WO2019113248A1 - Anti-upar antibody-drug conjugates and methods of use thereof - Google Patents

Abstract

The present disclosure relates to antibody-drug conjugates ("ADCs") that bind to and modulate the activity of urokinase plasminogen activator receptor (uPAR/CD87), compositions comprising the agents, and methods involving use of the compositions.

Description

ANTI-UPAR ANTIBODY-DRUG CONJUGATES AND METHODS OF USE THEREOF

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under grant no. P41

CA196276, awarded by the National Institutes of Health. The government has certain rights in the invention.

INTRODUCTION

[0002] The urokinase plasminogen activator receptor (uPAR or CD87 (Cluster of

Differentiation 87)) is a glycosylated protein of 45-55 kDa consisting of three homologous cysteine-rich domains. The protein is localized to the extracellular leaf of the plasma membrane through a glycosylphosphatidylinositol anchor. UPAR mediates a wide variety of cellular processes including inflammation, metastasis and invasion, tissue remodeling, angiogenesis, and cell adhesion.

[0003] Many of these processes are initiated by the highly specific binding of various ligands to membrane-bound uPAR at the cell surface. One such interaction is between uPAR and uPA, which mediates both extracellular and intracellular signaling events.

[0004] Binding of extracellular pro-uPA to uPAR facilitates its activation. In turn, uPA activates proteases, such as plasmin, which directly and indirectly degrade the extracellular matrix (ECM). Furthermore, plasmin can activate pro-uPA leading to a positive feedback loop that accelerates ECM degradation. uPAR is also able to act intracellularly by activating proliferative signal transduction pathways. uPAR is believed to directly associate with integrin family adhesion receptors in complexes that mediate RGD-independent cell signaling and migration. Accordingly, uPAR plays a role in the development of cancer and the metastasis of cancer.

SUMMARY

[0005] The present disclosure relates to antibody-drug conjugates (“ADCs”) that bind to and modulate the activity of urokinase plasminogen activator receptor (uPAR/CD87), compositions comprising the agents, and methods involving use of the compositions. [0006] Aspects of the present disclosure include a conjugate comprising at least one modified amino acid residue with a side chain of formula (I):

wherein

Z is CR⁴ or N;

R¹ 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;

R 2 and R 3 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, acyl amino, amino acyl, 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;

each R⁴ 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker comprising -(T¹-V¹)_a-(T²-V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e- , wherein a, b, c, d and e are each independently 0 or 1, where the sum of a, b, c, d and e is 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from (Ci-Ci₂)alkyl, substituted (Ci- C_l2)alkyl, (EDA)_W, (PEG)_n, (AA)_P, -(CR¹³OH)_h-, piperidin-4-amino (4AP), para-amino- benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol or a modified polyethylene glycol, and AA is an amino acid residue, 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;

V , V , V , V and V are each independently selected from the group consisting of a covalent bond, -CO-, -NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H4)-, -CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, - OC(O)-, -0-, -S-, -S(O)-, -S0₂-, -S0₂NR¹⁵-, -NR¹⁵S0₂- and -P(0)OH-, wherein q is an integer from 1 to 6;

each R is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl;

each R¹⁵ 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;

W¹ is a chemical entity; and

W is an anti-uP AR antibody.

[0007] In some embodiments, the conjugate includes the following features:

T¹ is selected from a (Ci-Ci₂)alkyl and a substituted (Ci-Ci₂)alkyl;

T², T³, T⁴ and T⁵ are each independently selected from (EDA)_W, (PEG)_n, (Ci-Ci₂)alkyl, substituted (Ci-Ci₂)alkyl, (AA)_P , -(CR OH)_h-, 4-amino-piperidine (4AP), para-amino- benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a hydrazine, and an ester; and

V , V , V , V and V are each independently selected from the group consisting of a covalent bond, -CO-, -NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H4)-, -CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, - OC(O)-, -0-, -S-, -S(O)-, -S0₂- , -S0₂NR¹⁵-, -NR¹⁵S0₂-, and -P(0)OH-;

wherein:

integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:

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

4-amino-piperidine

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

R is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl.

[0008] In some embodiments, the chemical entity is a drug.

[0009] In some embodiments, the drug is selected from the group consisting of a maytansinoid and an auristatin.

[0010] In some embodiments, the maytansinoid is of the formula:

where indicates the point of attachment between the maytansinoid and L.

[0011] In some embodiments, the auristatin is MMAE.

[0012] In some embodiments, the anti-uPAR antibody comprises a sequence of the formula (II):

X ¹ (FGly’ )X²Z²⁰X³Z³⁰ (II)

wherein

FGly’ is the modified amino acid residue of formula (I);

Z²⁰ is either a proline or alanine residue;

Z³⁰ is a basic amino acid or an aliphatic amino acid; X¹ may be present or absent and, when present, can be any amino acid, with the proviso that when the sequence is at the N-terminus of the conjugate, X¹ is present; and

X and X are each independently any amino acid.

[0013] In some embodiments, the anti-uPAR antibody includes the following features:

Z³⁰ is selected from R, K, H, A, G, L, V, I, and P;

X^I is selected from L, M, S, and V; and

X and X are each independently selected from S, T, A, V, G, and C.

[0014] In some embodiments, the sequence is L(FGly’)TPSR.

[0015] In some embodiments, the modified amino acid residue is positioned at a C- terminus of a heavy chain constant region of the anti-uPAR antibody. In some embodiments, the heavy chain constant region comprises the sequence SLSLSLGSL(FGly’)TPSRGS

[0016] In some embodiments, the modified amino acid residue is positioned in a CH1 region of the anti-uPAR antibody. In some embodiments, the CH1 region comprises the sequence WN S G AL(FGly’ )TPS RG VHTFP A .

[0017] In some embodiments, the modified amino acid residue is positioned in a light chain constant region of the anti-uPAR antibody.

[0018] In some embodiments, the modified amino acid residue is positioned in a heavy chain CH2 region of the anti-uPAR antibody.

[0019] In some embodiments, the modified amino acid residue is positioned in a heavy chain CH3 region of the anti-uPAR antibody.

[0020] Aspects of the present disclosure include a pharmaceutical composition that includes a conjugate as disclosed herein, and a pharmaceutically acceptable excipient.

[0021] Aspects of the present disclosure include a method comprising administering to a subject an effective amount of a conjugate as disclosed herein.

[0022] Aspects of the present disclosure include a method of treating cancer in a subject, where the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate as disclosed herein, wherein the

administering is effective to treat cancer in the subject.

[0023] Aspects of the present disclosure include a method of delivering a drug to a target site in a subject, where the method includes administering to the subject a pharmaceutical composition comprising a conjugate as disclosed herein, wherein the administering is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject.

BRIEF DESCRIPTION OF FIGURES

[0024] FIG. 1 shows a schematic illustration of two types of anti-uPAR antibodies (2G10 and 3C6), which are antagonists and compete against uPAR interaction with urokinase plasminogen activator and b integrins.

[0025] FIG. 2 shows a schematic illustration of an anti-uPAR antibody (e.g., 2G10 or

3C6) conjugated to a cytotoxic agent, according to embodiments of the present disclosure. FIG. 2 (panel A) shows the components of a site- specifically modified uPAR ADC. FIG. 2 (panel B) shows a schematic illustration of the production of an aldehyde-tagged antibody using formylglycine generating enzyme (FGE).

[0026] FIG. 3 shows graphs demonstrating that anti-uPAR antibodies 2G10 and 3C6 are therapeutically effective in an in vitro triple-negative breast cancer (TNBC) model, according to embodiments of the present disclosure.

[0027] FIG. 4 shows a schematic illustration of the mechanism of action of an anti-uPAR

ADC, according to embodiments of the present disclosure.

[0028] FIG. 5 shows images of intracellular trafficking of anti-uPAR Fabs, according to embodiments of the present disclosure.

[0029] FIG. 6 (panel A) shows a table of anti-uPAR ADCs that were tested in an in vivo

TNBC model, according to embodiments of the present disclosure. The drug-antibody ratio (DAR) for each of the ADC tested was about 2. FIG. 6 (panel B) shows graphs of % change in cell number relative to untreated cells after 110 hours of treatment with the ADC, according to embodiments of the present disclosure.

[0030] FIG. 7 shows a graph of tumor volume relative to initial implants (%) vs. time

(days) for an in vivo TNBC model used to test the efficacy of anti-uPAR ADCs, where an anti- uPAR antibody (e.g., 2G10 or 3C6) was conjugated to a drug, e.g., maytansine or MMAE, according to embodiments of the present disclosure.

[0031] FIG. 8 shows a graph of body weight (g) vs. time (days) for the mice tested in the experimental example corresponding to FIG. 7. [0032] FIG. 9 is a table showing different anti-uPAR 2G10 ADCs that were made and characterized according to embodiments of the present disclosure.

[0033] FIG. 10 shows a graph of tumor volume relative to initial implants (%) vs time

(days) for various anti-uPAR ADCs tested in an in vivo mouse MDA-MB-231 xenograft model of TNBC, according to embodiments of the present disclosure.

[0034] FIG. 11 shows a graph of body weight (g) vs. time (days) for the mice tested in the experimental example corresponding to FIG. 10.

[0035] FIG. 12 shows a schematic of patient-derived xenografts (PDX) model development and the workflow for testing anti-uPAR ADCs in vivo.

[0036] FIG. 13 shows characteristics of grown PDX.

[0037] FIG. 14 shows morphology of cells isolated from a tumor (panel A) and a histogram of cell count vs. log uPAR level (panel B).

[0038] FIG. 15 shows a graph of tumor volume (%) vs. time (days) for an anti-uPAR

3C6-MMAE ADC tested in an in vivo mouse PDX model of pancreatic adenocarcinoma (PD AC), according to embodiments of the present disclosure.

[0039] FIG. 16 shows a graph of body weight (%) vs. time (days) for an anti-uPAR 3C6-

MMAE ADC tested in an in vivo mouse PDX model of PD AC, according to embodiments of the present disclosure.

[0040] FIG. 17 shows a graph of tumor volume (%) vs. time (days) for anti-uPAR 2G10-

MMAE ADCs tested in an in vivo mouse PDX model of PD AC, according to embodiments of the present disclosure.

[0041] FIG. 18 shows a graph of body weight (%) vs. time (days) for anti-uPAR 2G10-

MMAE ADCs tested in an in vivo mouse PDX model of PD AC, according to embodiments of the present disclosure.

DETAILED DESCRIPTION

[0042] The present disclosure relates to agents (e.g., antibody-drug conjugates

(“ADCs”)) that bind to urokinase plasminogen activator receptor (uPAR), compositions comprising the agents, and methods involving use of the compositions. The agents disclosed herein and methods of use can modulate uPAR by disrupting binding of uPAR to other proteins. [0043] Kits containing one or more compositions of the present disclosure, as well as those with instructions for use in a method of the present disclosure also are provided.

[0044] Before the present invention and specific embodiments of the invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. That the upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0047] 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. Thus, for example, reference to“an antigen” includes a plurality of such antigens and reference to“the peptide” includes reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0048] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DEFINITIONS

[0049] The following terms have the following meanings unless otherwise indicated.

Any undefined terms have their art recognized meanings.

[0050] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from

1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH₃-), ethyl (CH₃CH₂-), n-propyl (CH₃CH₂CH₂-), isopropyl ((CH₃)₂CH-), n-butyl

(CH₃CH₂CH₂CH₂-), isobutyl ((CH₃)₂CHCH₂-), sec-butyl ((CH₃)(CH₃CH₂)CH-), t-butyl

((CH₃)₃C-), n-pentyl (CH₃CH₂CH₂CH₂CH₂ ), and neopentyl ((CH₃)₃CCH₂-).

[0051] The term“substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the Ci carbon atom) have been optionally replaced with a heteroatom such as -0-, -N-, -S-, -S(0)_n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- aryl, -SO-heteroaryl, -S0₂-alkyl, -S0₂-aryl, -S0₂-heteroaryl, and -NR^aR^b, wherein R and R may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0052] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from

1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -0-, -NR¹⁰-, -NR¹⁰C(O)-, -C(0)NR¹⁰- and the like. This term includes, by way of example, methylene (-CH₂-), ethylene (-CH₂CH₂-), n-propylene (-CH₂CH₂CH₂-), iso-propylene (-CH₂CH(CH₃)-), (-C(CH₃)₂CH₂CH₂-), (-C(CH₃)₂CH₂C(0)-), (-C(CH₃)₂CH₂C(0)NH-), (-CH(CH₃)CH₂-), and the like. [0053] “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of“substituted” below.

[0054] The term“alkane” refers to alkyl group and alkylene group, as defined herein.

[0055] The term“alkylaminoalkyl”,“alkylaminoalkenyl” and“alkylaminoalkynyl” refers to the groups R NHR - where R is alkyl group as defined herein and R is alkylene, alkenylene or alkynylene group as defined herein.

[0056] The term“alkaryl” or“aralkyl” refers to the groups -alkylene-aryl and

-substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0057] “Alkoxy” refers to the group -O-alkyl, wherein alkyl is as defined herein.

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

[0058] The term“substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0059] The term“alkoxyamino” refers to the group -NH-alkoxy, wherein alkoxy is defined herein.

[0060] The term“haloalkoxy” refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.

[0061] The term“haloalkyl” refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group. Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.

[0062] 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. [0063] The term“alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S- substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0064] “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-l-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.

[0065] The term“substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -S0₂-alkyl, -S0₂-substituted alkyl, -S0₂-aryl and - S0₂-heteroaryl.

[0066] “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-CºCH), and propargyl (-CH₂CºCH).

[0067] The term“substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -S0₂-alkyl, -S0₂-substituted alkyl, -S0₂-aryl, and - S0₂-heteroaryl. [0068] “Alkynyloxy” refers to the group -O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0069] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl- C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl- C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl- C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the“acetyl” group CH₃C(0)-

[0070] “Acylamino” refers to the groups -NR²⁰C(O)alkyl, -NR²⁰C(O)substituted alkyl, N

R²⁰C(O)cycloalkyl, -NR²⁰C(O)substituted cycloalkyl, -NR²⁰C(O)cycloalkenyl,

-NR²⁰C(O)substituted cycloalkenyl, -NR²⁰C(O)alkenyl, -NR²⁰C(O)substituted alkenyl,

-NR²⁰C(O)alkynyl, -NR²⁰C(O)substituted alkynyl, -NR²⁰C(O)aryl, -NR²⁰C(O)substituted aryl, -NR²⁰C(O)heteroaryl, -NR²⁰C(O)substituted heteroaryl, -NR²⁰C(O)heterocyclic, and

-NR C(0)substituted heterocyclic, wherein R 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, heterocyclic, and substituted heterocyclic are as defined herein.

[0071] “Aminocarbonyl” or the term“aminoacyl” refers to the group

wherein R and R independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R and R are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [0072] “Aminocarbonylamino’’ refers to the group -NR²¹C(0)NR²²R²³ where R²¹, R²², and R 23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.

[0073] The term“alkoxycarbonylamino” refers to the group -NRC(0)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0074] The term“acyloxy” refers to the groups alkyl-C(0)0-, substituted alkyl-C(0)0-, cycloalkyl-C(0)0-, substituted cycloalkyl-C(0)0-, aryl-C(0)0-, heteroaryl-C(0)0-, and heterocyclyl-C(0)0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0075] “Amino sulfonyl” refers to the group -S0₂NR²¹R²², wherein R²¹ and R²² independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R and R are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0076] “Sulfonylamino” refers to the group -NR²¹S0₂R²², wherein R²¹ and R²² independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R and R are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0077] “Aryl” or“Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S0₂-alkyl, -S0₂- substituted alkyl, -S0₂-aryl, -S0₂-heteroaryl and trihalomethyl.

[0078] “Aryloxy” refers to the group -O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.

[0079] “Amino” refers to the group -NH₂.

[0080] The term“substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.

[0081] The term“azido” refers to the group -N₃.

[0082] “Carboxyl,”“carboxy” or“carboxylate” refers to -C0₂H or salts thereof.

