have the following formula:

X ¹ (FGly′)X ² Z ²⁰ X ³ Z ³⁰ (II)

in

FGly′ is a modified amino acid residue of formula (I);

Z ²⁰ is a proline or alanine residue (which may also be represented by (P/A));

Z ³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), such as A, G, L, V, or I;

^Xi may be present or absent, and when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, V, S, or T, such as L, M, or V, provided that ^Xi is present when the sulfatase motif is at the N-terminus of the target polypeptide; and

^X2 and ^X3 independently can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G.

Modification site

As described above, the amino acid sequence of the antibody is modified to include a sulfatase motif containing a serine or cysteine residue that can be converted (oxidized) to a FGly residue by the action of FGE in vivo (e.g., when translating a protein containing an aldehyde tag in a cell) or in vitro (e.g., by contacting a protein containing an aldehyde tag with FGE in a cell-free system). The antibody used to produce the conjugate of the present disclosure includes at least an Ig constant region, such as an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and CH2 domain; a CH1, CH2 and CH3 domain; or a CH1, CH2, CH3 and CH4 domain), or an Ig light chain constant region. Such an Ig polypeptide is referred to herein as a "target Ig polypeptide" or "target antibody."

The site of introducing the sulfatase motif into the antibody can be any convenient site. As described above, in some cases, the degree of modification of the native amino acid sequence of the target polypeptide is minimized so as to minimize the number of amino acid residues inserted, deleted, substituted (alternated) and/or added (e.g., to the N- or C-terminus). Minimizing the degree of modification of the amino acid sequence of the target antibody can minimize the effects of such modifications on antibody function and/or structure.

The antibody heavy chain constant region may include an Ig constant region of any heavy chain isotype, a non-naturally occurring Ig heavy chain constant region (including a consensus Ig heavy chain constant region). The Ig constant region may be modified to include an aldehyde tag, wherein the aldehyde tag is present in or adjacent to a solvent accessible loop region of the Ig constant region. The Ig constant region may 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.

In some cases, the aldehyde-labeled antibody comprises an aldehyde-labeled Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and CH2 domain; a CH1, CH2 and CH3 domain; or a CH1, CH2, CH3 and CH4 domain). The aldehyde-labeled Ig heavy chain constant region may include a heavy chain constant region sequence of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3 or IgG4 isotype heavy chain or any allotypic variant thereof, such as a human heavy chain constant region sequence or a mouse heavy chain constant region sequence, a hybrid heavy chain constant region, a synthetic heavy chain constant region or a consensus heavy chain constant region sequence, etc., which is modified to include at least one sulfatase motif that can be modified by FGE to produce 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.

In some cases, the antibody of aldehyde labeling comprises the Ig light chain constant region of aldehyde labeling.The Ig light chain constant region of aldehyde labeling can include the constant region sequence of κ light chain, λ light chain, such as human κ or λ light chain constant region, hybrid light chain constant region, synthetic light chain constant region or common light chain constant region sequence, etc., which is modified to include at least one sulfatase motif, and the sulfatase motif can be modified by FGE to produce the antibody modified by FGly.Exemplary constant region includes human γ1 and γ3 regions.Except sulfatase motif, the modified constant region can have wild-type amino acid sequence, or it can 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 the wild-type amino acid sequence.

In some embodiments, the sulfatase motif is located at a position other than the C-terminus of the Ig polypeptide heavy chain. As described above, an isolated aldehyde-labeled antibody may comprise a heavy chain constant region modified to include a sulfatase motif as described above, wherein the sulfatase motif is in or adjacent to a surface accessible loop region of the antibody heavy chain constant region.

Sulfatase motifs can be provided within or near one or more of these amino acid sequences of such modification sites of the Ig heavy chain. For example, the Ig heavy chain polypeptide can be modified at one or more of these amino acid sequences (e.g., wherein the modification includes one or more amino acid residue insertions, deletions and/or substitutions) to provide sulfatase motifs adjacent to and at the N-terminus and/or adjacent to and at the C-terminus of these modification sites. Alternatively or additionally, the Ig heavy chain polypeptide can be modified at one or more of these amino acid sequences (e.g., wherein the modification includes one or more amino acid residue insertions, deletions and/or substitutions) to provide a sulfatase motif between any two residues of the Ig heavy chain modification site. In some embodiments, the Ig heavy chain polypeptide can be modified to include two motifs, which can be adjacent to each other, or can be separated by one, two, three, four or more (e.g., about 1 to about 25, about 25 to about 50, or about 50 to about 100 or more) amino acids. Alternatively or additionally, when the native amino acid sequence provides one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues at the modification site of the Ig heavy chain polypeptide amino acid sequence can be modified (e.g., when 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.

The antibodies used in the antibody-drug conjugates of the present disclosure may have any of a variety of antigen binding specificities, including, but not limited to, for example, antigens present on cancer cells; antigens present on autoimmune cells; antigens present on pathogenic microorganisms; antigens present on virus-infected cells (e.g., cells infected with human immunodeficiency virus); antigens present on diseased cells; and the like. For example, the antibody conjugate may bind to an antigen, wherein the antigen is present on the surface of a cell. ^The antibody conjugates of the present disclosure may bind to an antigen with a suitable binding affinity, such as a binding affinity of ^5x10-6 M to ^10-7 M, ^10-7 M to ^5x10-7 M, ^5x10-7 M to ^10-8 M, ^10-8 M to ^5x10-8 M, ^5x10-8 M to ^10-9 M, or greater than ^10-9 M.

As a non-limiting example, the antibody conjugates of the invention can bind to antigens present on cancer cells (e.g., tumor-specific antigens; antigens overexpressed on cancer cells; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, the subject antibody conjugates can be specific for antigens on cancer cells, wherein the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

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

Drugs for conjugation to peptides

The present disclosure provides drug-polypeptide conjugates (e.g., antibody-drug conjugates). Any of a number of drugs are suitable for use as or can be modified to be suitable for use as a reactive partner conjugated to an antibody. Examples of drugs include small molecule drugs and peptide drugs.

As used herein, "small molecule drug" refers to a compound, such as an organic compound, which exhibits a pharmaceutical activity of interest and generally has a molecular weight of 800 Da or less or 2000 Da or less, but can encompass molecules as large as 5 kDa and as large as 10 kDa. Small inorganic molecules refer to molecules that do not contain carbon atoms, while small organic molecules refer to compounds that contain at least one carbon atom.

For example, the drug or active agent can be maytansine. "Maytansine", "maytansine moiety", "maytansine active agent moiety" and "maytansine-like compound" refer to maytansine and its analogs and derivatives, as well as pharmaceutically active maytansine moieties and/or portions thereof. The maytansine conjugated to the polypeptide can be any of a variety of maytansine-like compound moieties, such as but not limited to maytansine and its analogs and derivatives described herein (e.g., deacylated maytansine). In other cases, the drug or active agent can be auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or a portion thereof. The auristatin conjugated to the polypeptide can be any of a variety of auristatin moieties, such as but not limited to auristatin and its analogs and derivatives described herein. 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. The duocarmycin conjugated to the polypeptide can be any of a variety of duocarmycin moieties, such as but not limited to duocarmycin and its analogs and derivatives described herein.

In certain embodiments, the drug is selected from the group consisting of a cytotoxin, a kinase inhibitor, an immunostimulant, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.

For example, a cytotoxin can include any compound that causes cell death (eg, necrosis or apoptosis) or a decrease in cell viability.

Kinase inhibitors may include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, b), Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandetanib, Vemurafenib, etc.

Immunostimulants may include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, etc. TLR agonists include, but are not limited to, imiquimod, resiquimod, etc.

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

Aptamer drugs include but are not limited to pentaganib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, etc.

Cytokines include, but are not limited to, interferon alpha-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, 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 α 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 CD40-ligand), Regramostim, Romiplostim, Sargramostim, Thrombopoietin, Tucotuzumab celmoleukin, Viral Macrophage-Inflammatory Protein, etc.

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

As used herein, "peptide drugs" refer to polymeric compounds containing amino acids, and are intended to cover naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, and peptide mimetics. Peptide drugs can be obtained by chemical synthesis or produced from a genetically encoded source (e.g., a recombinant source). The molecular weight range of a peptide drug can be 200Da to 10kDa or more. 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; antiviral peptides; peptides that inhibit viral fusion; peptides that increase the production of one or more lymphocyte populations; antimicrobial peptides; growth factors; growth hormone releasing factors; vasoactive peptides; anti-inflammatory peptides; peptides that regulate glucose metabolism; antithrombotic peptides; anti-nociceptive peptides; vasodilator peptides; platelet aggregation inhibitors; analgesics; and the like.

Examples of drugs that can be used in the conjugates and compounds described herein include, but are not limited to, terpilaxin M, calicheamicin, SN-38, exotecan, STAT3 inhibitors, α-amanitin, Aurora kinase inhibitors, belotecan, 9-aminocamptothecin (9-AC), and anthracycline antibiotics.

Other examples of drugs include small molecule drugs, such as cancer chemotherapeutics. For example, when the polypeptide is an antibody (or fragment thereof) that is specific to tumor cells, the antibody can be modified as described herein to include modified amino acids, which can then be conjugated to cancer chemotherapeutics. Cancer chemotherapeutics include non-peptide (i.e., non-protein) compounds that reduce cancer cell proliferation, and encompass cytotoxic agents and cell inhibitors. Non-limiting examples of chemotherapeutics include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds can also be used.

Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO 96/33212, WO 96/14856, and U.S. 6,323,315. For example, dolastatin 10 or auristatin PE may be included in the antibody-drug conjugates of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansine compounds and their active analogs and derivatives (see, e.g., EP 1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and their active analogs and derivatives (e.g., including synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepine (PBD).

Agents for reducing cell proliferation are known in the art and are widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates and triazenes, including but not limited to nitrogen mustards, cyclophosphamide (Cytoxan ^™ ), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (lomustine) (CCNU), semustine (semustine) (methyl-CCNU), streptozotocin, chlorozotocin, uracil nitrogen mustard, nitrogen mustard, ifosfamide, chlorambucil, pipobroman, triethylmelamine, triethylenethiophosphamide, busulfan, dacarbazine and temozolomide.

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

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, Docetaxel deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, such as etoposide, teniposide, etc.; antibiotics, such as anthracycline antibiotics, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin, and morpholino derivatives Biologicals, etc.; phenoxyhydrazone bicyclic peptides, such as dactinomycin; basic glycopeptides, such as bleomycin; anthraquinone glycosides, such as plicamycin (mithramycin); anthracenediones, such as mitoxantrone; aziridine pyrroloindoledione, such as mitomycin; macrocyclic immunosuppressants, such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide and droloxafine.

Microtubule-affecting agents with antiproliferative activity are also suitable for use and include, but are not limited to, isocolchicine (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 Derivatives, docetaxel Thiocolchicine (NSC 361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to epothilone A, epothilone B, estramustine, nocodazole, etc.

Suitable hormone modulators and steroids (including synthetic analogs) include, but are not limited to, adrenocortical hormones, such as prednisone, dexamethasone, etc.; estrogens and progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, etc.; and adrenocortical inhibitors, such as aminoglutethimide; 17α-ethinyl estradiol; diethylstilbestrol, testosterone, etc. erone), fluoxymesterone, drostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chloranthene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

Other suitable chemotherapeutic agents include metal complexes, such as cis-DDP, carboplatin, etc.; ureas, such as hydroxyurea; and hydrazines, such as N-methylhydrazine; epipodophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; folinic acid; tegafur; etc. Other antiproliferative agents of interest include immunosuppressants, such as mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); and the like.

Taxanes are suitable for use. "Taxane" includes paclitaxel and any active taxane derivative or prodrug. "Paclitaxel" (which is understood herein to include analogs, formulations and derivatives, such as docetaxel, TAXOL ^™ , TAXOTERE ^™ (formulations of docetaxel), 10-deacetyl analogs of paclitaxel and 3'N-debenzoyl-3'N-tert-butoxycarbonyl analogs of paclitaxel) can be readily prepared using 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 from a variety of commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 is from Taxus brevifolia; or T-1912 is from Taxus yannanensis).

Paclitaxel should be understood to refer not only to the common chemically available form of paclitaxel, but also to analogs and derivatives (eg, Taxotere ^™ docetaxel as described above) and paclitaxel conjugates (eg, paclitaxel-PEG, paclitaxel-dextran or paclitaxel-xylose).

The term "taxane" also includes a variety of known derivatives, including hydrophilic derivatives and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazine and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451 and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and paclitaxel derivatives described in U.S. Pat. No. 5,415,869. It also includes prodrugs of paclitaxel, including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Applicable biological response modifiers 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 specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony stimulating factor; and (9) angiogenesis inhibitors.

Examples of drugs include small molecule drugs, such as cancer chemotherapeutics. For example, when the polypeptide is an antibody (or fragment thereof) that is specific to tumor cells, the antibody can be modified as described herein to include modified amino acids, which can then be conjugated to cancer chemotherapeutics, such as microtubule-affecting agents. In certain embodiments, the drug is a microtubule-affecting agent with antiproliferative activity, such as a maytansine compound.

In certain embodiments, the drug is a maytansine compound having the following structure:

in represents the connection point between the midanine compound and the second linker ^L2 in the conjugates and compounds described herein. "Connection point" refers to The symbol represents the bond between N of the maytansine compound and the second linker ^L2 in the conjugates and compounds described herein. For example, in formula (I), ^W1 can be a maytansine compound, such as a maytansine compound of the above structure, wherein represents the connection point between the maytansine compound and the second linker ^L2 . In some cases, the maytansine compound of the above structure can be referred to as deacylated maytansine.

In certain embodiments, the drug is an anti-mitotic agent, such as auristatin or an active auristatin analog or derivative thereof (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). In certain embodiments, the drug is MMAE, which has the following structure:

For example, an MMAE active agent can be included in an antibody-drug conjugate as follows:

in represents the point of connection between auristatin and the second linker ^L2 in the conjugates and compounds described herein. For example, The symbol represents the bond between N of auristatin and the second linker ^L2 , for example, as shown in formula (I). For example, in formula (I), ^W1 can be auristatin, such as MMAE, wherein in the above structure represents the connection point between MMAE and the second linker ^L2 .

In certain embodiments, the drug is a DNA alkylating agent, such as duocarmycin. Examples of duocarmycins include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065. In certain embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.

In some cases, the duocarmycin is a compound having the following structure:

For example, a duocarmycin active agent can be included in the following antibody-drug conjugate:

in = represents the point of attachment between the duocarmycin and the second linker ^L2 in the conjugates and compounds described herein. For example, The symbol represents the bond between the duocarmycin and the second linker L ² , for example, as shown in formula (I). For example, in formula (I), W ¹ can be a duocarmycin, such as the duocarmycin shown above, wherein represents the connection point between duocarmycin and the second linker ^L2 .

As described above, in certain embodiments, ^L2 is a second linker described by the formula -( ^L21 ) _e- ( ^L22 ) _f- ( ^L23 ) _g- ( ^L24 ) _h- , wherein ^L21 , ^L22 , ^L23 and ^L24 are each independently a second linker subunit. In certain embodiments, ^L21 is connected to the first cleavable moiety. In certain embodiments, ^L21 (if present) is also connected to ^W1 (drug). In certain embodiments, ^L22 (if present) is connected to ^W1 (drug). In certain embodiments, ^L23 (if present) is connected to ^W1 (drug). In certain embodiments, ^L24 (if present) is connected to ^W1 (drug).

As described above, in certain embodiments, the second linker -(L ²¹ ) _e -(L ²² ) _f -(L ²³ ) _g -(L ²⁴ ) _h - is described by the formula -(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -, wherein e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4. In certain embodiments, as described above, L ²¹ is connected to the first cleavable moiety. Thus, in certain embodiments, T ⁵ is connected to the first cleavable moiety. In certain embodiments, V ⁵ is connected to W ¹ (drug). In certain embodiments, as described above, L ²² (if present) is connected to W ¹ (drug). Thus, in certain embodiments, T ⁶ (if present) is connected to W ¹ (drug), or V ⁶ (if present) is connected to W ¹ (drug). In certain embodiments, as described above, L ²³ (if present) is connected to W ¹ (drug). Thus, in certain embodiments, T ⁷ (if present) is connected to W ¹ (drug), or V ⁷ (if present) is connected to W ¹ (drug). In certain embodiments, as described above, L ²⁴ (if present) is connected to W ¹ (drug). Thus, in certain embodiments, T ⁸ (if present) is connected to W ¹ (drug), or V ⁸ (if present) is connected to W ¹ (drug).

Embodiments of the present disclosure include conjugates, wherein the antibody is conjugated to one or more drug moieties, such as 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, or 10 or more drug moieties. The drug moiety can be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugate has an average drug to antibody ratio (DAR) (molar ratio) in the range of 0.1 to 10, or 0.5 to 10, or 1 to 10, such as 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In certain embodiments, the average DAR of the conjugate is 1 to 2, such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2. In certain embodiments, the conjugate has an average DAR of 1 to 1.5. In certain embodiments, the conjugate has an average DAR of 1.5 to 2. Average refers to the arithmetic mean.