[0083] “Carboxyl ester” or“carboxy ester” or the terms“carboxy alkyl” or

“carboxylalkyl” refers to the groups -C(0)0-alkyl, -C(0)0-substituted alkyl, -C(0)0-alkenyl, -C(0)0-substituted alkenyl, -C(0)0-alkynyl, -C(0)0-substituted alkynyl, -C(0)0-aryl, -C(0)0-substituted aryl, -C(0)0-cycloalkyl, -C(0)0-substituted cycloalkyl,

-C(0)0-cycloalkenyl, -C(0)0-substituted cycloalkenyl, -C(0)0-heteroaryl, -C(0)0-substituted heteroaryl, -C(0)0-heterocyclic, and -C(0)0-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0084] “(Carboxyl ester)oxy” or“carbonate” refers to the groups -0-C(0)0-alkyl,

-0-C(0)0-substituted alkyl, -0-C(0)0-alkenyl, -0-C(0)0-substituted alkenyl, -0-C(0)0- alkynyl, -0-C(0)0-substituted alkynyl, -0-C(0)0-aryl, -0-C(0)0-substituted aryl, -0-C(0)0- cycloalkyl, -0-C(0)0-substituted cycloalkyl, -0-C(0)0-cycloalkenyl, -0-C(0)0-substituted cycloalkenyl, -0-C(0)0-heteroaryl, -0-C(0)0-substituted heteroaryl, -0-C(0)0-heterocyclic, and -0-C(0)0-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0085] “Cyano” or“nitrile” refers to the group -CN.

[0086] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0087] The term“substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy,

thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -S O-heteroaryl, -S0₂-alkyl, -S0₂-substituted alkyl, -S0₂-aryl and -S0₂-heteroaryl.

[0088] “Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds. [0089] The term“substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -S0₂-alkyl, -S0₂-substituted alkyl, -S0₂-aryl and - S0₂-heteroaryl.

[0090] “Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.

[0091] “Cycloalkoxy” refers to -O-cycloalkyl.

[0092] “Cycloalkenyloxy” refers to -O-cycloalkenyl.

[0093] “Halo” or“halogen” refers to fluoro, chloro, bromo, and iodo.

[0094] “Hydroxy” or“hydroxyl” refers to the group -OH.

[0095] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N- oxide (N 0), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -S O-heteroaryl, -S0₂-alkyl, -S0₂-substituted alkyl, -S0₂-aryl and -S0₂-heteroaryl, and trihalomethyl.

[0096] The term“heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0097] “Heteroaryloxy” refers to -O-heteroaryl.

[0098] “Heterocycle,”“heterocyclic,”“heterocycloalkyl,” and“heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, where, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or - S0₂- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein.

[0099] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, l,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b] thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), l,l-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[00100] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,

hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -S O-heteroaryl, -S0₂-alkyl, -S0₂- substituted alkyl, -S0₂-aryl, -S0₂-heteroaryl, and fused heterocycle.

[00101] “Heterocyclyloxy” refers to the group -O-heterocyclyl.

[00102] The term“heterocyclylthio” refers to the group heterocyclic-S-.

[00103] The term“heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.

[00104] The term“hydroxyamino” refers to the group -NHOH.

[00105] “Nitro” refers to the group -N0₂.

[00106] “Oxo” refers to the atom (=0).

[00107] “Sulfonyl” refers to the group S0₂-alkyl, S0₂-substituted alkyl, S0₂-alkenyl, S0₂- substituted alkenyl, S0₂-cycloalkyl, S0₂-substituted cylcoalkyl, S0₂-cycloalkenyl, S0₂- substituted cylcoalkenyl, S0₂-aryl, S0₂- substituted aryl, S0₂-heteroaryl, S0₂-substituted heteroaryl, S0₂-heterocyclic, and S0₂-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-S0₂-, phenyl-S0₂-, and 4-methylphenyl-S0₂-.

[00108] “Sulfonyloxy” refers to the group -OS0₂-alkyl, OS0₂-substituted alkyl, OS0₂- alkenyl, OS0₂-substituted alkenyl, OS0₂-cycloalkyl, OS0₂-substituted cylcoalkyl, OS0₂- cycloalkenyl, OS0₂-substituted cylcoalkenyl, OS0₂-aryl, OS0₂-substituted aryl, OS0₂- heteroaryl, OS0₂-substituted heteroaryl, OS0₂-heterocyclic, and OS0₂ substituted

heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [00109] The term“aminocarbonyloxy” refers to the group -0C(0)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[00110] “Thiol” refers to the group -SH.

[00111] “Thioxo” or the term“thioketo” refers to the atom (=S).

[00112] “Alkylthio” or the term“thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

[00113] The term“substituted thioalkoxy” refers to the group -S-substituted alkyl.

[00114] The term“thioaryloxy” refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.

[00115] The term“thioheteroaryloxy” refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.

[00116] The term“thioheterocyclooxy” refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.

[00117] In addition to the disclosure herein, the term“substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[00118] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =0, =NR , =N-OR , =N₂ or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R⁶⁰, halo, =0, -OR⁷⁰, -SR⁷⁰, -NR⁸⁰R⁸⁰, trihalomethyl, -CN, -OCN, -SCN, -NO, -N0₂, =N₂, -N₃, -S0₂R⁷°, -S0₂0 M⁺, -S0₂0R⁷°, -OS0₂R⁷⁰, -0S0₂0 M⁺, -OS0₂OR⁷⁰, -P(0)(0 )₂(M⁺)₂, -P(O)(OR⁷⁰)O M⁺, -P(O)(OR⁷⁰) ₂, -C(0)R⁷⁰, -C(S)R⁷⁰, -C(NR⁷⁰)R⁷⁰, -C(0)0 M⁺, -C(0)OR⁷⁰, -C(S)OR⁷⁰, -C(0)NR⁸⁰R⁸⁰,

-C(NR⁷⁰)NR⁸⁰R⁸⁰, -OC(0)R⁷⁰, -OC(S)R⁷⁰, -OC(0)O M⁺, -OC(0)OR⁷⁰, -OC(S)OR⁷⁰,

-NR⁷⁰C(O)R⁷⁰, -NR⁷⁰C(S)R⁷⁰, -NR^7,IC0₂ M⁺, -NR⁷⁰C0₂R⁷⁰, -NR⁷⁰C(S)OR⁷⁰,

-NR⁷⁰C(O)NR⁸⁰R⁸⁰, -NR⁷⁰C(NR⁷⁰)R⁷⁰ and -NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl,

heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R is independently hydrogen or R⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, two R⁸⁰ s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M⁺ is a counter ion with a net single positive charge. Each M⁺ may independently be, for example, an alkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; or an alkaline earth ion, such as [Ca²⁺]o_.s, [Mg²⁺]o_.s, or [Ba²⁺]o_.s (“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR R is meant to include -NH₂, -NH-alkyl, N- pyrrolidinyl, N- piperazinyl, 4/V-methyl-piperazin-l-yl and /V-morpholinyl.

[00119] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in“substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R⁶⁰, halo, -O M⁺, -OR⁷⁰, -SR⁷⁰, -S M⁺, -NR⁸⁰R⁸⁰, trihalomethyl, -CF₃, -CN, -OCN, -SCN, -NO, -NO2, -N₃, -SO2R⁷⁰, -S0₃ M⁺, -S0₃R⁷⁰, -0S0₂R⁷°, -OS0₃ M⁺, -0S0₃R⁷⁰, -P0_3- ²(M⁺)₂, -P(O)(OR⁷⁰)O M⁺, -P(O)(OR⁷⁰)₂, -C(0)R⁷⁰, -C(S)R⁷⁰, -C(NR⁷⁰)R⁷⁰, -C0₂ M⁺, -C0₂R⁷⁰, -C(S)OR⁷⁰, -C(0)NR⁸⁰R⁸⁰, -C(NR⁷⁰)NR⁸⁰R⁸⁰, -OC(0)R⁷⁰, -OC(S)R⁷⁰, -OC0₂ M⁺, -OC0₂R⁷⁰, -OC(S)OR⁷⁰, -NR⁷⁰C(0)R⁷⁰, -NR⁷⁰C(S)R⁷⁰, -NR⁷⁰CO₂ M⁺, -NR⁷⁰C0₂R⁷⁰,

-NR⁷⁰C(S)OR⁷⁰, -NR⁷⁰C(0)NR⁸⁰R⁸⁰, -NR⁷⁰C(NR⁷⁰)R⁷⁰ and -NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -0 M⁺, -OR⁷⁰, -SR⁷⁰, or -S M⁺.

[00120] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in“substituted” heteroalkyl and

cycloheteroalkyl groups are, unless otherwise specified, -R⁶⁰, -0 M⁺, -OR⁷⁰, -SR⁷⁰, -S M⁺, -NR⁸⁰R⁸⁰, trihalomethyl, -CF₃, -CN, -NO, -N0₂, -S(0)₂R⁷°, -S(0)₂O M⁺, -S(0)₂0R⁷°,

-OS(0)₂R⁷⁰, -OS(0)₂O M⁺, -OS(0)₂OR⁷⁰, -P(0)(0 )₂(M⁺)₂, -P(0)(OR⁷⁰)O M⁺,

-P(O)(OR⁷⁰)(OR⁷⁰), -C(0)R⁷⁰, -C(S)R⁷⁰, -C(NR⁷⁰)R⁷⁰, -C(0)OR⁷⁰, -C(S)OR⁷⁰, -C(0)NR⁸⁰R⁸⁰, -C(NR⁷⁰)NR⁸⁰R⁸⁰, -OC(0)R⁷⁰, -OC(S)R⁷⁰, -OC(0)OR⁷⁰, -OC(S)OR⁷⁰, -NR⁷⁰C(0)R⁷⁰,

-NR⁷⁰C(S)R⁷⁰, -NR⁷⁰C(0)OR⁷⁰, -NR⁷⁰C(S)OR⁷⁰, -NR⁷⁰C(0)NR⁸⁰R⁸⁰, -NR⁷⁰C(NR⁷⁰)R⁷⁰ and -NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previously defined.

[00121] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[00122] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[00123] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-0-C(0)-.

[00124] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[00125] The term“pharmaceutically acceptable salt” means a salt which is acceptable for administration to a patient, such as a mammal (salts with counterions having acceptable mammalian safety for a given dosage regime). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, and the like.

[00126] The term“salt thereof’ means a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation and the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts of

intermediate compounds that are not intended for administration to a patient. By way of example, salts of the present compounds include those wherein the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[00127] “Solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, NN-d\ methyl formamidc, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate._

[00128] “Stereoisomer” and“stereoisomers” refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[00129] “Tautomer” refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.

[00130] It will be appreciated that the term“or a salt or solvate or stereoisomer thereof’ is intended to include all permutations of salts, solvates and stereoisomers, such as a solvate of a pharmaceutically acceptable salt of a stereoisomer of subject compound.

[00131] “Pharmaceutically effective amount” and“therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[00132] “Patient” refers to human and non-human subjects, especially mammalian subjects.

[00133] The terms“polypeptide,”“peptide,” and“protein” are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless specifically indicated otherwise,“polypeptide,”“peptide,” and“protein” can include genetically coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, proteins which contain at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant bacterial host cell); immunologically tagged proteins; and the like.

[00134] “Native amino acid sequence” or“parent amino acid sequence” are used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include a modified amino acid residue.

[00135] The terms“amino acid analog,”“unnatural amino acid,” and the like may be used interchangeably, and include amino acid-like compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include a- hydroxy acids, and a-amino acids, and the like. [00136] The terms“amino acid side chain” or“side chain of an amino acid” and the like may be used to refer to the substituent attached to the a-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. An amino acid side chain can also include an amino acid side chain as described in the context of the modified amino acids and/or conjugates described herein.

[00137] The term“carbohydrate” and the like may be used to refer to monomers units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to the smaller carbohydrates, such as monosaccharides, disaccharides. The term“carbohydrate derivative” includes compounds where one or more functional groups of a carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemistry) or absent (e.g., eliminated or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compounds and conjugates.

[00138] The term“antibody” is used in the broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and

multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single-chain antibodies, chimeric antibodies, antibody fragments (e.g., Fab fragments), and the like. An antibody is capable of binding a target antigen. (Janeway, C., Travers, P., Walport, M.,

Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen can have one or more binding sites, also called epitopes, recognized by complementarity determining regions (CDRs) formed by one or more variable regions of an antibody.

[00139] The term“natural antibody” refers to an antibody in which the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies.

[00140] The term“humanized antibody” or“humanized immunoglobulin” refers to a non human (e.g., mouse or rabbit) antibody containing one or more amino acids (in a framework region, a constant region or a CDR, for example) that have been substituted with a

correspondingly positioned amino acid from a human antibody. In general, humanized antibodies produce a reduced immune response in a human host, as compared to a non-humanized version of the same antibody. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et ah, Protein Engineering 7(6):805-8l4 (1994); Roguska. et ah, PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann et ah, Nature 332:323 (1988)).

Additional methods for humanizing antibodies contemplated for use in the present invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO 98/45331 and WO 98/45332. In particular embodiments, a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art.

[00141] The term“chimeric antibodies” refer to antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although domains from other mammalian species may be used.

[00142] An immunoglobulin polypeptide immunoglobulin light or heavy chain variable region is composed of a framework region (FR) interrupted by three hypervariable regions, also called“complementarity determining regions” or“CDRs”. The extent of the framework region and CDRs have been defined (see,“Sequences of Proteins of Immunological Interest,” E. Rabat et ah, U.S. Department of Health and Human Services, 1991). The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. [00143] Throughout the present disclosure, the numbering of the residues in an immunoglobulin heavy chain and in an immunoglobulin light chain is that as in Kabat et ah, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference.

[00144] A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence which lacks an aldehyde-tagged constant region as described herein. The parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (such as additions, deletions and/or substitutions).

[00145] In the context of an Ig polypeptide, the term“constant region” is well understood in the art, and refers to a C-terminal region of an Ig heavy chain, or an Ig light chain. An Ig heavy chain constant region includes CH1, CH2, and CH3 domains (and CH4 domains, where the heavy chain is a m or an e heavy chain). In a native Ig heavy chain, the CH1, CH2, CH3 (and, if present, CH4) domains begin immediately after (C-terminal to) the heavy chain variable (VH) region, and are each from about 100 amino acids to about 130 amino acids in length. In a native Ig light chain, the constant region begins begin immediately after (C-terminal to) the light chain variable (VL) region, and is about 100 amino acids to 120 amino acids in length.

[00146] As used herein, the term“CDR” or“complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol.

196:901-917 (1987); and MacCallum et al., /. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. Table 1: CDR Definitions

Residue numbering follows the nomenclature of Rabat et al., supra

² Residue numbering follows the nomenclature of Chothia et al., supra

³ Residue numbering follows the nomenclature of MacCallum et al., supra

[00147] By“genetically-encodable” as used in reference to an amino acid sequence of polypeptide, peptide or protein means that the amino acid sequence is composed of amino acid residues that are capable of production by transcription and translation of a nucleic acid encoding the amino acid sequence, where transcription and/or translation may occur in a cell or in a cell- free in vitro transcription/translation system.

[00148] The term“control sequences” refers to DNA sequences that facilitate expression of an operably linked coding sequence in a particular expression system, e.g. mammalian cell, bacterial cell, cell-free synthesis, etc. The control sequences that are suitable for prokaryote systems, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cell systems may utilize promoters, polyadenylation signals, and enhancers.

[00149] A nucleic acid is“operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate the initiation of translation. Generally,“operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Linking is accomplished by ligation or through amplification reactions. Synthetic oligonucleotide adaptors or linkers may be used for linking sequences in accordance with conventional practice.

[00150] The term“expression cassette” as used herein refers to a segment of nucleic acid, usually DNA, that can be inserted into a nucleic acid (e.g., by use of restriction sites compatible with ligation into a construct of interest or by homologous recombination into a construct of interest or into a host cell genome). In general, the nucleic acid segment comprises a

polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to facilitate insertion of the cassette in the proper reading frame for transcription and translation. Expression cassettes can also comprise elements that facilitate expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

[00151] As used herein the term“isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs.“Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.

[00152] As used herein, the term“substantially purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated.

[00153] The term“physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

[00154] By‘‘reactive partner” is meant a molecule or molecular moiety that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include a cysteine or serine of a sulfatase motif and Formylglycine Generating Enzyme (FGE), which react to form a reaction product of a converted aldehyde tag containing a formylglycine (FGly) in lieu of cysteine or serine in the motif. Other exemplary reactive partners include an aldehyde of an fGly residue of a converted aldehyde tag (e.g., a reactive aldehyde group) and an “aldehyde-reactive reactive partner”, which comprises an aldehyde-reactive group and a moiety of interest, and which reacts to form a reaction product of a modified aldehyde tagged polypeptide having the moiety of interest conjugated to the modified polypeptide through a modified fGly residue. [00155] “N-terminus” refers to the terminal amino acid residue of a polypeptide having a free amine group, which amine group in non-N-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.

[00156] “C-terminus” refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, which carboxyl group in non-C-terminus amino acid residues normally forms part of the covalent backbone of the polypeptide.

[00157] By‘‘internal site” as used in referenced to a polypeptide or an amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or at the C-terminus.