Drugs coupled to polypeptides can be modified to incorporate reactive partners that react with polypeptides. When the drug is a peptide drug, the reactive part (e.g., aminooxy or hydrazide) can be located at the position of the N-terminal region, N-terminal, C-terminal region, C-terminal or peptide interior. For example, the example of the method relates to the synthesis of peptide drugs with aminooxy groups. In this embodiment, the peptide is synthesized by a precursor protected by Boc. The amino group of the peptide can react with a compound comprising a carboxylic acid group and an oxy-N-Boc group. For example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidine-1-yloxy) propionic acid. Other variants of compounds comprising carboxylic acid groups and oxy-N-protecting groups can include substituents on different numbers of carbons and alkylene joints in alkylene joints. The reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N-protecting group occurs by standard peptide coupling chemistry. Examples of peptide coupling reagents that can be used include, but are not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazole diethyl phosphate-1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethylfluoroformamidine hexafluorophosphate), DPPA (diphenyl phosphate), BOP (benzotriazole-1-oxotri(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-methylmethylammonium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinyl chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)-tris(pyrrolidinyl)phosphonium tetrafluorophosphate), BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP (bromotris(tetrahydropyrrole)phosphonium hexafluorophosphate). As non-limiting examples, HOBt and DIC can be used as peptide coupling reagents.

Deprotection is performed on peptides containing N-protecting groups to expose amino-oxy functional groups. Deprotection of N-oxysuccinimide groups occurs, for example, according to standard deprotection conditions for cyclic amide groups. Deprotection conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd edition, 1999, John Wiley & Sons, NY and Harrison, etc. Certain deprotection conditions include hydrazine reagents, amino reagents or sodium borohydride. Deprotection of Boc protecting groups can be performed with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride and methylamine. Products and intermediates can be purified by conventional methods, such as HPLC purification.

It will be appreciated by those of ordinary skill that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to the reactive partner of interest) are important. Modifying the reaction conditions to provide optimal conjugation conditions is entirely within the skill of those of ordinary skill and is routine in the art. When coupling with a polypeptide present in or on a living cell, physiologically compatible conditions are selected. For example, pH can be temporarily reduced to a time sufficient to allow the reaction to occur, but within a time period (e.g., about 30 minutes to 1 hour) tolerated by the cell. Physiological conditions for carrying out polypeptide modification on the cell surface can be similar to those used in ketone-azide reactions when modifying cells carrying cell surface azides (see, e.g., U.S. 6,570,040).

Small molecule compounds containing or modified to contain α-nucleophilic groups that serve as reactive partners with the compounds or conjugates disclosed herein are also expected to be used as drugs in the polypeptide-drug conjugates of the present disclosure. General methods for chemical synthesis protocols and conditions for synthesizing compounds of interest are known in the art (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).

preparation

The conjugates of the present disclosure can be formulated in a variety of different ways. Generally, when the conjugate is an antibody-drug conjugate, the conjugate is formulated in a manner that is compatible with the drug, the antibody, the condition to be treated, and the route of administration to be used.

In some embodiments, provided are pharmaceutical compositions comprising any of the conjugates of the present disclosure and a pharmaceutically acceptable excipient.

The conjugate (e.g., antibody-drug conjugate) can be provided in any suitable form, such as in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, such as oral, topical or parenteral administration. When the conjugate is provided in a liquid injectable form (e.g., in those embodiments administered intravenously or directly into a tissue), the conjugate can be provided as a ready-to-use dosage form, or as a reconstituted storage-stable powder or liquid consisting of a pharmaceutically acceptable carrier and excipient.

Methods for formulating the conjugate can be modified from those readily available. For example, the conjugate can be provided in a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to a mammal, such as those suitable for administration to a human.

Treatment

The antibody-drug conjugates of the present disclosure are used to treat a condition or disease in a subject that is amenable to treatment by administration of the parent drug (ie, the drug prior to conjugation to the antibody).

In some embodiments, methods are provided comprising administering to a subject an effective amount (eg, a therapeutically effective amount) of any of the conjugates of the present disclosure.

In certain aspects, a method for delivering a drug to a target site in a subject is provided, the method comprising administering to a subject a pharmaceutical composition comprising any conjugate of the present disclosure, wherein the administration 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, the antibody-drug conjugate of the present disclosure may include a cleavable linker, such as an enzymatically cleavable linker including a first enzymatically cleavable portion and a second enzymatically cleavable portion. In some cases, the cleavable linker may be cleaved under appropriate conditions to separate or release the drug from the antibody at the desired target site of action of the drug. For example, a second cleavable linker that protects the first cleavable linker from cleavage may be cleaved to allow the first cleavable portion to be cleaved, which results in the cleavage of the cleavable linker into two or more parts, thereby releasing the drug from the antibody-drug conjugate at the desired site of action.

In certain embodiments, the first cleavable moiety can be an enzymatically cleavable moiety. In some cases, the enzyme that promotes the 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, the first enzyme can be administered before, simultaneously with, or after administration of the antibody-drug conjugate described herein.

In certain embodiments, the second cleavable moiety can be an enzymatically cleavable moiety. In some cases, the enzyme that promotes the cleavage of the second cleavable moiety is an enzyme administered to the subject to be treated (i.e., exogenous to the subject to be treated). For example, the second enzyme can be administered before, simultaneously with, or after the administration of the antibody-drug conjugate described herein. In certain embodiments, the first enzyme and the second enzyme are different enzymes.

In other cases, the first enzyme that promotes the cleavage of the first cleavable portion is an enzyme present in the subject to be treated (i.e., endogenous to the subject to be treated). For example, the first enzyme may be present at the desired site of action of the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate can specifically target the desired site of action (e.g., can specifically bind to the antigen present at the desired site of action), wherein the desired site of action also has the first enzyme. In some cases, the first enzyme is present in excess at the desired site of action compared to other regions in the subject to be treated. For example, the first enzyme may be overexpressed at the desired site of action compared to other regions in the subject to be treated. In some cases, due to the first enzyme being located in a specific region or position, the first enzyme is present in excess at the desired site of action. For example, the first enzyme may be associated with a certain structure such as a lysosome in the desired site of action. In some cases, the first enzyme is present in excess in a lysosome compared to other regions in the subject. In some embodiments, a lysosome comprising the first enzyme is found at the desired site of action of the drug of the antibody-drug conjugate, such as a site of a cancer or tumor to be treated with a drug. In certain embodiments, the first enzyme is a protease, such as a human protease (eg, cathepsin B).

In certain embodiments, the second enzyme that promotes the cleavage of the second cleavable portion is an enzyme present in the subject to be treated (i.e., endogenous to the subject to be treated). For example, the second enzyme may be present at the desired site of action of the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate can specifically target the desired site of action (e.g., can specifically bind to the antigen present at the desired site of action), wherein the desired site of action also has a second enzyme. In some cases, the second enzyme is present in excess at the desired site of action compared to other regions in the subject to be treated. For example, the second enzyme may be overexpressed at the desired site of action compared to other regions in the subject to be treated. In some cases, due to the second enzyme being located in a specific region or position, the second enzyme is present in excess at the desired site of action. For example, the second enzyme may be associated with a certain structure such as a lysosome in the desired site of action. In some cases, the second enzyme is present in excess in a lysosome compared to other regions in the subject. In some embodiments, a lysosome comprising a second enzyme is found at the desired site of action of the drug of the antibody-drug conjugate, such as a site of a cancer or tumor to be treated with a drug. In certain embodiments, the second enzyme is a glycosidase, such as galactosidase, glucosidase, mannosidase, fucosidase, or the like.

Any suitable enzyme can be used for cleaving the first cleavable part and the second cleavable part of the antibody-drug conjugate described herein. Other enzymes may also be suitable for cleaving the first cleavable part and the second cleavable part of the antibody-drug conjugate described herein, such as but not limited to enzymes from other vertebrates (e.g., primates, mice, rats, cats, pigs, quail, goats, dogs, etc.).

In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions.Substantially stable refers to 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 the second enzyme as described above. For example, as described above, the second cleavable part can protect the first cleavable part from cleavage, and therefore the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the second enzyme as described above. For example, the cleavable linker of the antibody-drug conjugate can be substantially stable, so that 25% or less of the antibody-drug conjugate is cleaved in the absence of the first enzyme and/or the 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, so 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 the second enzyme, but can be cleaved in the presence of the first enzyme and the second enzyme. For example, the antibody-drug conjugate can be substantially stable after being administered 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 present at the desired site of action of the second enzyme, the second cleavable portion can be cleaved from the cleavable linker, so that the first cleavable portion is subsequently cleaved by the first enzyme, and the first enzyme then releases the drug at the desired site of action. In certain embodiments, after administration to a subject, the antibody-drug conjugate is stable in the absence of the first enzyme and/or the second enzyme for an extended period of time, for example, 1 hour or longer, or 2 hours or longer, or 3 hours or longer, or 4 hours or longer, or 5 hours or longer, or 6 hours or longer, or 7 hours or longer, or 8 hours or longer, or 9 hours or longer, or 10 hours or longer, or 15 hours or longer. Or 20 hours or longer, or 24 hours (1 day) or longer, or 2 days or longer, or 3 days or longer, or 4 days or longer, or 5 days or longer, or 6 days or longer, or 7 days (1 week) or longer. In certain embodiments, the antibody-drug conjugate is stable in the absence of the first enzyme and/or the second enzyme over a range of pH values for an extended period of time, for example, in a pH range of 2 to 10, or 3 to 9, or 4 to 8, or 5 to 8, or 6 to 8, or 7 to 8.

As described above, the antibody-drug conjugates of the present disclosure are used to treat a condition or disease in a subject, which is suitable for treatment by administering the parent drug. "Treatment" refers to at least achieving an improvement in the symptoms associated with the condition that plagues the host, wherein in a broad sense, improvement refers to at least reducing the magnitude of a parameter (e.g., symptom) associated with the condition being treated. Therefore, treatment also includes situations in which the pathological condition or at least the symptoms associated therewith are completely suppressed (e.g., prevented from occurring) or stopped (e.g., terminated) so that the host no longer suffers from the condition or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, i.e., reducing the risk of clinical symptom development, including preventing clinical symptoms from developing, such as preventing the disease from progressing to a harmful state; (ii) inhibition, i.e., preventing the development or further development of clinical symptoms, such as alleviating or completely inhibiting active disease; and/or (iii) relief, i.e., causing regression of clinical symptoms.

The subject to be treated can be a subject in need of treatment, wherein the subject to be treated is a subject suitable for treatment with the parent drug. Therefore, a variety of subjects can be subject to treatment using the antibody-drug conjugate disclosed herein. Typically, such subjects are "mammals", and humans are of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs and rats, for example, as in animal models of disease) and non-human primates (e.g., chimpanzees and monkeys).

The amount of antibody-drug conjugate administered can initially be determined based on the dosage of the parent drug and/or the guidance of the dosing regimen. In general, the antibody-drug conjugate can provide targeted delivery and/or enhanced serum half-life of the bound drug, thereby achieving at least one of reduced dose or reduced administration in the dosing regimen. Therefore, relative to the parent drug before conjugation in the antibody-drug conjugate of the present disclosure, the antibody-drug conjugate can achieve reduced dose and/or reduced administration in the dosing regimen.

Furthermore, as described above, because antibody-drug conjugates can achieve controlled stoichiometry of drug delivery, the dosage of the antibody-drug conjugate can be calculated based on the number of drug molecules provided on a per antibody-drug conjugate basis.

In some embodiments, multiple doses of antibody-drug conjugates are administered. The frequency of administration of the antibody-drug conjugate may vary according to any of a variety of factors, such as the severity of the symptoms, the condition of the subject, etc. For example, in some embodiments, the antibody-drug conjugate is administered once a month, twice a month, three times a month, every other week, once a week (qwk), twice a week, three times a week, four times a week, five times a week, six times a week, every other day, every day (qd/℃d), twice a day (bds/bid) or three times a day (tds/tid), etc.

Methods for treating cancer

The present disclosure provides methods comprising delivering the conjugates of the present disclosure to individuals suffering from cancer. The methods can be used to treat a variety of cancers, including carcinomas, sarcomas, leukemias and lymphomas. In the context of cancer, the term "treatment" includes one or more (e.g., each) of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing total tumor burden, and improving one or more symptoms associated with cancer.

Cancers that may be treated using the subject methods include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), phospholipid cell carcinoma (various tissues), bladder cancer (including transitional cell carcinoma (a malignant tumor of the bladder)), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell carcinoma and non-small cell carcinoma of the lung), adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteoblastic cancer, epithelial cancer, and nasopharyngeal cancer, among others.

Sarcomas that may be treated using the subject methods include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that may be treated using the subject methods include, but are not limited to, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas.

Leukemias that can be treated using the subject methods include, but are not limited to: a) chronic myeloproliferative syndrome (a neoplastic disorder of multipotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of multipotent hematopoietic stem cells or hematopoietic cells of restricted lineage potential); c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas that can be treated using the methods of the invention include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; non-Hodgkin's B-cell lymphoma; and the like.

In certain aspects, methods of treating cancer in a subject are provided, such methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any of the conjugates of the present disclosure, wherein the administration is effective to treat the cancer in the subject.

Examples

The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the 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 that follow are all or the only experiments that were performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. "Average" refers to the arithmetic mean. Standard abbreviations may be used, for example, bp, base pair; kb, kilobase; p1, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; i.m, intramuscular; i.p, intraperitoneal; s.c, subcutaneous; and the like.

General Synthesis Methods

Many general references are available that provide commonly known chemical synthesis schemes and conditions for synthesizing the disclosed compounds (see, for example, 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).

The compounds described herein can be purified by any purification scheme 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 phase and reverse phase and ionic resins. In certain embodiments, the disclosed compounds are purified by silica gel and/or alumina chromatography. See, for example, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley & Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969.

In any process for preparing the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules involved. This can be achieved by conventional protecting groups as described in standard works, for example 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, 3rd ed., Wiley, New York 1999, in The Peptides; Vol. 3 (eds. E.Gross and J.Meienhofer), Academic Press, London and New York 1981, in Methoden der organischen Chemie, Houben-Weyl, 4th ed., Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, Amino Acids, Peptides, Proteins, Verlag Chemie, Weinheim, Deerfield Beach and Basel 1982, and/or in Jochen Lehmann, Chemie der Kohlenhydrate: Monosaccharides and Derivates, Georg Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient subsequent stage using methods known in the art.

The subject compounds can be synthesized using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Various examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the following schemes.

Real 1

Galactoside and glucoside dual cleavage linkers for antibody-drug conjugates

introduce

The utility of glucuronide-dipeptide dual cleavage linkers has been demonstrated, for example, in Scheme 1 below, where MMAE construct 4 carries a Val-Ala-PABC linker having a glucuronide moiety attached to a self-immolative unit, which, when conjugated to an antibody, provides an antibody-drug conjugate (ADC) with superior stability compared to the generic dipeptide MMAE drug-linker but with similar efficacy in vivo. As shown in Scheme 1, the glucuronide-based MMAE construct was synthesized in 10 synthetic steps and good overall yields. The synthesis started with commercially available fully protected β-glucuronic acid bromide. The desired overall deprotection of all acetate groups in the glucuronide moiety of intermediate 3 and the cleavage of the methyl ester were accomplished by hydrolysis under strong alkaline conditions (LiOH in methanol).

Scheme 1. P1′-glucuronide double cleavage of MMAE construct 4.

Although these strongly alkaline conditions are compatible with MMAE drugs (not particularly sensitive to strong bases), the need for alkaline hydrolysis in the final step of the synthesis significantly limits the application of glucuronide-based dual-cleavage linkers. In fact, a variety of cytotoxins frequently used to produce antibody-drug conjugates contain base-labile functional groups (Figure 1). For example, the primary alkyl chloride and amide groups in duocarmycin DM 5 are highly sensitive to alkaline hydrolysis, as are the acetate groups in terpyrimidine M 6 and the ester functional groups in calicheamicin 7. Maytansine compounds 8 and 9 are particularly unstable under alkaline conditions because their structures contain esters and epoxides. Similarly, lactones in DNA topoisomerase I inhibitors such as SN-3810 and isotecan 11 are known to hydrolyze even under mild alkaline conditions.

Despite the above compatibility issues, we have previously shown that certain maytansine compounds, such as 4AP-maytansine 9 (Figure 1), can still be paired with glucuronide-based linkers, however, the synthesis of such constructs is laborious and inefficient (17 steps, 1.8% overall yield, Scheme 2). In this synthetic route, the glucuronide linker 12 must be prepared separately and enter the synthetic sequence in a fully deprotected form at a later stage to react with the separately synthesized maytansine derivative 13.

Scheme 2. Synthesis of P1′-glucuronide 4AP-maytansine construct 16.

Although this particular maytansine compound (9) is somewhat amenable to synthetic modification, the more attractive NMC3-maytansine (8) is unlikely to be used in a similar manner due to its structural unit. If the synthetic strategy used for 9 (Scheme 2) is used for 8, the selectivity problem of distinguishing the two carboxyl groups in the glucuronic acid intermediate 18 (Scheme 3) will inevitably be faced in the later stages of the synthesis, and alternative and lengthy protection-deprotection methods will be required. Scheme 3. Synthesis of P1′-glucuronide NMC3-maytansine construct 19 (not feasible due to the presence of free carboxylic acid in the glucuronide moiety).

Results and discussion

In contrast to the examples described in the introduction, a wider range of cytotoxins can be successfully used in the synthesis when different biologically relevant glycosides are used in the double cleavage linker. Thus, the use of galactosides instead of glucuronides allows the use of NMC3-maytansine (8, Figure 1) and produces P1′-galactoside construct 30 (Scheme 4) in 11 synthetic steps with a good overall yield (7.5%). Due to the absence of a carboxyl group (as in compound 29, Scheme 4), the galactoside moiety is in its completely unprotected form, does not cause chemical selectivity problems and can be successfully used with a large number of base-sensitive ADC payloads. The same is expected for glucosides and mannosides, as their structures are not significantly different from galactosides.

Scheme 4. Synthesis of P1′-galactoside NMC3-maytansine construct 30.