[00158] It is further noted that the claims may be drafted to exclude any optional or alternative element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as“solely”,“only” and the like in connection with the recitation of claim elements, or the use of a“negative” limitation.

[00159] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent a definition of a term set out in a document incorporated herein by reference conflicts with the definition of a term explicitly defined herein, the definition set out herein controls.

[00160] Examples of methods and compositions employable therein are described first in greater detail, followed by a review of the various specific compositions, formulations, kits and the like that may find use in the methods of the present disclosure, as well as a discussion of representative applications in which the methods and compositions of the present disclosure find use. ANTI-UPAR ANTIBODIES

[00161] The present disclosure provides anti-uPAR antibody-drug conjugates (“uPAR

ADCs”). uPAR, the target of the subject agents, is also known as urokinase plasminogen activator receptor, urokinase receptor, uPA receptor, or CD87 (Cluster of Differentiation 87). UPAR is composed of three different domains of the Ly-6/uPAR/alpha-neuro toxin family. All three domains are involved in high affinity binding of the primary ligand, urokinase. Besides the primary ligand urokinase, uPAR interacts with several other proteins, including vitronectin, the uPAR associated protein (uPARAP) and the integrin family of membrane proteins.

[00162] As used herein,“uPAR” refers to urokinase plasminogen activator receptor, including those whose amino acid sequences that are at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of a naturally-occurring allelic variant and/or isoform thereof. Variants can also include mutations whose expression is associated with cancer. Many mammalian uPARs and their corresponding isoforms are known in the art. For example, the amino acid sequence of the longest human isoform is available as GenBank Accession No. NP_002650.l and UniProt Accession No. Q03405.

[00163] Binding of ligands and/or integrins to uPAR is involved in signaling that can lead to proliferation. Certain signaling cascades that are initiated by activated uPAR mediate the regulation of cellular shape, adhesion, and mobility, and thus play a role in cell invasion.

Accordingly, preventing ligands such as uPA and/or integrins (e.g., bΐ integrins, such as a5b1 or a3b1) from binding to uPAR can reduce the effects of proliferative signaling cascades and those signals leading to angiogenesis. A subject binding agent can exhibit features that allow not only competitive binding with proteins (e.g., integrins and/or ligands) that bind to uPAR but also potent inhibition of uPAR-mediated cell signaling.

[00164] UPAR-binding agents of the present disclosure can find use in a variety of applications, including use in various methods of treating a host suffering from a disease or condition associated with uPAR signaling, as well as in diagnosis of various diseases and conditions associated with uPAR expression. For example, a subject agent, such as an antibody, is specific for the integrin-binding site on uPAR and may be used to inhibit the proliferation or metastasis of cancer cells. More uses of a subject agent will be described later. [00165] uPAR-expressing cells can serve as targets for the uPAR antibodies of the present disclosure. For example, uPAR-binding agents (e.g., antibodies) of the present disclosure can be used to bind human cells that express surface exposed uPAR. The binding may be specific so that cells that express uPAR are labeled using the subject antibody but cells that do not express uPAR are not. The uPAR expressed in cells may be endogenous, recombinants, naturally- occurring variants and isoforms, and/or a homolog of human uPAR (murine, rat, bovine, primates, etc.). Particularly, uPAR molecules that are expressed by cancer cells can be bound by the subject antibody. Such antibody may be useful in specifically labeling cancer cells (e.g., uPAR-positive cancer) for use in a diagnostic method, described in more detail below.

[00166] As a reference, an amino acid sequence of uPAR is provided below and can also be found in RSCB Protein Data Bank identified as 3BT1. Numbering system used in the present disclosure to refer to an amino acid residue position in uPAR would be in the context of the following amino acid sequence:

LRCMQCKTNGDCRVEECALGQDLCRTTIVRLWEEGEELELVEKSCTHSEKTNRTLSYRT GLKITSLTEVVCGLDLCNQGNSGRAVTYSRSRYLECISCGSSDMSCERGRHQSLQCRSPE EQCLDVVTHWIQEGEEGRPKDDRHLRGCGYLPGCPGSNGFHNNDTFHFLKCCNTTKCN EGPILELENLPQN GRQC Y S CKGN S THGCS S EETFLIDCRGPMN QCL V ATGTHEPKN QS Y MVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYR (SEQ ID NO: l)

[00167] The present disclosure provides uPAR antibodies that compete with and/or disrupt integrin binding to uPAR. Integrins encompass bΐ integrins, such as a5b1 or a3b1. The agents thus find use in inhibiting integrin binding to cells (e.g., human cells expressing uPAR). For example, antibodies of clone 3C6 inhibit a5b1 and a3b1 integrin binding to uPAR. This inhibition may be due to the binding of the antibody to an epitope involved in the interaction between integrin and uPAR (e.g., integrin binding site) or to an epitope outside of the binding site so that uPAR is modified in a way to decrease uPAR’s affinity to integrin (e.g., allosteric site). As such, a uPAR antibody of the present disclosure can compete with an antibody that binds to an epitope located in the integrin-binding site (e.g., a5b1 and/or a3b1 integrin binding site). One or more epitopes of an antibody of the present disclosure can be found in domain III, which corresponds to the amino acid sequence of uPAR from about amino acid residue position 192 to about position 275. Other epitopes outside of domain III may also contribute to the binding affinity of integrin or an antibody of the present disclosure to uPAR. [00168] Antibodies of the ADCs of the present disclosure include those that can compete with an antibody that binds to an epitope including one or more of the following residues: D262, E208, E230, H249, and S 156, all of which are located in domain III except for S 156, which is located in domain II. For example, an antibody can bind to an epitope or compete with an antibody that binds to an epitope including residue E208. In another example, the epitope can include residue H249 and D262. Alternatively, the epitope includes E230 or S 156.

[00169] The present disclosure also provides antibodies that compete with and/or inhibit uPA binding to uPAR. Urokinase-type plasminogen activator (uPA, also known as urokinase), an endogenous ligand of uPAR, is a member of a family of enzymes that exhibit protease activity described as EC 3.4.21.73 according to the IUMBM enzyme nomenclature. UPAR antibodies can decrease binding of uPA to uPAR by competitive inhibition, where the antibody binds to the same site of uPAR as uPA binds or at a different site outside of the uPA binding site (e.g., allosteric site), or by noncompetitive inhibition. Examples of antibodies that can inhibit uPA binding to uPAR include antibodies from clone 2E9 and antibodies from clone 2G10.

[00170] As such, a uPAR antibody of the ADCs of the present disclosure can compete with an antibody that binds to an epitope located in the uPA-binding site. One or more epitopes of a uPA-binding site can be found in domain I and/or domain II of uPAR. Domain I corresponds to an amino acid sequence of uPAR from about amino acid residue position 1 to about position 80. Domain II corresponds to an amino acid sequence of uPAR from about amino acid residue position 91 to about position 191.

[00171] As noted above, antibody affinity for uPAR may be described by the dissociation constant, K_D- Antibodies of the present disclosure, for example, include those having a K_D for uPAR of less than about lOOOnM, less than about 500nM, less than about 300nM, less than about 200nM, less than about lOOnM, less than about 80 nM, less than about 60 nM, less than about 55 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 5nM, less than about 2nM, less than about lnM, less than about 750pM, less than about 500pM, less than about 300pM, less than about 200pM, less than about lOOpM, or less than about 50pM. For example, the divalent IgG antibody derived from clone 2G10 has a K_D of about 40.5 nM. Other examples of antibodies of the present disclosure are described in WO 2011/100620, the disclosure of which is incorporated herein by reference. [00172] uPAR antibodies of the ADCs of the present disclosure include antibodies that facilitate a decrease in cellular signaling associated with uPAR ligand or integrin binding. Such antibodies can find use in, for example, decreasing cellular proliferation by binding to uPAR. Cellular signaling effects can be assessed by modulation of (e.g., a decrease in) phosphorylation levels of kinases associated with uPAR signaling, such as extracellular signal-regulated kinases (ERKs), mitogen activated kinases (MAPK), and/or microtubule-associated protein kinase. For example, antibodies of the present disclosure include those that can inhibit uPAR-dependent ERK phosphorylation and in turn, inhibit ERK activation. Antibodies of the present disclosure include those that can inhibit fibronectin-dependent ERK phosphorylation. Antibodies of the present disclosure include those that can facilitate inhibition of proliferation of cells by binding to cell- surface uPAR.

[00173] Antibodies of the ADCs of the present disclosure include those that can facilitate a decrease in invasion of uPAR-expressing cells into extracellular matrix and/or facilitate a decrease in adhesion of uPAR-expressing cells (e.g., fibronectin- or vitronecting-dependent adhesion). The ability of cells to invade is a phenotype correlated with the metastatic potential of cancer cells. For example, antibodies having the CDRs of clones 2G10 or 3C6 can facilitate inhibition of cancer cell invasion. Antibodies having the CDRs of clone 3C6 can also facilitate a decrease in fibronectin-or vitronectin-dependent cell adhesion. Antibodies of the present disclosure include those that can find use in reducing migration of uPAR-expressing cancer cells.

Amino acid sequences

[00174] uPAR antibodies of the ADCs of the present disclosure include antibodies that bind an epitope in the ligand-binding region and/or integrin-binding region of uPAR.

[00175] Antibodies of the ADCs of the present disclosure include antibodies having one, two, or three heavy chain CDRs about 85%, 90%, 95%, 98%, 99%, or 100% identical to V_H CDR1 , V_H CDR2, or V_H CDR3, of Table 2 below. Antibodies of the ADCs of the present disclosure include antibodies having one, two, or three light chain CDRs about 85%, 90%, 95%, 98%, 99%, or 100% identical to V_L CDR1, V_L CDR2, or V_L CDR3 of Table 2. All CDRs may be derived from the same antibody or be independently selected from different antibodies listed in Table 2. [00176] The V_H and V_L CDRS are separated by framework regions (FR). Amino acid sequences for FRs are exemplified by the FRs of the uPAR antibodies disclosed herein. uPAR antibodies include those containing FRs or other linkers having amino acid sequence that are different from the framework regions disclosed herein. Conservative amino acid substitutions may also be contemplated for any amino acid residue of CDR, framework regions, or linker regions. Other substitutions may be contemplated based on alignments, for example as described in WO 2011/100620, the disclosure of which is incorporated herein by reference.

[00177] Optional linkers within a heavy chain or light chain polypeptide of an antibody may comprise amino acid residues or non-peptide polymers. The linkers may have a length of from about 1 to about 100 monomers, e.g., from about 2 to about 5, from about 7 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 30, from about 30 to about 50, from about 50 to about 75, or from about 75 to about 100 monomers.

[00178] Examples of uPAR antibodies of the ADCs of the present disclosure include an antibody comprising a light chain polypeptide having an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of the amino acid sequence set forth as 3C6 V_L.

[00179] Examples of uPAR antibodies of the ADCs of the present disclosure include an antibody comprising a light chain polypeptide having an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of the amino acid sequence set forth as 2G10 V_L.

[00180] Examples of uPAR antibodies of the ADCs of the present disclosure include an antibody comprising a heavy chain polypeptide having an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of the amino acid sequence set forth as 3C6 V_H.

[00181] Examples of uPAR antibodies of the ADCs of the present disclosure include an antibody comprising a heavy chain polypeptide having an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of the amino acid sequence set forth as 2G10 V_H·

[00182] Examples of uPAR antibodies of the ADCs of the present disclosure include an antibody comprising a light or a heavy chain polypeptide sequence as depicted in any of the 2G10 and/or 3C6 antibodies listed below. Such antibodies can also include any CDRs and framework regions (FRs) as the antibodies 2G10 and/or 3C6 antibodies as described below.

[00183] Examples of uPAR antibodies of the present disclosure include an antibody comprising a light chain polypeptide comprising one or more CDRs (CDR1, CDR2 or CDR3) of the variable region of a light chain polypeptide of 2G10 and a heavy chain polypeptide comprising one or more CDRs (CDR1, CDR2, or CDR3) of the variable region of any heavy chain polypeptide 2G10. Examples of uPAR antibodies of the present disclosure include an antibody comprising a light chain polypeptide comprising one or more CDRs (CDR1, CDR2 or CDR3) of the variable region of a light chain polypeptide of 3C6 and a heavy chain polypeptide comprising one or more CDRs (CDR1, CDR2, or CDR3) of the variable region of any heavy chain polypeptide 3C6. One or more amino acid residues in one or more of the CDRs set forth above may be deleted, inserted, or substituted in the subject antibody. Conservative substitutions may also be present.

[00184] uPAR antibodies of the present disclosure may be of any subclass (e.g., IgG, IgE, IgD, IgA, or IgM). The antibody may be fully human or may be a humanized monoclonal antibody. Chimeric antibodies composed of human and non-human amino acid sequences are also contemplated by the present disclosure. Antibodies of the present disclosure encompass antibodies and antibody fragments that are capable of exhibiting immunological binding properties of the antibodies described herein, e.g., antibodies that compete for binding of an epitope bound by any of the antibodies exemplified herein. Example of antibody fragments include, but are not limited to, Fab, Fab' and F(ab')₂, Fd, single-chain Fvs (scFv), single-chain immunoglobulins (e.g., wherein a heavy chain, or portion thereof, and light chain, or portion thereof, are fused), disulfide-linked Fvs (sdFv), diabodies, triabodies, tetrabodies, scFv, affibodies, minibodies, Fab minibodies, and dimeric scFv and any other fragments comprising a VL and a VH domain in a conformation such that a specific antigen binding region is formed. Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: a heavy chain constant domain, or portion thereof, e.g., a CH1, CH2, CH3, transmembrane, and/or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g., a Ckappa or Ci_ambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1, CH2, CH3, C_kappa, Ci_ambda, transmembrane and cytoplasmic domains. One or more fragments of the antibody may also be provided as cyclized forms.

Amino acid and nucleic acid sequences

[00185] uPAR-binding antibodies of the uPAR ADCs of the present disclosure can comprise a VH CDR1 , VH CDR2, or VH CDR3, of a 2G10 or 3C6 VH polypeptide as provided below. uPAR-binding antibodies of the uPAR ADCs of the present disclosure can comprise a VL CDR1, V_L CDR2, or V_L CDR3 of CDRs of a 2G10 or 3C6 VL polypeptide as provided below.

As discussed above, CDRs can be as defined by Rabat et al., J. Biol. Chem. 252:6609-6616 (1977); Rabat et al., U.S. Dept of Health and Human Services,“Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and

MacCallum et al., J. Mol. Biol. 262:732-745 (1996). The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in Table 1 above as a comparison.

[00186] uPAR-binding antibodies can comprise a contiguous amino acid sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or 100%) to a contiguous sequence of any of the amino acid sequences of 2G10 and 3C6 VH and V_L polypeptides listed below.

[00187] 2G10 V_H:

Q V QLQQS GPGLVRPS QTLS LTC AIS GDS V S S NS A A WNWIRQS PS RGLEWLGRT Y YRS R W YND Y A V S VRS RIIINPDTS RN QF S LQLN S VTPEDT A V Y Y C ARDPGGPLDD S FDIWGQG TM VT V S S AS TRGPS VFPLAPS S RS TS GGT A ALGCLVRD YFPEP VT VS WN S GALT S G VHT FP A VLQS S GLY S LS S V VT VPS S S LGTQT YICN VNHRPS NTRVDRRVEPRS C (SEQ ID NO://)

[00188] 2G10 V_L:

LD V VMTQS PLS LP VTPGEP AS IS CRS S QS LLRS N G YN YLD W YLQRPGQS PQLLIYLGS IRA S G VPDRF S GS GS GTDFTLRIS RVE AED VG V Y YCMQ ALQTPFTFGQGTRLEIRRT V A APS V FIFPPS DEQLKS GT AS V VCLLNNF YPRE AKV QWKVDN ALQS GN S QES VTEQDS KDS T Y S LS S TLTLS KAD YEKHKV Y ACE VTHQGLS S P VTKS FNRGEC (SEQ ID NO://)

[00189] 3C6 V_H:

Q V QLQQW G AGLLKPS ETLS LTC A V Y GGS FS G Y YWS WIRQPPGKGLEWIGEINHS GS TN YNPS LKS RVTIS VDT S KN QF S LKLS S VT A ADT A V Y Y C ARGRRFGDFD YW GQGTLVT V S S AS TKGPS VFPLAPS S KS TS GGT A ALGCLVKD YFPEP VT VS WN S GALT S G VHTFP A VLQS S GLY S LS S V VT VPS S S LGT QT YICN VNHKPS NTKVDKKVEPKS C (SEQ ID NO://)

[00190] 3C6 V_L:

QP VLTQPPS VS V APGKT ARITC GGNNIGS KS VHW Y QQKPGQ AP VLV V YDDS DRPPGIPE RFS GS NS GNT ATLTIS RVE AGDE AD Y Y CQ V WDS S S DHS PFGT GTK VT VLGQPKANPT VT LFPPS S EELQ ANKATL VCLIS DF YPG A VT V A WKADGS P VKAG VETTKPS KQS NNKY A AS S YLS LTPEQWKS HRS Y S CQ VTHEGS T VEKT V APTEC S (SEQ ID NO://)

Table 2: Complementarity determining regions of 3C6 and 2G10 according to the Kabat database.