Indeed, similar to galactosides, other monosaccharide derivatives can be prepared for the production of base-sensitive payloads for ADCs. Thus, the double-cleavable glucoside linker was successfully paired with the NMC3-maytansine payload to provide the corresponding construct 44 in good overall yield (11 steps, 4.9%), as shown in Scheme 5.

Scheme 5. Synthesis of P1′-glucoside NMC3-maytansine construct 44.

Furthermore, it was found that the use of galactosides or glucosides allowed for consistently more efficient and high yield synthesis of drug-linkers compared to their glucuronide counterparts. Thus, the synthetic route for the P1′-galactoside MMAE construct 33 shown in Scheme 6 produced the desired compound in excellent overall yield (9.4% vs. 4.2% for the glucuronide construct, Table 1).

Scheme 6. Synthesis of P1′-galactoside MMAE construct 33.

Similarly, the glucosidic homologue 46 was prepared in a more efficient manner following an improved synthetic route (Scheme 7) starting from the corresponding monosaccharide derivative 41 (7.6% yield vs. 4.2% for the glucuronide construct, Table 1) when compared to the glucuronide-bearing compound 4.

Scheme 7. Synthesis of glucoside MMAE construct 46.

Furthermore, a comparison of material costs showed that the required galactose and glucose derivatives were on average 10 to 20 times cheaper than the corresponding glucuronic acid derivatives (see Table 1).

Table 1. Comparison of synthesis efficiency and cost according to the type of glycoside used.

*Cost of acetate-protected bromide derivatives of glucuronic acid, galactose, and glucose (AKScientific catalog, September 2020)

When incorporated into the linker of the adjacent enzymatically cleavable dipeptide, the glycoside can be used as a temporary protecting group to prevent the antibody-drug conjugate dipeptide linker from premature cleavage in the circulation. Once the ADC is transported to the lysosomal compartment of the cell, the glycoside portion must be cleaved by the lysosomal glycosidase to expose the dipeptide linker, which in turn undergoes enzyme-mediated cleavage to release the payload. There are many human glycosidases with different substrate preferences and different cell and tissue expression patterns. Although we have previously shown that the glucuronide portion can be effectively removed in a variety of cell types as the first step to achieve payload release, it is unclear whether the galactoside or glucoside portion can be processed similarly. To address this issue, constructs 30 and 33 were conjugated to aldehyde-labeled anti-HER2 and anti-CD79b antibodies using HIPS, and constructs 44 and 46 were conjugated to aldehyde-labeled anti-FITC, anti-HER2 and anti-TROP-2 antibodies using HiPS (Fig. 2). The analytical characterization of the resulting conjugates is shown in Figures 3 to 10 and 19 to 30.

The in vitro activity of the obtained conjugates was tested using HER2+ (NCI-N87, Sk-Br-3), CD79b+ (Granta-519, Ramos-RA) and TROP-2+ (MDA-MB-468, BxPC3) cancer cell lines. In some studies, the efficacy of the ADC conjugated with maytansine carrying a galactoside-modified double cleavage linker (30) was compared with the activity of the corresponding ADC produced by a single cleavage linker-payload 35 (construct 35 shown in Figure 11, in vitro efficacy data are shown in Figures 12 to 15). In other studies, the efficacy of the ADC conjugated with maytansine carrying a galactoside-modified double cleavage linker (30) was compared with the activity of the corresponding ADC produced using a glucoside-modified double cleavage linker 44 (in vitro efficacy data are shown in Figures 31 to 34). For MMAE-conjugated ADCs, the in vitro potency of galactoside-modified (33) or glucoside-modified (46) dual-cleavage linkers was compared to that of glucuronide-modified dual-cleavage linker 34 (construct 34 shown in FIG11 , in vitro potency data are shown in FIGS16 to 18 , 35 , and 36 ). In summary, the results show that ADCs prepared with mono- and dual-cleavage linkers (including glucuronide, galactoside, and glucoside variants) provide similar levels of potency in cell lines representing solid tumors (gastric, pancreatic, and breast cancer) and hematological tumor indications. The data demonstrate that despite unknown glycosidase expression patterns and substrate specificity, removal of galactoside and glucoside moieties is robust and efficient, and overall ADC catabolism and payload release are comparable for all tested linkers.

Materials and methods

Overview

Synthetic reagents were purchased from Sigma-Aldrich, Acros, AK Scientific or other commercial sources and used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Acetyl bromide-α-D-galactose 21, acetyl bromide-α-D-glucose 36, MMAE 2 and maytansine 17 were purchased from commercial sources. HIPS linker compound 32 was commercially available from Shanghai Medicilon and used without purification. Pentafluoro-ester 28 was synthesized from 32 using standard literature procedures. The synthesis of compounds 34 and 35 was as described above. In all cases, the solvent was removed under reduced pressure using a Buchi rotary evaporator R-114 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed with a Biotage Isolera chromatography system. Preparative HPLC purification was performed using a Waters preparative HPLC unit equipped with a Phenomenex Kinetex 5 μm EVO C18 150x21.2 mm column. HPLC analysis was performed on an Agilent 1100 series analytical HPLC equipped with a G1322A degasser, a G1311A quadrupole pump, a G1329A autosampler, a G1314 variable wavelength detector, an Agilent Poroshell 120SB C18, 4.6 mm x 50 mm, at room temperature using a 10-100% gradient of water and acetonitrile containing 0.1% formic acid. HPLC was monitored at 254 nm.

Synthesis of constructs 30 and 33

Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(5-formyl-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (21a)

A 50 mL round bottom flask was charged with 204 mg (1.22 mmol) of 3-hydroxy-4-nitrobenzaldehyde 20 and acetyl bromide-α-D-galactose 21 (500 mg, 1.22 mmol) followed by 20 mL of anhydrous acetonitrile. The solution was treated with silver (I) oxide (986 mg, 4.3 mmol) and the resulting mixture was vigorously stirred at room temperature in the dark for 24 hours. After the reaction mixture was concentrated under vacuum, the residue was purified on silica gel using ethyl acetate-hexane mixture as eluent (0-100% gradient) to give 420 mg (0.85 mmol, 70% yield) of the product as a light yellow solid. LRMS (ESI): m/z 520.0 [M+Na] ⁺ , calculated for C ₂₁ H ₂₃ NO ₁₃ m/z 520.1.

Preparation of (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-amino-5-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (22)

To a solution of acetate protected galactoside intermediate 21a (350 mg, 0.70 mmol) in 3 mL of ethyl acetate was added 30 mg of palladium/carbon (10 wt %) and 30 μL of triethylamine. After deaeration, the flask was equipped with a hydrogen balloon and the reaction mixture was stirred at room temperature for 48 hours. After filtering through a pad of celite, the solvent was removed under vacuum and the residue was dried under high vacuum to give 300 mg of crude product 22 as a white solid, which was further used in the synthesis without additional purification. LRMS (ESI): m/z 470.1 [M+H] ⁺ , calculated for C ₂₁ H ₂₇ NO ₁₁ m/z 470.2.

Preparation of (2R,3R,4S5S,6S)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyramido)propionamido)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (26)

Boc-L-alanine (220 mg, 1.11 mmol) and aniline derivative 22 (520 mg, 1.16 mmol) were combined in 5 mL of anhydrous dichloromethane and 0.50 mL of anhydrous methanol. The resulting solution was treated with EEDQ (280 mg, 1.13 mmol) at room temperature. The reaction mixture was stirred in the dark for 1 hour, and then the solvent was removed in vacuo. The resulting crude compound 23 was dried under high vacuum for 1 hour and dissolved in 6 mL of 1: 1 TFA-DCM mixture at room temperature. The solution was allowed to stand for 1 hour, then the solvent was removed, and the resulting crude amine 24 was kept under high vacuum for several hours.

In a separate 20 mL scintillation vial, Fmoc-L-valine (375 mg, 1.1 mmol) was mixed with HATU (420 mg, 1.1 mmol) and DIPEA (375 μL, 2.15 mmol) in 3 mL of anhydrous DMF. The mixture was stirred at room temperature for 30 minutes, combined with crude compound 24, and stirring continued overnight until the reaction was judged complete by LCMS analysis. The reaction mixture was directly purified on a reverse phase C18 column (CH ₃ CN-H ₂ O/0.05% TFA, 0-80% gradient) to afford compound 25 as a mixture with minor impurities. The product was thoroughly dried under high vacuum and carried to the next step without additional purification.

Compound 25 was dissolved in 6 mL of anhydrous DCM and treated with DIPEA (375 μL, 2.15 mmol), followed by the addition of 686 mg (2.26 mmol) of bis(4-nitrophenyl)carbonate in one portion at room temperature. The reaction mixture was stirred for 1 hour, then the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (EtOAc-hexanes, 10-100% gradient) to give p-nitrophenyl carbonate product 26 (640 mg, 0.62 mmol, 56% yield over 4 steps) as a light yellow solid. LRMS (ESI): m/z 1049.3 [M+Na] ⁺ , calculated for C ₅₁ H ₅₄ N ₄ O ₁₉ m/z 1049.3.

Preparation of 4-((((4-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-3-(((2R,3R,4S,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(methyl)amino)butyric acid (27)

4-(Methylamino)butyric acid (61 mg, 0.52 mmol), 61 μL (0.35 mmol) of DIPEA and 1 mL of anhydrous DMF were added to a 20 mL scintillation vial. The resulting solution was combined with 180 mg (0.175 mmol) of PNP-carbonate 26 at room temperature. The reaction mixture was stirred for 1 hour until the PNP-carbonate was completely consumed as judged by LCMS analysis. The solvent was removed in vacuo and the residue was dissolved in 3 mL of methanol. The resulting solution was cooled to 0°C and treated with 3 mL of 1M aqueous lithium hydroxide solution. The reaction mixture was stirred for 30 minutes, then warmed to room temperature and concentrated under vacuum. The residue was directly purified on a reverse phase C18 column (CH ₃ CN-H ₂ O/0.05% TFA, 0-50% gradient) to obtain 90 mg (0.146 mmol, 83% yield over 2 steps) of compound 27 as a yellow oil. LRMS (ESI): m/z 615.3 [M+H] ⁺ , calculated for C ₂₇ H ₄₂ N ₄ O ₁₂ m/z 615.3.

Preparation of (2S,5S,18R)-1-((4-((((4-(((((S)-1-(((1 ⁴ S,1 ⁶ S,3 ² R,3 ³ R,2R,4S,10E,12E,14R)-86-chloro- ^{1 4} -hydroxy8 ⁵ ,14-dimethoxy-3 ³ ,2,7,10-tetramethyl-1 ² ,6-dioxo-7-aza-1(64)-oxazinane-3(2,3)-oxiran-8(1,3)-benzocyclotetradec-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-4-oxobutyl)(methyl)carbamoyl)oxy)methyl)-2-(((2R,3R,4S,5R,6S)-3,4,5-trihydroxy 6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propionylamino)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonane-19-sulfonic acid (30)

Compound 27 (84 mg, 0.14 mmol), 1 mL of anhydrous DMF and DIPEA (48 μL, 0.28 mmol) were added to a 20 mL scintillation vial, followed by 130 mg (0.14 mmol) of pentafluoroester 28. The resulting mixture was stirred for 30 minutes until the coupling was judged to be complete (LCMS). Maytansine 17 (89 mg, 0.14 mmol) was added directly to the reaction mixture as a solution in 0.5 mL of DMF, followed by HATU (52 mg, 0.14 mmol). After 30 minutes, the reaction mixture was purified on a reverse phase C18 column (CH ₃ CN-H ₂ O/0.05% TFA, 10-95% gradient). After complete removal of the solvent, the resulting intermediate 29 was dissolved in 3 mL of DMF and treated with 150 μL of piperidine at room temperature. After 30 min, the reaction mixture was directly purified by reverse phase preparative HPLC (C18 column, _CH3CN - _H2O /0.05% TFA, 0-50% gradient). Pure fractions were lyophilized to afford 58 mg (0.032 mmol, 23% yield over 3 steps) of compound 30. LRMS (ESI): m/z 1801.8 [M+H _] ⁺ _, calculated for _{C82H118CIN13O28S} m/ _z 1800.8.

Preparation of (2R, 3S, 4S, 5R, 6S)-2-(acetoxymethyl)-6-(2-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-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)tetrahydro-2H-pyran-3,4,5-triyl triacetate (31)

MMAE 2 (13.4 mg TFA salt, 0.016 mmol), DIPEA (3.8 μL, 0.022 mmol) and 1 mL anhydrous DMF were mixed in a 4 mL glass vial. The resulting solution was treated with PNP-carbonate 26 (15 mg, 0.015 mmol) and HOAt (1.0 mg, 0.008 mmol). After stirring overnight at room temperature, piperidine (30 μL) was added directly to the reaction mixture. After 30 minutes, the reaction mixture was purified by reverse phase preparative HPLC (C18 column, CH ₃ CN-H ₂ O/0.05% TFA, 0-50% gradient). The pure fractions were lyophilized to give 8 mg (0.006 mmol, 40% yield over 2 steps) of compound 31 as an off-white solid. LRMS (ESI): m/z 1383.5 [M+H] ⁺ , calculated for C ₆₉ H ₁₀₆ N ₈ O ₂₁ m/z 1383.8.

Preparation of (2S,5S,18R)-1-((4-((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-4,7,1 2-(((2R,3R,4S,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propionamido)-5-isopropyl 2-methyl 1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonane-19-sulfonic acid (33)

Compound 31 (8 mg, 0.006 mmol), HIPS linker compound 32 (4.5 mg, 0.006 mmol), DIPEA (1.5 μL, 0.009 mmol) and 2.3 mg (0.06 mmol) of HATU were combined in 1 mL of DMF in a 4 mL glass vial. The reaction mixture was stirred at room temperature for 30 minutes, then the DMF was removed in vacuo and the residue was dissolved in 1 mL of methanol. To this solution was added 1 mL of 1 M aqueous lithium hydroxide solution, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was directly purified on reverse phase preparative HPLC (C18 column, CH ₃ CN-H ₂ o/0.05% TFA, 10-60% gradient), and the pure fractions were lyophilized to give 6 mg (0.0033 mmol, 60% yield over 2 steps) of compound 33 as an off-white powder. LRMS (ESI): m/z 1770.9 [M+H] ⁺ , calculated for C ₈₄ H ₁₃₂ N ₁₄ O ₂₅ S m/z 1769.9.

Synthesis of constructs 44 and 46

Preparation of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-amino-5-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (37).

Silver oxide (I) (15.6 g, 87.9 mmol, 5 equivalents) was added to a mixture of compound 20 (5.0 g, 29.9 mmol, 1.7 equivalents) and compound 36 (7.23 g, 17.6 mmol, 1 equivalent) in anhydrous acetonitrile (100 mL). The mixture was stirred at 25 ° C in the dark under nitrogen for 24 hours. The reaction mixture was diluted with EtOAc (100 mL), filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-30% hexane-EtOAc). Palladium on carbon (10 weight %, 800 mg, 0.75 mmol) was added once to a mixture of intermediate aldehyde (5.61 g, 11.3 mmol) and triethylamine (2.5 mL) in EtOAc (80 mL). The reaction mixture was stirred at 25 ° C under _H2 atmosphere for 24 hours. The solid was filtered off and the resulting filtrate was concentrated to afford 5.2 g (11.1 mmol, 98% yield) of the product 37 as a white solid, which was used in the next step without further purification.

Preparation of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-((S)-2-((tert-butoxycarbonyl)amino)propionylamino)-5-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (38).

A mixture of compound 37 (5.20 g, 11.1 mmol), Boc-L-Ala-OH (1.75 g, 9.25 mmol) and EEDQ (2.3 g, 9.25 mmol) in anhydrous DCM (40 mL) and MeOH (4 mL) was stirred at room temperature in the dark for 1 hour. The reaction mixture was concentrated to give 5.5 g of crude product 38 as a yellow solid. The crude product was used in the next step without further purification.

Preparation of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-((S)-2-aminopropionamido)-5-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (39).

Crude compound 38 (5.5 g, 8.6 mmol) was dissolved in TFA (23 mL). The resulting solution was stirred at room temperature for 10 minutes and concentrated in vacuo. The residue was purified by reverse phase chromatography (C18 column, 0-75% acetonitrile-water containing 0.05% TFA). Pure fractions were combined and concentrated to give 4.0 g of product amine 39 as a yellow oil (7.5 mmol, 66% yield over 3 steps).

Preparation of (2S,3R,4S,5R,6R)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)-5-(hydroxymethyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (40).

At room temperature, HATU (3.5 g, 9.2 mmol) was added once to a mixture of Fmoc-L-valine (3.1 g, 9.2 mmol) and DIPEA (3.9 mL, 22.2 mmol) in anhydrous DMF (20 mL). The resulting solution was stirred at room temperature for 30 minutes and then combined with amine 39 (4.0 g, 7.5 mmol). The reaction mixture was stirred for 16 hours and concentrated in vacuo. The residue was purified by silica gel chromatography (hexane: EtOAc, 0-100%) to give compound 40 (4.5 g, 5.2 mmol, 70% yield) as a white solid.

Preparation of (2S,3R,4S,5R,6R)-2-(2-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propionamido)-5-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (41).

To a mixture of alcohol 40 (4.5 g, 5.2 mmol) and DIPEA (4.5 mL, 26.1 mmol, 5 equiv) in anhydrous THF (20 mL) was added bis(4-nitrophenyl)carbonate (7.9 g, 26.1 mmol, 5 equiv). The resulting mixture was stirred at room temperature for 24 h and concentrated under vacuum. The residue was purified by reverse phase chromatography (acetonitrile-water 0-70% with 0.05% TFA) to give 4-nitrophenyl carbonate product 41 as a white solid (3.9 g, 73% yield). LRMS (ESI): m/z 1027.3 [M+H] ⁺ , calculated for C ₅₁ H ₅₄ N ₄ O ₁₉ m/z 1027.3.