Nucleic acid

[00191] The present disclosure contemplates cells expressing a uPAR antibody as disclosed herein, e.g., by expression of heavy and light chain-encoding, or heavy and light chain fragment encoding, expression cassettes. Examples of encoding nucleic acids include a nucleic acid encoding a polypeptide comprising one or more CDRs at least about 85%, 90%, 95%, 98%, 99%, or 100% identical to those CDRs disclosed herein. In another example, the antibody has one or more light and heavy chain complementarity determining region (CDR) polypeptide sequences at least about 85%, 90%, 95%, 98%, 99%, or 100% identical to those light and heavy chain CDR polypeptide sequences disclosed herein.

[00192] Examples of nucleic acid sequence encoding a heavy chain of an antibody that binds to uPAR includes that of 2G10 and of 3C6. Example of nucleic acid sequence encoding a light chain of an antibody that binds to uPAR includes that of 2G10 and 3C6. The disclosure further contemplates recombinant host cells containing an exogenous polynucleotide encoding at least a CDR of a heavy chain polypeptide or at least a CDR of a light chain polypeptide of the subject antibody.

[00193] Wherein the subject agents are encoded by a nucleic acid (e.g., to produce a recombinant antibody), the nucleic acid can comprise a contiguous nucleic acid sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or 100%) to a contiguous sequence of any sequences listed below.

[00194] 2G10 V_H:

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTC

ACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTG

GATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGT

CCAAGTGGTATAATGATTATGCAGTATCCGTGAAAAGTCGAATAATTATCAACCCAG

ACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACACGG

CTGTGTATTACTGTGCAAGAGATCCGGGGGGGCCTCTCGATGATAGTTTTGATATCT

GGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTC

TTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGC

CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG

ACCAGCGGCGTCCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC

AGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA

CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT

GT (SEQ ID NO://)

[00195] 2G10 V_L:

CTTGATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCG

GCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCGTAGTAATGGATACAACTAT

TTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGT TCTATTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCGGGCACAGAT

TTTACACTGAGAATTAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATG

CAAGCTCTACAAACCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAGCG

AACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT

GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA

CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA

GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG

CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC

TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO://)

[00196] 3C6 V_H:

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTC

CCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCG

CCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCA

CCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAG

AACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTAC

TGTGCGAGAGGCAGAAGGTTCGGGGATTTTGACTACTGGGGCCAGGGAACCCTGGT

CACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC

CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC

CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTCCACACC

TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTG

CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG

CAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT (SEQ ID NO://)

[00197] 3C6 V_L:

CAGCCTGTGCTGACTCAGCCCCCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAG

GATTACCTGTGGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGA

AGCCAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCCCAGGG

ATCCCTGAGCGATTCTCTGGCTCCAATTCTGGGAACACGGCCACCCTGACCATCAGC

AGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAG

TGATCACTCCCCCTTCGGAACTGGGACCAAGGTCACCGTCCTAGGTCAGCCCAAGGC

CAACCCCACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAACAAGGC

CACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCCTGGAA GGCAGATGGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAAACCCTCCAAACAG AGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAA GTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGA CAGTGGCCCCTACAGAATGCTCT (SEQ ID NO://)

Recombinant antibody

[00198] The agents of the present disclosure may be an antibody produced by recombinant methods. Such antibodies can be produced by expression of a polynucleotide having a nucleotide sequence encoding a polypeptide that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 95%, at least 98%) to a contiguous sequence of any uPAR antibody disclosed herein and/or of any sequence listed above. The percent identity of nucleic acids is based on the shorter of the sequences compared. Well known programs such as BLASTN (2.0.8) (Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402) using default parameters and no filter may be employed to make a sequence comparison. Examples of nucleic acids encoding the antibodies of the present disclosure are discussed later below.

[00199] Methods for producing recombinant antibodies are known in the art. For example, the nucleic acids encoding the antibody, or at least a CDR of a heavy chain polypeptide or at least a CDR of a light chain polypeptide, are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded antibody. The recombinant antibody may be glycosylated by an endogenous glycosyl-transferase in the host cells, unglycosylated, or may have an altered glycosylation pattern.

[00200] Recombinant antibodies include chimeric antibodies. Chimeric antibodies are immunoglobulin molecules comprising human and non-human portions. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., murine), and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source. The chimeric antibody can have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large number of methods of generating chimeric antibodies are well known to those of skill in the art. An alternative approach is the generation of humanized antibodies by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et ah, Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989).

ANTIBODY-DRUG CONJUGATES

[00201] The present disclosure provides conjugates, e.g., an antibody-drug conjugate

(“ADC”)· By“conjugate” is meant a first moiety (e.g., an antibody, such as an anti-uPAR antibody) is stably associated with a second moiety (e.g., a chemical entity, such as a drug). By “stably associated” is meant that a moiety is bound to another moiety or structure under standard conditions. In certain embodiments, the first and second moieties are bound to each other through one or more covalent bonds. For example, an ADC may include a conjugate where a drug (e.g., a maytansinoid or auristatin active agent moiety) is stably associated with another moiety (e.g., the antibody, such as an anti-uPAR antibody).

[00202] In certain embodiments, the conjugate is a polypeptide conjugate, which includes a polypeptide conjugated to a second moiety. In certain embodiments, the moiety conjugated to the polypeptide can be a chemical entity selected from any of a variety of moieties of interest such as, but not limited to, a detectable label, a drug, a water-soluble polymer, or a moiety for immobilization of the polypeptide to a membrane or a surface.

[00203] In certain embodiments, the conjugate is a maytansine conjugate, where a polypeptide is conjugated to a maytansine or a maytansinoid active agent moiety. “Maytansine”, “maytansine moiety”,“maytansine active agent moiety” and“maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof. A maytansine conjugated to the polypeptide can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein.

[00204] In certain embodiments, the conjugate is an auristatin conjugate, where a polypeptide is conjugated to an auristatin active agent moiety, including analogs and derivatives thereof, and pharmaceutically active auristatin moieties and/or portions thereof.

[00205] The moiety of interest (e.g., chemical entity) can be conjugated to the polypeptide at any desired site of the polypeptide. Thus, the present disclosure provides, for example, a modified polypeptide having a moiety conjugated at a site at or near the C-terminus of the polypeptide. Other examples include a modified polypeptide having a moiety conjugated at a position at or near the N-terminus of the polypeptide. Examples also include a modified polypeptide 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). Combinations of the above are also possible where the modified polypeptide is conjugated to two or more moieties.

[00206] In certain embodiments, a conjugate of the present disclosure includes a drug conjugated to an amino acid reside of a polypeptide at the a-carbon of an amino acid residue. Stated another way, a drug conjugate includes a polypeptide where the side chain of one or more amino acid residues in the polypeptide have been modified to be attached to a chemical entity (e.g., attached to a drug through a linker as described herein). For example, a drug conjugate includes a polypeptide where the a-carbon of one or more amino acid residues in the polypeptide has been modified to be attached to a chemical entity (e.g., attached to a drug through a linker as described herein).

[00207] Embodiments of the present disclosure include conjugates where a polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, or 10 or more moieties. The moieties may be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of the polypeptide. In some cases, one moiety is conjugated to an amino acid residue of the polypeptide. In other embodiments, two moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first moiety is conjugated to a first amino acid residue of the polypeptide and a second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example where a polypeptide is conjugated to a first moiety at a first amino acid residue and conjugated to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, a polypeptide conjugated to first and second moieties at a first amino acid residue and conjugated to third and fourth moieties at a second amino acid residue, etc.

[00208] The one or more amino acid residues of the polypeptide that are conjugated to the one or more moieties may be naturally occurring amino acids, unnatural amino acids, or combinations thereof. For instance, the conjugate may include a moiety conjugated to a naturally occurring amino acid residue of the polypeptide. In other instances, the conjugate may include a moiety conjugated to an unnatural amino acid residue of the polypeptide. One or more moieties may be conjugated to the polypeptide at a single natural or unnatural amino acid residue as described above. One or more natural or unnatural amino acid residues in the polypeptide may be conjugated to the moiety or moieties as described herein. For example, two (or more) amino acid residues (e.g., natural or unnatural amino acid residues) in the polypeptide may each be conjugated to one or two moieties, such that multiple sites in the polypeptide are modified.

[00209] As described herein, a polypeptide may be conjugated to one or more moieties. In certain embodiments, the moiety of interest is a chemical entity, such as a drug or a detectable label. For example, a drug (e.g., a maytansine or an auristatin) may be conjugated to the polypeptide, or in other embodiments, a detectable label may be conjugated to the polypeptide. Thus, for instance, 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 a detectable label; a conjugate of two or more drugs and a polypeptide; a conjugate of two or more detectable labels and a polypeptide; and the like.

[00210] In certain embodiments, the polypeptide and the moiety of interest are conjugated through a coupling moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bonded) to the coupling moiety, thus indirectly binding the polypeptide and the moiety of interest (e.g., a drug, such as a maytansine or an auriststin) together through the coupling moiety. In some cases, the coupling moiety includes a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl compound. For instance, a general scheme for coupling a moiety of interest (e.g., a maytansine or an auriststin) to a polypeptide through a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety is shown in the general reaction scheme below. Hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl coupling moiety are also referred to herein as a hydrazino-Ao-Pictet-Spengler (HIPS) coupling moiety and an aza-hydrazino-Ao-Pictet- Spengler (azaHIPS) coupling moiety, respectively.

(polypeptidq)

[00211] In the reaction scheme above, R is the moiety of interest (e.g., a maytansine or an auriststin) that is conjugated to the polypeptide. As shown in the reaction scheme above, a polypeptide that includes a 2-formylglycine residue (fGly) is reacted with a drug (e.g., a maytansine or an auriststin) that has been modified to include a coupling moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety) to produce a polypeptide conjugate attached to the coupling moiety, thus attaching the drug to the polypeptide through the coupling moiety.

[00212] As described herein, the moiety can be any of a variety of moieties such as, but not limited to, chemical entity, such as a detectable label, or a drug (e.g., a maytansinoid or an auriststin). R’ and R” may each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z may be CR , NR , N, O or S, where R and R are each independently selected from any of the substituents described for R’ and R” above.

[00213] Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moieties may be modified to be attached (e.g., covalently attached) to a linker. As such, embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety attached to a drug (e.g., a maytansine or an auriststin) through a linker. Various embodiments of the linker that may couple the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety to the drug (e.g., a maytansine or an auriststin) are described in detail herein.

[00214] In certain embodiments, the polypeptide may be conjugated to a moiety of interest, where the polypeptide is modified before conjugation to the moiety of interest.

Modification of the polypeptide may produce a modified polypeptide that contains one or more reactive groups suitable for conjugation to the moiety of interest. In some cases, the polypeptide may be modified at one or more amino acid residues to provide one or more reactive groups suitable for conjugation to the moiety of interest (e.g., a moiety that includes a coupling moiety, such as a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety as described above). For example, the polypeptide may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be included in an“aldehyde tag” or“ald- tag”, which as used herein refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C/S)TPSR) that has been converted by action of a formylglycine generating enzyme (FGE) to contain a 2-formylglycine residue (referred to herein as“FGly”). The FGly residue generated by an FGE may also be referred to as a“formylglycine”. Stated differently, the term“aldehyde tag” is used herein to refer to an amino acid sequence that includes a“converted” sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to FGly by action of an FGE, e.g., L(FGly)TPSR). A converted sulfatase motif may be derived from an amino acid sequence that includes an“unconverted” sulfatase motif (i.e., a sulfatase motif in which the cysteine or serine residue has not been converted to FGly by an FGE, but is capable of being converted, e.g., an unconverted sulfatase motif with the sequence: L(C/S)TPSR). By

“conversion” as used in the context of action of a formylglycine generating enzyme (FGE) on a sulfatase motif refers to biochemical modification of a cysteine or serine residue in a sulfatase motif to a formylglycine (FGly) residue (e.g., Cys to FGly, or Ser to FGly). Additional aspects of aldehyde tags and uses thereof in site-specific protein modification are described in U.S. Patent No. 7,985,783 and U.S. Patent No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

[00215] In some cases, the modified polypeptide containing the FGly residue may be conjugated to the moiety of interest by reaction of the FGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety, as described above). For example, an FGly-containing polypeptide may be contacted with a reactive partner- containing drug under conditions suitable to provide for conjugation of the drug to the polypeptide. In some instances, the reactive partner-containing drug may include a hydrazinyl- indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety as described above. For example, a drug (e.g., a maytansine or an auriststin) may be modified to include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety. In some cases, the drug is attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl, such as covalently attached to a

hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl through a linker, as described in detail herein.

[00216] In certain embodiments, a conjugate of the present disclosure includes a polypeptide (e.g., an antibody, such as an anti-uPAR antibody) having at least one modified amino acid residue. The modified amino acid residue of the polypeptide may be coupled to a drug (e.g., a maytansine or an auriststin) containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl coupling moiety as described above. In certain embodiments, the modified amino acid residue of the polypeptide (e.g., anti-uPAR antibody) may be derived from a cysteine or serine residue that has been converted to an FGly residue as described above. In certain embodiments, the FGly residue is conjugated to a drug containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl coupling moiety as described above to provide a conjugate of the present disclosure where the drug is conjugated to the polypeptide through the hydrazinyl-indolyl or hydrazinyl- pyrrolo-pyridinyl coupling moiety. As used herein, the term FGly’ refers to the modified amino acid residue of the polypeptide (e.g., anti- uPAR antibody) that is coupled to the moiety of interest (e.g., a drug, such as a maytansinoid or an auriststin).

[00217] In certain embodiments, the conjugate includes at least one modified amino acid residue of the formula (I) described herein. For instance, the conjugate may include at least one modified amino acid residue with a side chain of the formula (I):

wherein

Z is CR⁴ or N;

R¹ 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;

R 2 and R 3 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, acyl amino, amino acyl, 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; each R⁴ 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker comprising -(T¹-V¹)_a-(T²-V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e- , wherein a, b, c, d and e are each independently 0 or 1, where the sum of a, b, c, d and e is 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from (Ci-Ci₂)alkyl, substituted (Ci- C_l2)alkyl, (EDA)_W, (PEG)_n, (AA)_P, -(CR¹³OH)_h-, piperidin-4-amino (4AP), para-amino- benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol or a modified polyethylene glycol, and AA is an amino acid residue, 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;

V , V , V , V and V are each independently selected from the group consisting of a covalent bond, -CO-, -NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H₄)-, -CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, -OC(O)-, -O- , -S-, -S(O)-, -S0₂-, -S0₂NR¹⁵-, -NR¹⁵S0₂- and -P(0)0H-, wherein q is an integer from 1 to 6; each R is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl;

each R¹⁵ 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;

W¹ is a chemical entity; and

W is an anti-uP AR antibody.

[00218] In certain embodiments, Z is CR⁴ or N. In certain embodiments, Z is CR⁴. In certain embodiments, Z is N. [00219] In certain embodiments, R¹ 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, R¹ is hydrogen. In certain embodiments, R¹ is alkyl or substituted alkyl, such as C_l-6 alkyl or C_l-6 substituted alkyl, or Ci_₄ alkyl or CM substituted alkyl, or C_l-3 alkyl or C_l-3 substituted alkyl. In certain embodiments, R¹ 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, R¹ is alkynyl or substituted alkynyl, 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, R¹ is aryl or substituted aryl, such as Cs_₈ aryl or Cs_₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ substituted aryl. In certain embodiments, R¹ is heteroaryl or substituted heteroaryl, such as C_5-8 heteroaryl or C_5-8 substituted heteroaryl, such as a C₅ heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments,

R¹ is cycloalkyl or substituted cycloalkyl, such as C₃_s cycloalkyl or C₃_s substituted cycloalkyl, such as a C_3-6 cycloalkyl or C_3-6 substituted cycloalkyl, or a C₃_s cycloalkyl or C₃_s substituted cycloalkyl. In certain embodiments, R¹ is heterocyclyl or substituted heterocyclyl, such as C₃_s heterocyclyl or C₃_s substituted heterocyclyl, such as a C_3-6 heterocyclyl or C_3-6 substituted heterocyclyl, or a C₃_s heterocyclyl or C₃_s substituted heterocyclyl.

[00220] In certain embodiments, R 2 and R 3 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, acyl amino, amino acyl, 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.

2

[00221] 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 2 is hydrogen. In certain embodiments, R 2 is alkyl or substituted alkyl, such as C_l-6 alkyl or C_l-6 substituted alkyl, or Ci_₄ alkyl or CM substituted alkyl, or Ci_₃ alkyl or Ci_₃ substituted alkyl. In certain embodiments, R 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. In certain embodiments, R is alkoxy or substituted alkoxy. In certain embodiments, R 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acyl amino or amino acyl. In certain embodiments, R is alkylamide or substituted alkylamide.

In certain embodiments, R 2 is sulfonyl. In certain embodiments, R 2 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R is aryl or substituted aryl, such as Cs_₈ aryl or Cs_₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ 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 a C₅ heteroaryl or C₅ substituted heteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as C_3-8 cycloalkyl or C₃_s substituted cycloalkyl, such as a C_3-6 cycloalkyl or C_3-6 substituted cycloalkyl, or a C₃_s cycloalkyl or C₃_s substituted cycloalkyl. In certain embodiments, R is heterocyclyl or substituted heterocyclyl, such as a C_3-6 heterocyclyl or C_3-6 substituted heterocyclyl, or a C₃_s heterocyclyl or C₃_s substituted heterocyclyl.

[00222] 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R is hydrogen. In certain embodiments, R is alkyl or substituted alkyl, such as C_l-6 alkyl or C_l-6 substituted alkyl, or Ci_₄ alkyl or CM substituted alkyl, or Ci_₃ alkyl or Ci_₃ substituted alkyl. In certain embodiments, R 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. In certain embodiments, R is alkoxy or substituted alkoxy. In certain embodiments, R 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acyl amino or amino acyl. In certain embodiments, R is alkylamide or substituted alkylamide.

In certain embodiments, R 3 is sulfonyl. In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R is aryl or substituted aryl, such as Cs_₈ aryl or Cs_₈ substituted aryl, such as a C₅ aryl or C₅ substituted aryl, or a C₆ aryl or C₆ 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 a C₅ heteroaryl or C₅ substituted heteroaryl, or a 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 a C_3-6 cycloalkyl or C_3-6 substituted cycloalkyl, or a C_3-5 cycloalkyl or C_3-5 substituted cycloalkyl. In certain embodiments, R is heterocyclyl or substituted heterocyclyl, such as C₃_s heterocyclyl or C₃_s substituted

heterocyclyl, such as a C_3-6 heterocyclyl or C _-6 substituted heterocyclyl, or a C₃_s heterocyclyl or C_3-5 substituted heterocyclyl.

[00223] In certain embodiments, R and R are optionally cyclically linked to form a 5 or

6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5 or

6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5- membered heterocyclyl. In certain embodiments, R and R are cyclically linked to form a 6- membered heterocyclyl.

[00224] In certain embodiments, each R⁴ 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[00225] The various possibilities for each R⁴ are described in more detail as follows. In certain embodiments, R⁴ is hydrogen. In certain embodiments, each R⁴ is hydrogen. In certain embodiments, R⁴ is halogen, such as F, Cl, Br or I. In certain embodiments, R⁴ is F. In certain embodiments, R⁴ is Cl. In certain embodiments, R⁴ is Br. In certain embodiments, R⁴ is I. In certain embodiments, R⁴ is alkyl or substituted alkyl, such as Ci_₆ alkyl or Ci_₆ substituted alkyl, or C_1-4 alkyl or Ci_₄ substituted alkyl, or C_1-3 alkyl or C_1-3 substituted alkyl. In certain

embodiments, R⁴ 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, R⁴ is alkynyl or substituted alkynyl. In certain embodiments, R⁴ is alkoxy or substituted alkoxy. In certain embodiments, R⁴ is amino or substituted amino. In certain embodiments, R⁴ is carboxyl or carboxyl ester. In certain embodiments, R⁴ is acyl or acyloxy.

In certain embodiments, R⁴ is acyl amino or amino acyl. In certain embodiments, R⁴ is alkylamide or substituted alkylamide. In certain embodiments, R⁴ is sulfonyl. In certain embodiments, R⁴ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R⁴ is aryl or substituted aryl, such as Cs_₈ aryl or Cs_₈ substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C₆ aryl or C₆ 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 a C5 heteroaryl or C5 substituted heteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R⁴ is cycloalkyl or substituted cycloalkyl, such as C₃_s cycloalkyl or C₃_s substituted cycloalkyl, such as a C_3-6 cycloalkyl or C_3-6 substituted cycloalkyl, or a C₃_5 cycloalkyl or C₃_s substituted cycloalkyl. In certain embodiments, R⁴ is heterocyclyl or substituted heterocyclyl, such as C₃_s heterocyclyl or C₃_s substituted heterocyclyl, such as a C_3-6 heterocyclyl or C_3-6 substituted heterocyclyl, or a C₃_s heterocyclyl or C₃_s substituted

heterocyclyl.

[00226] In certain embodiments, W¹ is a chemical entity, such as a drug or a detectable label. In certain embodiments, W¹ is a drug. In certain embodiments, W¹ is a detectable label.

[00227] In certain embodiments, W¹ is a drug, such as a maytansinoid or an auristatin. In certain embodiments, W¹ is a maytansinoid. In certain embodiments, W¹ is an auristatin.

Further description of maytansinoids is found in the disclosure herein. Further description of auriststins is found in the disclosure herein.

2

[00228] In certain embodiments, W is an anti-uPAR antibody. Further description of the anti-uPAR antibody is found in the disclosure herein.

[00229] In certain embodiments, the compounds of formula (I) include a linker, L. The linker may be utilized to bind a coupling moiety to one or more moieties of interest and/or one or more polypeptides. In some embodiments, the linker binds a coupling moiety to either a polypeptide or a chemical entity. The linker may be bound (e.g., covalently bonded) to the coupling moiety (e.g., as described herein) at any convenient position. For example, the linker may attach a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety to a drug (e.g., a maytansine or an auriststin). The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl coupling moiety may be used to conjugate the linker (and thus the drug, e.g., a maytansine or an auriststin) to a polypeptide, such as an anti-uPAR antibody.

[00230] In certain embodiments, L attaches the coupling moiety to W¹, and thus the coupling moiety is indirectly bonded to W¹ through the linker L. As described above, W¹ can be a drug, and thus L attaches the coupling moiety to a drug, e.g., the coupling moiety is indirectly bonded to the drug through the linker, L, thus producing an ADC.

[00231] Any convenient linkers may be utilized in the subject conjugates and compounds.

In certain embodiments, L includes a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, L includes an alkyl or substituted alkyl group. In certain embodiments, L includes an alkenyl or substituted alkenyl group. In certain embodiments, L includes an alkynyl or substituted alkynyl group. In certain embodiments, L includes an alkoxy or substituted alkoxy group. In certain embodiments, L includes an amino or substituted amino group. In certain embodiments, L includes a carboxyl or carboxyl ester group. In certain embodiments, L includes an acyl amino group. In certain embodiments, L includes an alkylamide or substituted alkylamide group. In certain embodiments, L includes an aryl or substituted aryl group. In certain embodiments, L includes a heteroaryl or substituted heteroaryl group. In certain embodiments, L includes a cycloalkyl or substituted cycloalkyl group. In certain embodiments, L includes a heterocyclyl or substituted heterocyclyl group.

[00232] In certain embodiments, L includes a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol,

methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where the

homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ethers, polyvinylpyrrolidone, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is a polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below. [00233] In some embodiments, L is a linker described by the formula -(L 1 )_a-(L2 )_b-(L 3 )_c- (L⁴)_d-(L⁵)_e-, wherein L¹, L² , L³, L⁴ and L⁵ are each independently a linker unit, and a, b, c, d and e are each independently 0 or 1, wherein the sum of a, b, c, d and e is 1 to 5.

[00234] In certain embodiments, the sum of a, b, c, d and e is 1. In certain embodiments, the sum of a, b, c, d and e is 2. In certain embodiments, the sum of a, b, c, d and e is 3. In certain embodiments, the sum of a, b, c, d and e is 4. In certain embodiments, the sum of a, b, c, d and e is 5. In certain embodiments, a, b, c, d and e are each 1. In certain embodiments, a, b, c and d are each 1 and e is 0. In certain embodiments, a, b and c are each 1 and d and e are each 0. In certain embodiments, a and b are each 1 and c, d and e are each 0. In certain embodiments, a is 1 and b, c, d and e are each 0.

[00235] In certain embodiments, L¹ is attached to the hydrazinyl-indolyl or the hydrazinyl- pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain

embodiments, L , if present, is attached to W . In certain embodiments, L , if present, is attached to W¹. In certain embodiments, L⁴, if present, is attached to W¹. In certain

embodiments, L⁵, if present, is attached to W¹.

[00236] Any convenient linker units may be utilized in the subject linkers. Linker units of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, cleavable linker groups, combinations thereof, and substituted versions thereof. In some embodiments, each of L , L , L , L and L (if present) comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine), and a cleavable moiety (e.g., a chemically cleavable moiety, an enzymatically cleavable moiety (such as, but not limited to, a protease cleavable moiety, a glucuronidase cleavable moiety, a beta-lactamase cleavable moiety, etc.), a photocleavable moiety, and the like).

[00237] In some embodiments, L¹ (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, a diamine, or a cleavable moiety. In some embodiments, L¹ comprises a polyethylene glycol. In some embodiments, L¹ comprises a modified polyethylene glycol. In some embodiments, L¹ comprises an amino acid residue. In some embodiments, L¹ comprises an alkyl group or a substituted alkyl. In some embodiments, L¹ comprises an aryl group or a substituted aryl group. In some embodiments, L¹ comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L¹ comprises a cleavable moiety.

2

[00238] In some embodiments, L (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, a diamine, or a cleavable moiety. In some embodiments, L comprises a polyethylene glycol. In some embodiments, L comprises a modified polyethylene glycol. In

2 2

some embodiments, L comprises an amino acid residue. In some embodiments, L comprises an alkyl group or a substituted alkyl. In some embodiments, L comprises an aryl group or a substituted aryl group. In some embodiments, L comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L comprises a cleavable moiety.

[00239]

[00240] In some embodiments, L (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, a diamine, or a cleavable moiety. In some embodiments, L comprises a polyethylene glycol. In some embodiments, L comprises a modified polyethylene glycol. In some embodiments, L 3 comprises an amino acid residue. In some embodiments, L 3 comprises an alkyl group or a substituted alkyl. In some embodiments, L comprises an aryl group or a substituted aryl group. In some embodiments, L comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L comprises a cleavable moiety.

[00241]

[00242] In some embodiments, L⁴ (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, a diamine, or a cleavable moiety. In some embodiments, L⁴ comprises a polyethylene glycol. In some embodiments, L⁴ comprises a modified polyethylene glycol. In some embodiments, L⁴ comprises an amino acid residue. In some embodiments, L⁴ comprises an alkyl group or a substituted alkyl. In some embodiments, L⁴ comprises an aryl group or a substituted aryl group. In some embodiments, L⁴ comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L⁴ comprises a cleavable moiety.

[00243] [00244] In some embodiments, L⁵ (if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, a diamine, or a cleavable moiety. In some embodiments, L⁵ comprises a polyethylene glycol. In some embodiments, L⁵ comprises a modified polyethylene glycol. In some embodiments, L⁵ comprises an amino acid residue. In some embodiments, L⁵ comprises an alkyl group or a substituted alkyl. In some embodiments, L⁵ comprises an aryl group or a substituted aryl group. In some embodiments, L⁵ comprises a diamine (e.g., a linking group comprising an alkylene diamine). In some embodiments, L⁵ comprises a cleavable moiety.

[00245] Any convenient cleavable moieties may be utilized as a cleavable linker unit in the subject conjugates and compunds. In certain embodiments, the cleavable moiety is a para- amino-benzyloxycarbonyl group (PABC), a meta-amino-benzyloxycarbonyl group (MABC), a para-amino-benzyloxy group (PABO), a meta-amino-benzyloxy group (MABO), para- aminobenzyl (PAB), an acetal group, a disulfide, a hydrazine, a protease-cleavable moiety (e.g., a Cat B cleavable moiety), a glucuronidase cleavable moiety, a beta-lactamase cleavable moiety, or an ester.

[00246] In some embodiments, L is a linker comprising -(L 1 )_a-(L 2 3 4 5

)b-(L )_C-(L )d-(L )_e-, where:

wherein T¹, T², T³, T⁴ and T⁵, if present, are tether groups;

V 1 , V 2 , V 3 , V 4 and V 5 , if present, are covalent bonds or linking functional groups; and a, b, c, d and e are each independently 0 or 1, wherein the sum of a, b, c, d and e is 1 to 5.

[00247] As described above, in certain embodiments, L¹ is attached to the hydrazinyl- indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). As such, in certain embodiments, T¹ is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, V is attached to W (the maytansinoid). In certain embodiments, L , if present, is attached to W 1. As such, in certain embodiments, T 2 , if present, is attached to W 1 , or V2 , if present, is attached to W 1. In certain embodiments, L3 , if present, is attached to W 1. As such, in certain embodiments, T , if present, is attached to W , or V , if present, is attached to W . In certain embodiments, L⁴, if present, is attached to W¹. As such, in certain embodiments, T⁴, if present, is attached to W¹, or V⁴, if present, is attached to W¹. In certain embodiments, L⁵, if present, is attached to W¹. As such, in certain embodiments, T⁵, if present, is attached to W¹, or V⁵, if present, is attached to W¹.

[00248] Regarding the tether groups, T¹, T², T³, T⁴ and T⁵, any convenient tether groups may be utilized in the subject linkers. In some embodiments, T , T , T , T and T each comprise one or more groups independently selected from a (Ci-Ci₂)alkyl, a substituted (Ci-Ci₂)alkyl, an (EDA)_W, (PEG)_n, (AA)_P, -(CR OH)_h-, piperidin-4-amino (4AP), para-amino-benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta- amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a disulfide, a hydrazine, and an ester, where 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.

[00249] In certain embodiments, when the sum of a, b, c, d and e is 2 and one of T^-V¹, T²-V², T³-V³, T⁴-V⁴ or T⁵-V⁵ is (PEG)_n-CO, then n is not 6. For example, in some instances, the linker may have the following structure:

where n is not 6.

[00250] In certain embodiments, when the sum of a, b, c, d and e is 2 and one of T^-V¹, T²-V², T³-V³ T⁴-V⁴ or T⁵-V⁵ is (Ci-Ci₂)alkyl-NR¹⁵, then (Ci-Ci₂)alkyl is not a C₅-alkyl. For example, in some instances, the linker may have the following structure:

where g is not 4.

[00251] In certain embodiments, the tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a (Ci-Ci₂)alkyl or a substituted (Ci-Ci₂)alkyl. In certain embodiments, (Ci-Ci₂)alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, (Ci-Ci₂)alkyl may be an alkyl or substituted alkyl, such as C₁-C₁₂ alkyl, or C₁-C₁₀ alkyl, or C ₁ -C₆ alkyl, or C₁-C₃ alkyl. In some instances, (Ci-Ci₂)alkyl is a C₂-alkyl. For example, (Ci-Ci₂)alkyl may be an alkylene or substituted alkylene, such as Ci-Ci₂ alkylene, or C₁-C₁₀ alkylene, or C _|-C₆ alkylene, or C₁-C₃ alkylene. In some instances, (Ci-Ci₂)alkyl is a C₂-alkylene.

[00252] In certain embodiments, substituted (Ci-Ci₂)alkyl is a straight chain or branched substituted alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, substituted (Ci-Ci₂)alkyl may be a substituted alkyl, such as substituted Ci-Ci₂ alkyl, or substituted C₁-C₁₀ alkyl, or substituted C ₁ -C₆ alkyl, or substituted C₁-C₃ alkyl. In some instances, substituted (Ci-Ci₂)alkyl is a substituted C₂-alkyl. For example, substituted (Ci-Ci₂)alkyl may be a substituted alkylene, such as substituted Ci-Ci₂ alkylene, or substituted C₁-C₁₀ alkylene, or substituted C ₁ -C₆ alkylene, or substituted C₁-C₃ alkylene. In some instances, substituted (Ci-Ci₂)alkyl is a substituted C₂-alkylene.

[00253] In certain embodiments, the tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether. In certain embodiments, (EDA)_W includes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5 or 6). The linked ethylene diamine (EDA) moieties may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the EDA moiety is described by the structure:

where y is an integer from 1 to 6, r is 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5 or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl. In certain embodiments, any two adjacent R groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R groups are an alkyl group, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

[00254] In certain embodiments, the tether group includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidin-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl or a substituted aryl. In certain embodiments, the 4AP moiety is described by the structure:

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

[00255] In certain embodiments, R 12 includes a polyethylene glycol moiety described by the formula: (PEG)_k , which may be represented by the structure:

where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, k is 2. In certain embodiments, R 17 is selected from OH, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R is COOH.