Preparation of 4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)propionamido)-3-(((2S,3R,4S5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(methyl)amino)butanoic acid (42).

DIPEA (190 μL, 1.11 mmol, 3 equiv.) was added to a mixture of p-nitrophenyl carbonate 41 (380 mg, 0.37 mmol, 1 equiv.) and 4-(methylamino)butyric acid (55 mg, 0.47 mmol, 1.25 equiv.) in anhydrous DMF (2 mL). The resulting mixture was stirred for 16 hours, concentrated in vacuo, and the residue was redissolved in THF (2 mL). 2 mL of 1 M LiOH aqueous solution was added to the THF solution at room temperature and stirred for 2 hours until the hydrolysis was judged to be complete by HPLC analysis. The reaction mixture was quenched with 1 M HCl (2 mL), diluted with DMF (1 mL), and purified by reverse phase chromatography (C18 column, acetonitrile-water 0-75% MeCN containing 0.05% TFA). Pure fractions were collected, concentrated under reduced pressure, and lyophilized to obtain compound 42 as a white solid (160 mg, 0.26 mmol, 70% yield over two steps). LRMS (ESI): m/z 615.3 [M+H] ⁺ , calculated for C ₂₇ H ₄₂ N ₄ O ₁₂ m/z 615.3.

Preparation of 4-((((4-((2S,5S,18R)-5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18-(sulfomethyl)-22-(2-((1,2,2-trimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)-10,13-dioxa-3,6,16,19-tetraazaeicosanamide)-3-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)(methyl)amino)butanoic acid (43).

To a solution of compound 32 (97 mg, 0.12 mmol, 1 equivalent) in DMF (2 mL) was added HATU (40 mg, 0.1 mmol, 0.85 equivalent) at room temperature, followed by DIPEA (63 μL, 0.36 mmol, 3 equivalents). The resulting mixture was stirred for 1 hour and then merged with a 0.1 M DMF solution of compound 42 (1.22 mL, 0.12 mmol, 1 equivalent). The reaction mixture was stirred for 30 minutes until all raw materials were consumed as determined by HPLC analysis. The mixture was directly purified by reverse phase chromatography (C18 column, acetonitrile-water 0-75% MeCN, containing 0.05% TFA). Pure fractions were collected and lyophilized to give product 43 as a white solid (107 mg, 0.08 mmol, 67% yield). LRMS (ESI): m/z 1391.6 [M+H] ⁺ , calculated for C ₆₅ H ₈₆ N ₁₀ O ₂₂ S m/z 1391.6.

Preparation of (2S,5S18R)-1-((4-((((4-(((S)-1-(((14S,16S,32R,33R,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(64)-oxazinane-3(2,3)-oxirane-8(1,3)-benzocyclotetradecane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-4-( ...4-(((S)-1-(((14S,16S,32R,33R,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(64)-oxazinane-3(2,3)-oxirane- -(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propionamido)-5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonane-19-sulfonic acid (44).

At room temperature, HATU (19mg, 51μmol, 2 equivalents) was added to the mixture of compound 43 (35mg, 25μmol) and maytansine 17 (16mg, 25μmol) in DMF (2mL), followed by DIPEA (13μL, 76μmol, 3 equivalents). The reaction mixture was stirred for 30 minutes, until all raw materials were consumed by HPLC analysis. Then at room temperature, piperidines (49μL, 0.5mmol, 20 equivalents) were directly added to the solution, and the reaction mixture was stirred for 30 minutes. The mixture was purified by preparative HPLC (C18 columns, acetonitrile-water 0-75%MeCN containing 0.05%TFA). Pure fractions were combined and freeze-dried to obtain compound 44, which was a white solid (21mg, 12μmol, 48% yield). LRMS (ESI): m/z 1800.7 [M+H] ⁺ , calculated for C ₈₂ H ₁₁₈ CIN ₁₃ O ₂₈ 8 1800.8.

Preparation of 4-((S)-2-((S)-2-amino-3-methylbutyramido)propionamido)-3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R) -tert-butyl 3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (45).

A solution of monomethyl auristatin A2 (50 mg, 70 μmol) in anhydrous DMF (2 mL) was treated with HOAt (9.5 mg, 70 μmol) and DIPEA (36 μL, 209 μmol) at room temperature. Compound 41 (72 mg, 70 μmol, 1 equivalent) in solid form was added to the mixture at once at room temperature. The reaction mixture was stirred for 4 hours until all the raw materials were consumed as determined by LCMS analysis. The solvent was removed in vacuo and the residue was redissolved in THF (2 mL). The THF solution was treated with 1 M LiOH solution (2 mL) at room temperature and stirred for 2 hours until hydrolysis was complete. The reaction mixture was quenched with 1 M HCl (2 mL), diluted with DMF (1 mL), and purified by reverse phase chromatography (C18 column, acetonitrile-water 0-75% MeCN containing 0.05% TFA). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to obtain product 45 as a white solid (68 mg, 56 μmol, 80% yield).

Preparation of (2S,5S,18R)-1-((4-((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-4,7,1 0-triazatetradecyl)-2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)amino)-18-(3-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridin-1-yl)propionamido)-5-isopropyl 2-methyl 1,4,7,17-tetraoxo-10,13-dioxa-3,6,16-triazanonane-19-sulfonic acid (46).

HATU (24 mg, 63 μmol) was added to a solution of carboxylic acid 32 (50 mg, 63 μmol) in DMF (2 mL) at room temperature, followed by DIPEA (33 μL, 189 μmol). The resulting mixture was stirred for 1 hour and merged with compound 45 (68 mg, 63 μmol). The reaction mixture was stirred for 5 hours until all raw materials were consumed as determined by HPLC analysis. Piperidine (110 μL, 20 equivalents) was then added to the solution at room temperature, and the reaction mixture was stirred for 30 minutes, diluted with pH 4.70.5M acetate buffer until the solution became acidic, and purified by preparative HPLC (C18 column, acetonitrile-water 0-75% MeCN containing 0.05% TFA). Pure fractions were collected and freeze-dried to obtain compound 46 as a white solid (47 mg, 43% yield). LRMS (ESI): m/z 1769.9 [M+H] ⁺ , calculated for C ₈₄ H ₁₃₂ N ₁₄ O ₂₅ S 1769.9.

Example 2

Bioconjugation, purification and HPLC analysis

method

C-terminal aldehyde-tagged antibodies (15 mg/mL) were conjugated to linker-payloads 30, 33, 44, and 46 at 0.85 mM (8 mol equivalent drug: antibody) in 20 mM sodium citrate, 50 mM sodium chloride pH 5.5 (20/50 buffer) containing 0.85% DMA at 37°C for 72 hours. Free drug was removed using a 30 kD MWCO 0.5 mL Amicon spin concentrator. The sample was added to a spin concentrator, centrifuged at 15,000 x g for 7 minutes, then diluted with 450 μL 20 mM sodium citrate, 50 mM sodium chloride pH 5.5 and centrifuged again. The process was repeated 10 times. To determine the DAR of the final product, the ADC was checked by analytical HIC (Tosoh #14947) with mobile phase A: 1.5 M ammonium sulfate, 25 mM sodium phosphate pH 7.0 and mobile phase B: 25% isopropanol, 18.75 mM sodium phosphate pH 7.0. To determine aggregation, samples were analyzed using analytical size exclusion chromatography (SEC; Tosoh #08541) with a mobile phase of 300 mM sodium chloride, 25 mM sodium phosphate pH 6.8.

result

αHER2 (trastuzumab) and αCD79b (pollotuzumab) antibodies modified to contain an aldehyde tag at the C-terminus (CT) of the heavy chain were conjugated to linker-payloads 30 and 33 carrying maytansine and MMAE, respectively. These reactions are high yields with nearly quantitative conjugation efficiency and >95% overall yield. The resulting ADC has a drug-to-antibody ratio (DAR) of 1.79 to 1.89 and is primarily monomeric. Figures 3 to 10 show representative ADCs for DAR determined by HIC and monomer integrity determined by SEC.

αHER2 (trastuzumab), aTROP-2 (sacitozumab) and anti-FITC antibodies modified to contain an aldehyde tag at the C-terminus (CT) of the heavy chain were conjugated to linkers-payloads 44 and 46 carrying maytansine and MMAE, respectively. These reactions are generally high yields. The resulting ADC has a drug-to-antibody ratio (DAR) of 1.15 to 1.79 and is primarily monomeric. Figures 19 to 30 record representative ADCs for DAR determined by PLRP and monomer integrity determined by SEC.