[00256] In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes (PEG)_n, where (PEG)_n is a polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG)_n is described by the structure:

where n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, n is 2. In some instances, n is 3. In some instances, n is 6. In some instances, n is 12.

[00257] In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes (AA)_P, where AA is an amino acid residue. Any convenient amino acids may be utilized. Amino acids of interest include but are not limited to, L- and D-amino acids, naturally occurring amino acids such as any of the 20 primary alpha-amino acids and beta-alanine, non-naturally occurring amino acids (e.g., amino acid analogs), such as a non-naturally occurring alpha-amino acid or a non-naturally occurring beta-amino acid, etc. In certain embodiments, p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

[00258] In certain embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a moiety described by the formula -(CR OH)_h-, where h is 0 or n is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R is hydrogen. In certain embodiments, R is alkyl or substituted alkyl, such as C_l-6 alkyl or C_l-6 substituted alkyl, or Ci_₄ alkyl or C_M substituted alkyl, or C_l-3 alkyl or C_l-3 substituted alkyl. In certain embodiments, R 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. 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 acyl amino or amino acyl. 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 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C₆ aryl or C₆ substituted aryl. In certain embodiments, R is heteroaryl or substituted heteroaryl, such as Cs_8 heteroaryl or Cs_8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R is cycloalkyl or substituted cycloalkyl, such as C₃_8 cycloalkyl or C₃_s substituted cycloalkyl, such as a C_3-6 cycloalkyl or C_3-6 substituted cycloalkyl, or a C₃_s cycloalkyl or C₃_s substituted cycloalkyl. In certain embodiments, R is heterocyclyl or substituted heterocyclyl, such as C₃_s heterocyclyl or C₃_s substituted

heterocyclyl, such as a C_3-6 heterocyclyl or C_3-6 substituted heterocyclyl, or a C₃_s heterocyclyl or C₃_5 substituted heterocyclyl.

[00259] In certain embodiments, R 13 is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13.

[00260] Regarding the linking functional groups, V , V , V , V and V , any convenient linking functional groups may be utilized in the subject linkers. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate,

thiophosphoraidate, and the like. In some embodiments, V , V , V , V and V are each independently selected from a covalent bond, -CO-, -NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H₄)-, - CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, -0C(0)-, -0-, -S-, -S(O)-, -S0₂-, -S0₂NR¹⁵-, -NR¹⁵S0₂- and - P(0)OH-, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. [00261] In some embodiments, each R¹⁵ 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl,

heterocyclyl, and substituted heterocyclyl.

[00262] The various possibilities for each R¹⁵ are described in more detail as follows. In certain embodiments, R¹⁵ is hydrogen. In certain embodiments, each R¹⁵ is hydrogen. In certain embodiments, R¹⁵ is alkyl or substituted alkyl, such as C_l-6 alkyl or C_l-6 substituted alkyl, or C_M alkyl or C_M substituted alkyl, or C_l-3 alkyl or C_l-3 substituted alkyl. In certain embodiments, R¹⁵ is alkenyl or substituted alkenyl, such as C_2-6 alkenyl or C_2-6 substituted alkenyl, or C_2-4 alkenyl or C₂_4 substituted alkenyl, or C_2-3 alkenyl or C_2-3 substituted alkenyl. In certain embodiments, R¹⁵ is alkynyl or substituted alkynyl. In certain embodiments, R¹⁵ is alkoxy or substituted alkoxy. In certain embodiments, R¹⁵ is amino or substituted amino. In certain embodiments, R¹⁵ is carboxyl or carboxyl ester. In certain embodiments, R¹⁵ is acyl or acyloxy. In certain embodiments, R¹⁵ is acyl amino or amino acyl. In certain embodiments, R¹⁵ is alkylamide or substituted alkylamide. In certain embodiments, R¹⁵ is sulfonyl. In certain embodiments, R¹⁵ is thioalkoxy or substituted thioalkoxy. In certain embodiments, R¹⁵ is aryl or substituted aryl, such as C5_8 aryl or Cs_₈ substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C₆ aryl or C₆ substituted aryl. In certain embodiments, R¹⁵ is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C₆ heteroaryl or C₆ substituted heteroaryl. In certain embodiments, R¹⁵ is cycloalkyl or substituted cycloalkyl, such as C₃_s cycloalkyl or C₃_s substituted cycloalkyl, such as a C_3-6 cycloalkyl or C_3-6 substituted cycloalkyl, or a C₃_s cycloalkyl or C₃_s substituted cycloalkyl. In certain embodiments, R¹⁵ is heterocyclyl or substituted heterocyclyl, such as C₃_s heterocyclyl or C₃_8 substituted heterocyclyl, such as a C_3-6 heterocyclyl or C_3-6 substituted heterocyclyl, or a C₃_s heterocyclyl or C₃_s substituted heterocyclyl.

[00263] In certain embodiments, each R¹⁵ 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, the 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 R¹⁵.

[00264] In some embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a MABC group described by the following structure:

[00265] In some embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a MABO group described by the following structure:

[00266] In some embodiments of the MABO and MABC tether structures shown above, R is selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some embodiments of the MABO and MABC tether structures shown above, R is a carbohydrate or carbohydrate derivative.

[00267] In some embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a PABC group described by the following structure:

[00268] In some embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a

PABO group described by the following structure:

[00269] In some embodiments, a tether group (e.g., T¹, T², T³, T⁴ and/or T⁵) includes a para-aminobenzyl (PAB) group described by the following structure:

[00270] In some embodiments of the PABO, PABC, MABO, MABC and PAB tether structures shown above, 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments of PABO, PABC, MABO, MABC and PAB, R¹²is selected from hydrogen, an alkyl, a substituted alkyl, an aryl and a substituted aryl. In certain

embodiments of PABO, PABC, MABO, MABC and PAB, R¹² is selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl- OH). In some embodiments, any of the PABO, PABC, MABO, MABC and PAB tether structures shown above may be further substituted with one or more convenient aryl and/or alkyl substituents. In certain embodiments of PABO, PABC, MABO, MABC and PAB, R¹² is hydrogen. The divalent PABO, PABC, MABO, MABC and PAB tether groups may be covalently bound to adjacent moieties via any convenient chemistries.

[00271] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, a glucuronidase cleavable moiety, a beta-lactamase cleavable moiety, or an ester. In some embodiments, the tether group is an acetal group. In some embodiments, the tether group is a disulfide. In some embodiments, the tether group is a hydrazine. In some embodiments, the tether group is a glucuronidase cleavable moiety. In some embodiments, the tether group is a beta-lactamase cleavable moiety. In some embodiments, the tether group is an ester.

[00272] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes an ester.

[00273] As described above, in some embodiments, L is a linker comprising -(T 1 -V1 )_a-(T 2 - V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e-, where a, b, c, d and e are each independently 0 or 1, where the sum of a, b, c, d and e is 1 to 5.

[00274] In some embodiments, in the subject linker:

T¹ is selected from a (Ci-Ci₂)alkyl and a substituted (Ci-Ci₂)alkyl;

T², T³, T⁴ and T⁵ are each independently selected from (Ci-Ci₂)alkyl, substituted ( - C_l2)alkyl, (EDA)_W, (PEG)_n, (AA)_P, -(CR¹³OH)_h-, 4-amino-piperidine (4AP), para-amino- benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a disulfide, a hydrazine, and an ester; and

V , V , V , V and V are each independently selected from a covalent bond, -CO-, - NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H₄)-, -CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, -0C(0)-, -0-, -S-, -S(O)-, - S0₂-, -S0₂NR¹⁵-, -NR¹⁵S0₂- and -P(0)OH-, wherein q is an integer from 1 to 6;

wherein:

integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

4-amino-piperidine

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

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

R is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl.

[00275] In certain embodiments, T¹, T², T³, T⁴ and T⁵ and V¹, V², V³, V⁴ and V⁵ are selected from the following table, e.g., one row of the following table:

[00276] In some embodiments, L is a linker comprising -(L 1 )_a-(L 2 )_b-(L 3 )_C-(L 4 )_d-(L 5 )_e,

[00277] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00278] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00279] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is absent, T³ is absent , V³ is absent , T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00280] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - NR¹¹-, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00281] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -NR¹¹-, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00282] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (EDA)_W, V² is -CO- , T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00283] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (Ci-Ci₂)alkyl, V² is -NR¹¹-, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00284] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (EDA)_W, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00285] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (EDA)_W, V² is absent, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00286] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (EDA)_W, V² is -CO- , T³ is (CR¹³OH)_h, V³ is -CONR¹¹-, T⁴ is (Ci-Ci₂)alkyl, V⁴ is -CO-, T⁵ is absent and V⁵ is absent.

[00287] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00288] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (AA)_P, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00289] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (EDA)_W, V² is -CO- , T³ is (CR¹³OH)_b, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent. [00290] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00291] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00292] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00293] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is (AA)_p-PABC-(AA)_p , where each p is independently 0, 1, 2, 3, 4 , 5 or 6, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00294] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is (AA)_p-PABO where p is 0, 1, 2, 3, 4 , 5 or 6, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00295] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -S0₂-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00296] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (AA)_P, V³ is absent, T⁴ is PABC-(AA)_p where p is 0, 1, 2, 3, 4, 5 or 6, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00297] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (EDA)_W, V² is -CO- , T³ is (CR¹³OH)_h, V³ is -CONR¹¹-, T⁴ is (PEG)_n, V⁴ is -CO-, T⁵ is absent and V⁵ is absent.

[00298] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is MABC-(AA)_P- where p is 0, 1, 2, 3, 4, 5 or 6, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00299] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is MABO, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00300] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is MABO, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00301] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is MABC, V³ is absent, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00302] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (CR¹³OH)_h, V² is - CO-, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent. [00303] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is substituted (Ci-Ci₂)alkyl, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00304] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -S0₂-, T² is (Ci-Ci₂)alkyl, V² is -CO-, T³ is absent, V³ is absent, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00305] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (AA)_P, V³ is absent, T⁴ is PABC, V⁴ is -NR¹¹-, T⁵ is absent and V⁵ is absent.

[00306] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (Ci-Ci₂)alkyl, V² is absent, T³ is (CR¹³OH)_h, V³ is -CONR¹¹-, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00307] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00308] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (AA)_P, V³ is absent, T⁴ is MABO, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00309] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (AA)_P, V³ is absent, T⁴ is PABO, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00310] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CONR¹¹-, T² is (PEG)_n, V² is - CO-, T³ is (AA)_P, V³ is absent, T⁴ is PABC, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00311] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is PABO, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00312] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is PABC, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00313] In some embodiments, L is a linker comprising -(L 1 )_a-(L 2 )_b-(L 3 )_C-(L 4 )_d-(L 5 )_e-, and-

[00314] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is (AA)r, V⁴ is absent, T⁵ is PABC-(AA)p, and V⁵ is absent.

[00315] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)_n, V³ is -CO-, T⁴ is (AA)r, V⁴ is absent, T⁵ is PABO, and V⁵ is absent.

[00316] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (PEG)_n, V² is -CO-, T³ is (AA)r, V³ is absent, T⁴ is PABC, V⁴ is absent, T⁵ is (AA)p, and V⁵ is absent. [00317] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is (AA)_P, V² is -NR¹¹-, T³ is (PEG)n, V³ is -CO-, T⁴ is MABC, V⁴ is absent, T⁵ is (AA)p, and V⁵ is absent.

[00318] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)r, V⁴ is absent, T⁵ is PABO, and V⁵ is -CO-.

[00319] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)r, V⁴ is absent, T⁵ is PABO, and V⁵ is absent.

[00320] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)r, V⁴ is absent, T⁵ is PABC-(AA)p, and V⁵ is absent.

[00321] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00322] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is absent, T³ is (PEG)_n, V³ is -CO-, T⁴ is absent, V⁴ is absent, T⁵ is absent and V⁵ is absent.

[00323] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is absent, T² is (AA)_P, V² is NR¹¹, T³ is (PEG)n, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is PABO and V⁵ is -CO-4AP-.

[00324] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is absent, T² is (AA)_P, V² is NR¹¹, T³ is (PEG)_n, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is PABO and V⁵ is -CO-.

[00325] In certain embodiments, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is absent, T³ is (PEG)n, V³ is -CO-, T⁴ is (AA)_P, V⁴ is absent, T⁵ is PABO and V⁵ is -CO-4AP-.

[00326] In certain embodiments, the linker is described by one of the following structures:

71

73

[00327] In certain embodiments of the linker structures depicted above, each f is independently 0 or an integer from 1 to 12; each y is independently 0 or an integer from 1 to 20; each n is independently 0 or an integer from 1 to 30; each p is independently 0 or an integer from 1 to 20; each h is independently 0 or an integer from 1 to 12; each R is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and each R’ is independently H, a sidechain of an amino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments of the linker structures depicted above, each f is independently 0, 1, 2, 3, 4, 5 or 6; each y is independently 0, 1, 2, 3, 4, 5 or 6; each n is independently 0, 1, 2, 3, 4, 5 or 6; each p is independently 0, 1, 2, 3, 4, 5 or 6; and each h is independently 0, 1, 2, 3, 4, 5 or 6. In certain embodiments of the linker structures depicted above, each R is independently H, methyl or - (CH₂)_m-OH where m is 1, 2, 3 or 4 (e.g., 2).

[00328] In certain embodiments of the linker, L, T¹ is (Ci-Ci₂)alkyl, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is (Ci-Ci₂)alkyl, V³ is -CO-, T⁴ is absent and V⁴ is absent. In certain embodiments, T¹ is ethylene, V¹ is -CO-, T² is 4AP, V² is -CO-, T³ is ethylene, V³ is -CO-, T⁴ is absent and V is absent. In certain embodiments, T is ethylene, V is -CO-, T is 4AP, V is - CO-, T³ is ethylene, V³ is -CO-, T⁴ is absent and V⁴ is absent, where T² (e.g., 4AP) has the following structure:

wherein

R 12 is a polyethylene glycol moiety (e.g., a polyethylene glycol or a modified polyethylene glycol).

[00329] In certain embodiments, the linker, L, includes the following structure:

wherein

each f is independently an integer from 1 to 12; and

n is an integer from 1 to 30.

[00330] In certain embodiments, f is 1. In certain embodiments, f is 2. In certain embodiments, one f is 2 and one f is 1.

[00331] In certain embodiments, n is 1. [00332] In certain embodiments, the left-hand side of the above linker structure is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety, and the right-hand side of the above linker structure is attached to a chemical entity (e.g., a drug).

[00333] Any of the chemical entities, linkers and coupling moieties set forth in the structures above may be adapted for use in the subject compounds and conjugates.

[00334] Additional disclosure related to hydrazinyl-indolyl and hydrazinyl-pyrrolo- pyridinyl compounds and methods for producing a conjugate is found in U.S. Application Publication No. 2014/0141025, filed March 11, 2013, and U.S. Application Publication No. 2015/0157736, filed November 26, 2014, the disclosures of each of which are incorporated herein by reference.

Modified constant region of an anti-uPAR antibody

[00335] As noted above, the amino acid sequence of an anti-uPAR antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to a 2-formylglycine (FGly) residue by action of a formylglycine generating enzyme (FGE) either in vivo (e.g., at the time of translation of an ald tag-containing protein in a cell) or in vitro (e.g., by contacting an ald tag-containing protein with an FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as an FGE-modification site.

[00336] The use of sulfatase motifs to introduce a 2-formylglycine, which can be used a biorthogonal chemical handle for site-specific conjugation, is described in, for example, U.S. Patent No. 7,985,783, the disclosure of which is incorporated herein by reference.

Implementation of sulfatase motifs for site-specific conjugation in the context of antibodies is described in, for example, U.S. Patent No. 9,540,438, the disclosure of which is incorporated herein by reference.

[00337] For example, the CH1 domain of anti-uPAR antibody can be modified to provide a sulfatase motif, e.g., so as to have the amino acid sequence SWNSGALCTPSRGVHTFPA, where“LCTPSR” represents an example of a sulfatase motif. Modification of this site formylglycine generating enzyme (FGE) would convert the cysteine residue to a 2-formyl glycine (“FGly”), thus providing the sequence WNSGAL(FGlv)TPSRGVHTFPA.

[00338] In another example, a C-terminal end of the heavy chain constant region is modified to provide a sulfatase motif. In one example, the C-terminal end of the heavy chain constant region is modified to provide the amino acid sequence SLSLSLGSLCTPSRGS, where “LCTPSR” represents an example of a sulfatase motif. Modification of this site formylglycine generating enzyme (FGE) would convert the cysteine residue to a 2-formyl glycine (“FGly”), thus providing the sequence S LS LS LGS L(FG1 y )TPS RGS .

Sulfatase motifs

[00339] A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 amino acid residues in length, usually no more than 6 amino acid residues in length. Sulfatase motifs provided in an Ig polypeptide are at least 5 or 6 amino acid residues, and can be, for example, from 5 to 16, 6- 16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 amino acid residues in length.

[00340] In certain embodiments, polypeptides of interest include those where one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide for a sequence of a sulfatase motif in the polypeptide. In certain embodiments, the polypeptide includes a modification (insertion, addition, deletion, and/or substitution/replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide. Where an amino acid sequence native to the polypeptide (e.g., anti-uPAR antibody) contains one or more residues of the desired sulfatase motif, the total number of modifications of residues can be reduced, e.g., by site- specification modification (insertion, addition, deletion, substitution/replacement) of amino acid residues flanking the native amino acid residues to provide a sequence of the desired sulfatase motif. In certain embodiments, the extent of modification of the native amino acid sequence of the target anti-uPAR polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target anti-uPAR polypeptide may minimize the impact such modifications may have upon anti-uPAR function and/or structure. [00341] It should be noted that while aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to a“consensus sulfatase motif’), it will be readily appreciated that longer aldehyde tags are both contemplated and encompassed by the present disclosure and can find use in the compositions and methods of the present disclosure. Aldehyde tags can thus comprise a minimal sulfatase motif of 5 or 6 residues, or can be longer and comprise a minimal sulfatase motif which can be flanked at the N- and/or C- terminal sides of the motif by additional amino acid residues. Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.

[00342] An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C- terminus of a native, wild-type Ig heavy chain. An aldehyde tag can be present within a CH1 domain of an Ig heavy chain. An aldehyde tag can be present within a CH2 domain of an Ig heavy chain. An aldehyde tag can be present within a CH3 domain of an Ig heavy chain. An aldehyde tag can be present in an Ig light chain constant region, e.g., in a kappa light chain constant region or a lambda light chain constant region.

[00343] In certain embodiments, the sulfatase motif used may be described by the formula:

where

Z¹⁰ is cysteine or serine (which can also be represented by (C/S));

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

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

X¹ is present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present; and X 2 and X 3 independently can be any amino acid, though usually an aliphatic amino acid, a polar, uncharged amino acid, or a sulfur containing amino acid (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[00344] The amino acid sequence of an anti-uPAR heavy and/or light chain can be modified to provide a sequence of at least 5 amino acids of the formula X 1 Z 10 X 2 Z 20 X 3 Z 30 , where Z¹⁰ is cysteine or serine;

Z²⁰ is a proline or alanine residue;

Z³⁰ is an aliphatic amino acid or a basic amino acid;

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

X and X are each independently any amino acid,

where the sequence is within or adjacent a solvent-accessible loop region of the Ig constant region, and wherein the sequence is not at the C-terminus of the Ig heavy chain.

[00345] The sulfatase motif is generally selected so as to be capable of conversion by a selected FGE, e.g., an FGE present in a host cell in which the aldehyde tagged polypeptide is expressed or an FGE which is to be contacted with the aldehyde tagged polypeptide in a cell-free in vitro method.

[00346] For example, where the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including a human FGE), the sulfatase motif can be of the formula:

X 'CX²PX³Z³⁰ (I”)

where

X^I may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present;

X and X independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V or G; and

Z is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I. [00347] 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 ID NO://), LCAPSA (SEQ ID NO://), LCVPSA (SEQ ID NO://), and LCGPSA (SEQ ID NO://).

FGly-containing sequences

[00348] Upon action of FGE on the modified anti-uPAR heavy and/or light chain, the serine or the cysteine in the sulfatase motif is modified to FGly. Thus, the FGly-containing sulfatase motif can be of the formula:

X¹(FGly)X²Z²⁰X³Z³⁰ (G”)

where

FGly is the formylglycine residue;

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

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

X¹ may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present; and

X and X independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[00349] As described above, the modified polypeptide containing the FGly residue may be conjugated to a drug (e.g., a maytansinoid or an auristatin) by reaction of the FGly with the drug (e.g., a drug containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety, as described above) to produce an FGly’ -containing sulfatase motif. As used herein, the term FGly’ refers to the modified amino acid residue of the sulfatase motif that is coupled to the drug, such as a maytansinoid (e.g., the modified amino acid residue of formula (I)). Thus, the FGly’- containing sulfatase motif can be of the formula:

where

FGly’ is the modified amino acid residue of formula (I);

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

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

X¹ may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present; and

X and X independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[00350] In certain embodiments, the modified amino acid residue of formula (I) is positioned at a C-terminus of a heavy chain constant region of the anti-uPAR antibody. In some instances, the heavy chain constant region comprises a sequence of the formula (II):

wherein

FGly’ is the modified amino acid residue of formula (I);

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

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

X^I may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present;

X and X independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[00351] In certain embodiments, the modified amino acid residue of formula (I) is positioned in a light chain constant region of the anti-uPAR antibody. In certain embodiments, the light chain constant region comprises a sequence of the formula (II):

wherein

FGly’ is the modified amino acid residue of formula (I);

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

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

X^I may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present;

X and X independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

[00352] In certain embodiments, the modified amino acid residue of formula (I) is positioned in a heavy chain CH1 region of the anti-uPAR antibody. In certain embodiments, the heavy chain CH1 region comprises a sequence of the formula (II):

wherein

FGly’ is the modified amino acid residue of formula (I);

Z is either a proline or alanine residue (which can 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), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I;

X¹ may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target polypeptide, X¹ is present;

X and X independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur- containing amino acid, or a polar, uncharged amino acid, (i.e., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G.

Site of modification

[00353] As noted above, the amino acid sequence of an anti-uPAR antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an FGly residue by action of an FGE either in vivo (e.g., at the time of translation of an ald tag-containing protein in a cell) or in vitro (e.g., by contacting an ald tag- containing protein with an FGE in a cell-free system). The anti-uPAR polypeptides used to generate a conjugate of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region. Such Ig polypeptides are referred to herein as“target Ig polypeptides” or “target anti-uPAR antibodies” or“target anti-uPAR Ig polypeptides.”

[00354] The site in an anti-uPAR antibody into which a sulfatase motif is introduced can be any convenient site. As noted above, in some instances, the extent of modification of the native amino acid sequence of the target anti-uPAR polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and/or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target anti-uPAR polypeptide may minimize the impact such modifications may have upon anti- uPAR function and/or structure.

[00355] An anti-uPAR antibody heavy chain constant region can include Ig constant regions of any heavy chain isotype, non-naturally occurring Ig heavy chain constant regions (including consensus Ig heavy chain constant regions). An Ig constant region can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent a solvent-accessible loop region of the Ig constant region. An Ig constant region can be modified by insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above.

[00356] In some cases, an aldehyde-tagged anti-uPAR antibody comprises an aldehyde- tagged Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain). The aldehyde-tagged Ig heavy chain constant region can include heavy chain constant region sequences of an IgA, IgM, IgD, IgE, IgGl, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant of same, e.g., human heavy chain constant region sequences or mouse heavy chain constant region sequences, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an FGly-modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4.

[00357] In some cases, an aldehyde-tagged anti-uPAR antibody comprises an aldehyde- tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region can include constant region sequences of a kappa light chain, a lambda light chain, e.g., human kappa or lambda light chain constant regions, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an FGly-modified anti-uPAR antibody polypeptide. Exemplary constant regions include human gamma 1 and gamma 3 regions. With the exception of the sulfatase motif, a modified constant region may have a wild-type amino acid sequence, or it may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to a wild type amino acid sequence.

[00358] In some embodiments the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain. As noted above, an isolated aldehyde- tagged anti-uPAR polypeptide can comprise a heavy chain constant region modified to include a sulfatase motif as described above, where the sulfatase motif is in or adjacent a surface- accessible loop region of the anti-uPAR polypeptide heavy chain constant region. [00359] A sulfatase motif can be provided within or adjacent one or more of these amino acid sequences of such modification sites of an Ig heavy chain. For example, an Ig heavy chain polypeptide can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif adjacent and N-terminal and/or adjacent and C-terminal to these modification sites. Alternatively or in addition, an Ig heavy chain polypeptide can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modifications sites. In some embodiments, an Ig heavy chain polypeptide may be modified to include two motifs, which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids.

Alternatively or in addition, where a native amino acid sequence provides for one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues of the modification sites of an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide a sulfatase motif at the modification site.

Drugs for Conjugation to a Polypeptide

[00360] Any of a number of drugs are suitable for use, or can be modified to be rendered suitable for use, as a reactive partner to conjugate to a polypeptide. Examples of drugs include small molecule drugs and peptide drugs. Thus, the present disclosure provides drug-polypeptide conjugates.

[00361] “Small molecule drug” as used herein refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa. A small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.

[00362] “Peptide drug” as used herein refers to amino-acid containing polymeric compounds, and is meant to encompass naturally-occurring and non-naturally-occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics. The peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source). Peptide drugs can range in molecular weight, and can be from 200 Da to 10 kDa or greater in molecular weight.

[00363] In some cases, the drug is a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be modified as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce proliferation of cancer cells, and encompass cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds can also be used.

[00364] The present disclosure provides drug-polypeptide conjugates. Examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where the polypeptide is an antibody (or fragment thereof) that has specificity for a tumor cell, the antibody can be modified as described herein to include a modified amino acid, which can be

subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agents.

[00365] In certain embodiments, the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid. In certain embodiments, the drug is a maytansinoid, which as the following structure:

where indicates the point of attachment between the maytansinoid and the linker, L, in formula (I). By“point of attachment” is meant that the symbol indicates the bond between the N of the maytansinoid and the linker, L, in formula (I). For example, in formula (I), W¹ is a maytansinoid, such as a maytansinoid of the structure above, where indicates the point of attachment between the maytansinoid and the linker, L. For instance, in some embodiments, the maytansinoid is deacyl-maytansine.

[00366] Suitable cancer chemotherapeutic agents also include dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure.

[00367] In certain embodiments, the drug is an auristatin, such as MMAE. For example, the auristatin may have the following structure:

where indicates the point of attachment between the auristatin and the linker, L, in formula (I). The symbol indicates the bond between the N of the auristatin and the linker, L, in formula (I). For example, in formula (I), W¹ is an auristatin, such as an auristatin of the structure above, where indicates the point of attachment between the auristatin and the linker, L.

[00368] As described above, in certain embodiments, L is a linker described by the formula -(L )_a-(L )_b-(L )_C-(L )_d-(L )_e-, wherein L , L , L , L and L are each independently a linker unit. In certain embodiments, L¹ is attached to the coupling moiety, such as a hydrazinyl- indolyl or a hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, L , if present, is attached to W (the drug). In certain embodiments, L , if present, is attached to W¹ (the drug). In certain embodiments, L⁴, if present, is attached to W¹ (the drug). In certain embodiments, L⁵, if present, is attached to W¹ (the drug).

[00369] As described above, in certain embodiments, the linker -(L 1 )_a-(L2 )_b-(L 3 )_C-(L 4 )_d- (L⁵)_e- is described by the formula -(T¹-V¹)_a-(T²-V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e-, wherein a, b, c, d and e are each independently 0 or 1, where the sum of a, b, c, d and e is 1 to 5. In certain embodiments, as described above, L¹ is attached to the hydrazinyl-indolyl or the hydrazinyl- pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). As such, in certain embodiments, T¹ is attached to the hydrazinyl-indolyl or the hydrazinyl-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, V¹ is attached to W 1 (the drug). In certain embodiments, as described above, L 2 , if present, is attached to W 1 (the drug). As such, in certain embodiments, T 2 , if present, is attached to W 1 (the drug), or V2 , if present, is attached to W (the drug). In certain embodiments, as described above, L , if present, is attached to W 1 (the drug). As such, in certain embodiments, T 3 , if present, is attached to W 1 (the drug), or V , if present, is attached to W (the drug). In certain embodiments, as described above, L⁴, if present, is attached to W¹ (the drug). As such, in certain embodiments, T⁴, if present, is attached to W¹ (the drug), or V⁴, if present, is attached to W¹ (the drug). ). In certain embodiments, as described above, L⁵, if present, is attached to W¹ (the drug). As such, in certain embodiments, T⁵, if present, is attached to W¹ (the drug), or V⁵, if present, is attached to W¹ (the drug).

[00370] Embodiments of the present disclosure include conjugates where a polypeptide (e.g., anti-uPAR antibody) is conjugated to one or more drug moieties (e.g., a maytansinoid or an auristatin), 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 moieties may be conjugated to the polypeptide at one or more sites in the polypeptide, as described herein. In certain embodiments, the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 10, or from 0.5 to 10, or from 1 to 10, such as from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, the conjugates have an average DAR from 1 to 4. By average is meant the arithmetic mean.

[00371] Suitable cancer chemotherapeutic agents also include duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1- TM1); and benzodiazepines and active analogs and derivatives thereof (e.g.,

pyrrolobenzodiazepine (PBD).

Methods for modification of drugs to contain a reactive partner

[00372] Drugs to be conjugated to a polypeptide may be modified to incorporate a reactive partner for reaction with the polypeptide. Where the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide can be positioned at an N-terminal region, the N-terminus, a C- terminal region, the C-terminus, or at a position internal to the peptide. For example, an example of a method involves synthesizing a peptide drug having an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N-Boc group. As an example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidin-l-yloxy)propanoic acid. Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N-protecting group occurs through standard peptide coupling chemistry. Examples of peptide coupling reagents that can be used include, but not limited to, DCC

(dicyclohexylcarbodiimide), DIC (diisoprop ylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1- benzotriazole diethylphosphate-l-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (l-(3- dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPPA

(diphenylphosphorazidate), BOP (benzotriazol- l-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (0-benzotriazol-l-yl-N,N,N’,N’-tetramethyluronium

hexafluorophosphate), TBTU (O-benzotriazol- l-yl-N,N,N’ ,N’-tetramethyluronium

tetrafluoroborate), TSTU (0-(N-succinimidyl)-N,N,N’,N’-tetramethyluronium

tetrafluoroborate), HATU (N-[(dimethylamino)-l-H-l,2,3-triazolo[4,5,6]-pyridin-l- ylmethylene]- -N-methylmethanaminium hexafluorophosphate N-oxide), BOP-C1 (bis(2-oxo-3- oxazolidinyl)phosphinic chloride), PyBOP ((l-H-l,2,3-benzotriazol-l-yloxy)- tris(pyrrolidino)phosphonium tetrafluorophopsphate), BrOP

(bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one) PyBrOP

(bromotris(pyrrolidino)phosphonium hexafluorophosphate). As a non-limiting example, HOBt and DIC can be used as peptide coupling reagents.

[00373] Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group. Deprotection of the N-oxysuccinimide group, for example, occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wi_lts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. The product and intermediates can be purified by conventional means, such as HPLC purification.

[00374] The ordinarily skilled artisan will appreciate that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to reaction with a reactive partner of interest) are of importance, Modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with a polypeptide present in or on a living cell, the conditions are selected so as to be physiologically compatible. For example, the pH can be dropped temporarily for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (e.g., from about 30 min to 1 hour). Physiological conditions for conducting modification of polypeptides on a cell surface can be similar to those used in a ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040).

[00375] Small molecule compounds containing, or modified to contain, an oc-nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the polypeptide-drug conjugates of the present disclosure. General methods are known in the art for chemical synthetic schemes and conditions useful for synthesizing a compound of interest (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

Detectable labels

[00376] The conjugates and methods of the present disclosure can be used to conjugate a detectable label to polypeptide. Examples of detectable labels include, but are not limited to, fluorescent molecules (e.g., autofluorescent molecules, molecules that fluoresce upon contact with a reagent, etc.), radioactive labels (e.g., ^mIn, ¹²⁵I, ¹³¹I, ²¹²B, ⁹⁰Y, ¹⁸⁶Rh, and the like), biotin (e.g., to be detected through reaction of biotin and avidin), fluorescent tags, imaging reagents, and the like. Detectable labels also include peptides or polypeptides that can be detected by antibody binding, e.g., by binding of a detectably labeled antibody or by detection of bound antibody through a sandwich-type assay. Further examples of detectable labels include, but are not limited to, dye labels (e.g., chromophores, fluorophores, such as, but not limited to, Alexa Fluor® fluorescent dyes (e.g., Alexa Fluor® 350, 405, 430, 488, 532, 546, 555, 568, 594, 595, 610, 633, 635, 647, 660, 680, 700, 750, 790, and the like), coumarins, rhodamines (5- carboxyrhodamine and sulfo derivates thereof, e.g., 5-carboxy-disulfo-rhodamine, carbopyranins and oxazines, such as ATTO dyes (e.g., ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 61 IX, 620, 633, 635, 637, 647, 647N, 655, 665, 680, 700, 725 or 740), biophysical probes (spin labels, nuclear magnetic resonance (NMR) probes), Forster Resonance Energy Transfer (FRET)-type labels (e.g., at least one member of a FRET pair, including at least one member of a fluorophore/quencher pair), Bioluminescence Resonance Energy Transfer (BRET)- type labels (e.g., at least one member of a BRET pair), immunodetectable tags (e.g., FLAG, His(6), and the like), localization tags (e.g., to identify association of a tagged polypeptide at the tissue or molecular cell level (e.g., association with a tissue type, or particular cell membrane), and the like.