Example 3

In vitro cytotoxicity

method

CD79b positive B cell lymphoma cell lines (Granta 519 and Ramos-RA), HER2+ gastric cancer and breast cancer cell lines (NCI-N87 and SKBR3) and TROP-2+ pancreatic cancer and breast cancer cell lines (BxPC3 and MDA-MB-468) were obtained from DSMZ and ATCC cell banks. Cells were maintained in growth medium according to the supplier's recommendations. 24 hours before plating, cells were passaged to ensure logarithmic growth. On the day of plating, 5000 cells/well were inoculated into 100 μL of normal growth medium in 96-well plates. Cells were treated with 20 μL of diluted analytes at various concentrations, and the plates were incubated at 37°C in an atmosphere of 5% CO _2. After 5 days, 100 μL/well of Cell Titer-Glo reagent (Promega) was added, and luminescence was measured using a Molecular Devices SpectraMax M5 plate reader. Data analysis was performed using GraphPad Prism software.

result

Galactoside- and glucoside-modified dual-cleavage linkers exhibited potent (subnanomolar) in vitro cytotoxicity against antigen-positive cell lines, with activities comparable to those of free payload and ADCs carrying either single-cleavage linkers or glucuronide-modified dual-cleavage linkers ( FIGS. 12 to 1 and 31 to 36 ).

Although the present invention has been described with reference to specific embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, and process steps to the purpose, spirit, and scope of the present invention. All of these modifications are within the scope of the appended claims.

Claims (51)

1. A conjugate, comprising:

an antibody;

a drug; and

a cleavable linker connecting the antibody to the drug and comprising a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, said second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of: galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

2. The conjugate of claim 1, wherein the conjugate is of formula (I):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

R ¹ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

R ² and R is ³ Each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedAlkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ² And R is ³ Optionally linked in an cyclic manner to form a 5-or 6-membered heterocyclyl;

each R ⁴ Independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

Each R ⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl;

each R ⁶ Independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising the second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint;

W ¹ is the medicament; and is also provided with

W ² Is the antibody.

3. The conjugate of claim 2, wherein:

k is 2; and is also provided with

The conjugate has the formula (Ia):

4. a conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a galactoside.

5. A conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a glucoside.

6. A conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a mannoside.

7. A conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a fucoside.

8. The conjugate of any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises O-GlcNAc.

9. The conjugate of any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises O-GalNAc.

10. A conjugate according to claim 3, wherein the conjugate has formula (Ib):

11. the conjugate of claim 3, wherein the conjugate is of formula (Ic):

12. the conjugate of claim 3, wherein the conjugate has the formula (Id):

13. a conjugate according to claim 3, wherein the conjugate has formula (Ie):

14. a conjugate according to claim 3, wherein the conjugate has formula (If):

15. the conjugate of claim 3, wherein the conjugate has the formula (Ig):

16. the conjugate of any one of claims 2 to 15, wherein L ¹ Comprising:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -，

wherein the method comprises the steps of

a. b, c and d are each independently 0 or 1;

T ¹ 、T ² 、T ³ and T ⁴ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or 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;

V ¹ 、V ² 、V ³ and V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein each q is an integer from 1 to 6;

each R ¹³ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and is also provided with

Each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

17. The conjugate of any one of claims 2 to 16, wherein L ² Comprising:

-(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -，

wherein the method comprises the steps of

e. f, g and h are each independently 0 or 1;

T ⁵ 、T ⁶ 、T ⁷ and T ⁸ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or 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;

V ⁵ 、V ⁶ 、V ⁷ and V ⁸ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein each q is an integer from 1 to 6;

each R ¹³ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and is also provided with

Each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

18. The conjugate of claim 16, wherein:

T ¹ selected from (C) ₁ -C ₁₂ ) Alkyl and substituted (C) ₁ -C ₁₂ ) An alkyl group;

T ² 、T ³ and T ⁴ Each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(C ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, (AA) _p 、-(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, and esters; and is also provided with

V ¹ 、V ² 、V ³ And V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-;

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) as

Each R ¹² And R is ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moieties, aryl, and substituted aryl, wherein any two adjacent R ¹² The groups can be connected in a cyclic manner to form a piperazinyl ring; and is also provided with

R ¹³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.

19. The conjugate of any one of claims 16 to 18, wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is an amino acid analog, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0.

20. The conjugate of any one of claims 16 to 19, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CO-; and is also provided with

f. g and h are 0.

21. The conjugate of any one of claims 16 to 19, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl, and V ⁶ is-CO-; and is also provided with

g and h are 0.

22. The conjugate of any one of claims 1 to 21, wherein the drug is selected from the group consisting of: cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids and peptides.

23. The conjugate of any one of claims 1 to 21, wherein the drug is selected from the group consisting of: australian statin (auristatin), maytansine (maytansine) and duocarmycin (duocarmycin).

24. The conjugate of any one of claims 1 to 21, wherein the drug is selected from the group consisting of: terpirtine M, calicheamicin, SN-38, irinotecan, STAT3 inhibitor, alpha-amanitine, aurora kinase inhibitor, belotecan, 9-aminocamptothecin (9-AC) and anthracyclines.

25. A compound, comprising:

a cleavable linker for linking an antibody to a drug, wherein the cleavable linker comprises a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of: galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

26. The compound of claim 25, wherein the compound has formula (II):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

R ² And R is ³ Each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R ² And R is ³ Optionally linked in an cyclic manner to form a 5-or 6-membered heterocyclyl;

each R ⁴ Independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedAmino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

Each R ⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

each R ⁶ Independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising the second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint; and is also provided with

W1 is a drug.

27. A compound according to claim 26, wherein:

k is 2; and is also provided with

The compounds have the formula (IIa):

28. the compound of any one of claims 25-27, wherein the second cleavable moiety comprises a galactoside.

29. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises a glucoside.

30. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises a mannoside.

31. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises a fucoside.

32. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises O-GlcNAc.

33. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises O-GalNAc.

34. The compound of claim 27, wherein the compound is of formula (IIb):

35. the compound of claim 27, wherein the compound is of formula (IIc):

36. the compound of claim 27, wherein the compound has formula (IId):

37. the compound of claim 27, wherein the compound is of formula (IIe):

38. the compound of claim 27, wherein the compound has formula (IIf):

39. the compound of claim 27, wherein the compound has formula (IIg):

40. the compound of any one of claims 26 to 39, wherein L ¹ Comprising:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -，

wherein the method comprises the steps of

a. b, c and d are each independently 0 or 1;

T ¹ 、T ² 、T ³ and T ⁴ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, andand AA is an amino acid residue or 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;

V ¹ 、V ² 、V ³ and V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein each q is an integer from 1 to 6;

each R ¹³ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and is also provided with

Each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

41. The compound of any one of claims 26 to 40, wherein L ² Comprising:

-(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -，

wherein the method comprises the steps of

e. f, g and h are each independently 0 or 1;

T ⁵ 、T ⁶ 、T ⁷ And T ⁸ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino group-piperidine (4 AP), acetal groups, hydrazine, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analogue, 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;

V ⁵ 、V ⁶ 、V ⁷ and V ⁸ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein each q is an integer from 1 to 6;

each R ¹³ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and is also provided with

Each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

42. The compound of claim 40, wherein:

T ¹ Selected from (C) ₁ -C ₁₂ ) Alkyl and substituted (C) ₁ -C ₁₂ ) An alkyl group;

T ² 、T ³ and T ⁴ Each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(C ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, (AA) _p 、-(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, and esters; and is also provided with

V ¹ 、V ² 、V ³ And V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-;

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) as

Each R ¹² And R is ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moieties, aryl, and substituted aryl, wherein any two adjacent R ¹² The groups can be connected in a cyclic manner to form a piperazinyl ring; and is also provided with

R ¹³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.

43. The compound of any one of claims 40 to 42, wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is an amino acid analog, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0.

44. The compound of any one of claims 40 to 43, wherein:

T ⁵ Is a covalent bond, and V ⁵ is-CO-; and is also provided with

f. g and h are 0.

45. The compound of any one of claims 40 to 43, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl, and V ⁶ is-CO-; and is also provided with

g and h are 0.

46. The compound of any one of claims 25 to 45, wherein the drug is selected from the group consisting of: cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids and peptides.

47. The compound of any one of claims 25 to 45, wherein the drug is selected from the group consisting of: auristatin, maytansine, and sesquialter.

48. The compound of any one of claims 25 to 45, wherein the drug is selected from the group consisting of: terpirtine M, calicheamicin, SN-38, irinotecan, STAT3 inhibitor, alpha-amanitine, aurora kinase inhibitor, belotecan, 9-aminocamptothecin (9-AC) and anthracyclines.

49. A pharmaceutical composition comprising:

the conjugate of any one of claims 1 to 24; and

pharmaceutically acceptable excipients.

50. A method for administering a conjugate to a subject, the method comprising:

administering the conjugate of any one of claims 1 to 24 to a subject.

51. A method for treating cancer in a subject, the method comprising:

administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the conjugate of any one of claims 1 to 24, wherein the administration is effective to treat cancer in the subject.