Attachment of moieties for delivery to a target site

[00377] Embodiments of the present disclosure also include a polypeptide conjugated to one or more moieties, such as, but not limited to, a drug (e.g., a small molecule drug), toxin, or other molecule for delivery to a target site (e.g., a cell) and which can provide for a

pharmacological activity or can serve as a target for delivery of other molecules.

[00378] Also contemplated are conjugates that include one of a pair of binding partners (e.g., a ligand, a ligand-binding portion of a receptor, a receptor-binding portion of a ligand, etc.). For example, the conjugate can include a polypeptide that serves as a viral receptor and, upon binding with a viral envelope protein or viral capsid protein, facilitates attachment of virus to the cell surface on which the modified polypeptide is expressed. Alternatively, the conjugate may include an antigen that is specifically bound by an antibody (e.g., monoclonal antibody), to facilitate detection and/or separation of host cells expressing the modified polypeptide.

Attachment of target molecules to a support

[00379] The methods can provide for conjugation of a polypeptide to a moiety to facilitate attachment of the polypeptide to a solid substrate (e.g., to facilitate assays), or to a moiety to facilitate easy separation (e.g., a hapten recognized by an antibody bound to a magnetic bead). In some embodiments, the methods are used to provide for attachment of a protein to an array (e.g., chip) in a defined orientation. For example, a polypeptide modified at a selected site (e.g., at or near the N-terminus) can be generated, and the methods, conjugates and compounds used to deliver a moiety to the modified polypeptide. The moiety can then be used as the attachment site for affixing the polypeptide to a support (e.g., solid or semi-solid support, such as a support suitable for use as a microchip in high-throughput assays).

Water-soluble polymers

[00380] In some cases, a conjugate includes a covalently linked water-soluble polymer. A moiety of particular interest is a water-soluble polymer. A“water-soluble polymer” refers to a polymer that is soluble in water and is usually substantially non-immunogenic, and usually has an atomic molecular weight greater than 1,000 Daltons. The methods, conjugates and

compounds described herein can be used to attach one or more water-soluble polymers to a polypeptide. Attachment of a water-soluble polymer (e.g., PEG) to a polypeptide, such as a pharmaceutically active (e.g., therapeutic) polypeptide can be desirable as such modification can increase the therapeutic index by increasing serum half-life as a result of increased proteolytic stability and/or decreased renal clearance. Additionally, attachment of one or more polymers (e.g., PEGylation) can reduce immunogenicity of protein pharmaceuticals.

[00381] In some embodiments, the water-soluble polymer has an effective hydrodynamic molecular weight of greater than 5,000 Da, greater than 10,000 Da, greater than 20,000 to 500,000 Da, greater than 40,000 Da to 300,000 Da, greater than 50,000 Da to 70,000 Da, such as greater than 60,000 Da. In some embodiments, the water-soluble polymer has an effective hydrodynamic molecular weight of from 10 kDa to 20 kDa, from 20 kDa to 25 kDa, from 25 kDa to 30 kDa, from 30 kDa to 50 kDa, or from 50 kDa to 100 kDa. By“effective hydrodynamic molecular weight” is intended the effective water- solvated size of a polymer chain as determined by aqueous-based size exclusion chromatography (SEC). When the water-soluble polymer contains polymer chains having polyalkylene oxide repeat units, such as ethylene oxide repeat units, each chain can have an atomic molecular weight of 200 Da to 80,000 Da, or 1,500 Da to 42,000 Da, including 2,000 to 20,000 Da. Unless referred to specifically, molecular weight is intended to refer to atomic molecular weight. Linear, branched, and terminally charged water soluble polymers (e.g., PEG) may be used.

[00382] Polymers useful as moieties to be attached to a polypeptide can have a wide range of molecular weights, and polymer subunits. These subunits may include a biological polymer, a synthetic polymer, or a combination thereof. Examples of such water-soluble polymers include: dextran and dextran derivatives, including dextran sulfate, P-amino cross linked dextrin, and carboxymethyl dextrin, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and dextrines, and derivatives and hydroylactes of starch, polyalklyene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, heparin and fragments of heparin, polyvinyl alcohol and polyvinyl ethyl ethers, polyvinylpyrrolidone, aspartamide, and

polyoxyethylated polyols, with the dextran and dextran derivatives, dextrine and dextrine derivatives. It will be appreciated that various derivatives of the specifically recited water-soluble polymers are also contemplated.

[00383] Water-soluble polymers such as those described above include polyalkylene oxide based polymers, such as polyethylene glycol“PEG” (See. e.g.,“Poly(ethylene glycol)

Chemistry: Biotechnical and Biomedical Applications”, J. M. Harris, Ed., Plenum Press, New York, N.Y. (1992); and“Poly(ethylene glycol) Chemistry and Biological Applications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); and International Patent Applications: WO 90/13540, WO 92/00748, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28937, WO 95/11924, WO 96/00080, WO 96/23794, WO 98/07713, WO 98/41562, WO 98/48837, WO 99/30727, WO 99/32134, WO 99/33483, WO 99/53951, WO 01/26692, WO 95/13312, WO 96/21469, WO 97/03106, WO 99/45964, and U.S. Pat. Nos. 4,179,337;

5,075,046 5,089,261 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564;

5,275,838 5,281,698 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756;

5,405,877 5,455,027 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460;

5,614,549 5,618,528 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110;

5,730,990 5,739,208 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500;

5,880,131 5,900,461 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566;

5,985,263 5,990,237 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087;

6,180,095; 6,194,580; 6,214,966).

[00384] Examples of polymers of interest include those containing a polyalkylene oxide, polyamide alkylene oxide, or derivatives thereof, including polyalkylene oxide and polyamide alkylene oxide comprising an ethylene oxide repeat unit of the formula -(CH₂-CH₂-0)-. Further examples of polymers of interest include a polyamide having a molecular weight greater than 1,000 Daltons of the formula -[C(0)-X-C(0)-NH-Y-NH]n- or -[NH-Y-NH-C(0)-X-C(0)]_n-, where X and Y are divalent radicals that may be the same or different and may be branched or linear, and n is a discrete integer from 2-100, such as from 2 to 50, and where either or both of X and Y comprises a biocompatible, substantially non-antigenic water-soluble repeat unit that may be linear or branched. Further examples of water-soluble repeat units comprise an ethylene oxide of the formula -(CH₂-CH₂-0)- or -(0-CH₂-CH₂)- . The number of such water-soluble repeat units can vary significantly, with the number of such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, for example from 2 to 50. An example of an embodiment is one in which one or both of X and Y is selected from: -((CH₂) _nl-(CH₂-CH₂-0)_n2-(CH₂)- or -((CH₂)_ni-(0-CH₂-CH₂)_n2- (CH₂) _n_i-), where nl is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. A further example of an embodiment is one in which X is -(CH₂-CH₂)-, and where Y is -(CH₂-(CH₂-CH₂-0)₃-CH₂-CH₂-CH₂)- or -(CH₂-CH₂-CH₂-(0-CH₂-CH₂)₃-CH₂)-.

[00385] The polymer can include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties comprising one or more repeat units employed in a water-soluble polymer, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which can contain, for example, up to 18 carbon atoms or even an additional polymer chain.

[00386] The polymer moiety, or one or more of the spacers or linkers of the polymer moiety when present, may include polymer chains or units that are biostable or biodegradable. For example, polymers with repeat linkages have varying degrees of stability under

physiological conditions depending on bond lability. Polymers with such bonds can be categorized by their relative rates of hydrolysis under physiological conditions based on known hydrolysis rates of low molecular weight analogs, e.g., from less stable to more stable, e.g., polyurethanes (-NH-C(O)-O-) > polyorthoesters (-0-C((0R)(R’))-0-) > polyamides (-C(O)- NH-). Similarly, the linkage systems attaching a water-soluble polymer to a target molecule may be biostable or biodegradable, e.g., from less stable to more stable: carbonate (-O-C(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)-O-) > orthoester (-0-C((0R)(R’))-0-) > amide (-C(O)- NH-). In general, it may be desirable to avoid use of a sulfated polysaccharide, depending on the lability of the sulfate group. In addition, it may be less desirable to use polycarbonates and polyesters. These bonds are provided by way of example, and are not intended to limit the types of bonds employable in the polymer chains or linkage systems of the water-soluble polymers useful in the modified aldehyde tagged polypeptides disclosed herein.

THERAPEUTIC METHODS

[00387] The uPAR ADCs of the present disclosure can find use as therapeutic for treatment of proliferative disorders that are mediated by uPAR-expressing cells. For example, one or more uPAR-binding agents (e.g. antibody) can be used in a therapy for a uPAR- expressing cancer (including prevention and post-diagnosis therapy) or diagnostics for cancers in which cancer cells express uPAR. Subjects having, suspected of having, or at risk of developing a uPAR-expressing cancer are contemplated for therapy and diagnosis described herein. Samples obtained from such subject are likewise suitable for use in the methods of the present disclosure.

[00388] By‘‘treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease, e.g., so as to decrease tumor load, and/or to decrease the cancer metastases. Such treatment also includes situations where the pathological condition, or the progression of a pathological condition towards a more advanced disease state, or at least symptoms associated therewith, is reduced, or slowed down. In some cases, treatment includes situations wherein the mean time for survival between a patient population undergoing treatment comprising the administration of one or more subject antibodies and a control population not undergoing treatment is greater. In some cases, the increase in mean time for survival may be statistically significant. [00389] A variety of hosts are treatable according to the methods. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans. In some cases, the host may be a rodent (e.g. mouse, rat, or guinea pig) that is athymic, nude, or otherwise immune impaired. In some cases, the host may represent a xenotrophic cancer model in which human or other mammalian cancer cells from another species are introduced into the host, and then one or more subject antibodies are administered to treat the resulting tumor.

[00390] In the methods of cancer treatment, administering of one or more agents (e.g. antibodies) specific for uPAR facilitates a reduction in proliferation of cancerous cells and/or in inhibition of metastasis of cancer cells exposed to the antibody. The method involves

administering to the subject an effective amount of a pharmaceutically acceptable formulation that contains one or more agents (e.g. antibodies) specific for uPAR. The agent can have the effect of retarding or otherwise arresting cell growth and/or metastasis. The effects of the agent on cancer cells can be dose dependent, and thus adjustable.

[00391] In a related embodiment, the subject being treated has cells expressing overly active or overly abundant uPAR relative to a noncancerous cell. The uPAR can be expressed on the cell surface, such as on a cancer cell. This aspect can be beneficial in the context of the methods of the present disclosure in that cells expressing or presenting uPAR can be more amenable to treatment with a binding- antibody of the present disclosure. The antibody can be administered to a subject, for example, where therapy is initiated at a point where presence of the uPAR is not detectable, and thus is not intended to be limiting. It is also possible to initiate antibody therapy prior to the first sign of disease symptoms, at the first sign of possible disease, or prior to or after diagnosis of a disease.

Cancer

[00392] The uPAR ADC compositions may be used in an anti-cancer therapy in treatment of cancers that express uPAR on an extracellularly accessible cell surface.

[00393] The presence of uPAR and other members of the plasminogen activation system in normal human tissue appear to be transient and low abundance. It is prevalent only in abnormal cells, such as cancer cells including metastasizing cancer cells. Since expression of high levels of uPAR exists predominantly in cancer cells, treatment with subject compositions can be used to detect the presence and localize cancer growth and can block cancer growth. It should be noted that while uPAR may be expressed at higher levels on a cancer cell compared to a non-cancerous cell, this is not a limitation of the therapies disclosed herein.

[00394] The subject compositions described herein can be administered to a subject (e.g. a human patient) to, for example, reduce proliferation cancerous cells, e.g., to reduce tumor size, reduce tumor load, decrease metastatic potential (e.g. reduce cancer cell migration) and/or improve the clinical outcome in patients. In other words, the compositions can be used to reduce cell growth, cell division, and/or decrease the invasiveness of cancer cells, e.g., by decreasing any signaling events leading up to cancer metastasis. Some ways of decreasing cancer invasiveness involve reducing the ability of cancer cells to leave the original cancerous site, reducing the ability of cancer cells to migrate, and the ability of cancer cells to adhere to areas of the body after migration.

[00395] Cancers particularly amenable to antibody therapy can be identified by examining markers of cellular proliferation (e.g., Ki-67 antigen) and/or by examining the presence / accessibility of the uPAR bound by one or more subject antibodies (e.g. 3C6, 2G10, 2E9) or by other antibodies specific for uPAR (e.g., as in an in vitro assay).

Types of cancer

[00396] Where the anti-cancer therapy comprises administration of an antibody composition described previously, the anti-cancer therapy can be particularly directed to cancer cells. For example, one or more subject antibodies (e.g. 3C6, 2G10) may bind a uPAR- expressing cancer cell.

[00397] Examples of cancers presenting uPAR include but not limited to cancer cells of epithelial origin. Some examples are squamous carcinomas, hematological neoplasms, gastric cancer, lymph node, colorectal cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma), hepatic cancer, and immunological disorders. Other more specific examples of cancer include breast (e.g. triple negative breast tumor), ovarian, prostate, lung, leukaemias, fibrosarcomas, glioblastomas, and prostate cancer, as discussed above. Combination therapies

[00398] The therapeutic methods described herein can include administration of a uPAR agent (e.g., antibody) in combination with one or more other therapies. The combination therapy below can provide for additive or synergistic benefits relative to a regimen in which only one therapy is administered.

[00399] An example of combination therapy involves administering more than one type of agent (e.g., antibody) to a subject. As described above for pharmaceutical compositions, the therapeutic method may involve administering at least one, at least two, at least three or more different types of antibodies simultaneously or sequentially, including for example one or more subject antibodies. The antibodies may differ in the epitopes of uPAR to which they bind. The method, for example, may involve administering and ADC having CDRs of 3C6 or 2G10 to a subject in need of therapy. The antibodies may also bind the same or overlapping epitopes of uPAR. The method for example may involve administering two or more antibodies that each inhibit the interaction between uPAR and uPA, or two or more antibodies that each inhibit the interaction between uPAR and an integrin, or two or more antibodies that each inhibit the interaction between uPAR and vitronectin, or two or more antibodies that each inhibit the interaction between uPAR and uPARAP, or the method may involve administering two or more antibodies that bind to uPAR but do not inhibit one or more of the foregoing interactions, or any combination thereof.

[00400] The combination therapy method can treat cancer in various ways. As noted above for the subject composition, the subject method can employ one or more agents that inhibit one or more uPAR signaling pathways. Where more than one signaling pathways are targeted by the agents, there can be a synergistic inhibition of cell adhesion, proliferation, and/or migration of cancer cells. For example, one signaling pathway that can be inhibited by a binding agent is mediated by uPA binding to uPAR, while another pathway is mediated by integrin (e.g., a bΐ integrin, such as such as a5b1 or a3b1) binding to uPAR.

[00401] Additional standard anti-cancer therapeutics that may or may not be administered in conjunction with a subject antibody, include but not limited to immunotherapy,

chemotherapeutic agents and surgery. In addition, therapeutic administration of a subject antibody can also be post-therapeutic treatment of the subject with an anti-cancer therapy, where the anti-cancer therapy can be, for example, surgery, radiation therapy, administration of chemotherapeutic agents, and the like. Cancer therapy using a subject antibody in combination with immunotherapy that employs uPAR ADCs is of particular interest.

Dosage

[00402] In the methods, an effective amount of an agent (e.g., a uPAR antibody or a uPAR ADC) is administered to a subject in need thereof. For example, in some embodiments, a uPAR- binding agent can facilitate inhibition of growth and/or proliferation of a uPAR-expressing cancer cell. The amount administered can vary depending upon the goal of the administration, the health and physical condition of the individual to be treated, age, the degree of resolution desired, the formulation of a subject agent, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. For example, the amount of subject agent employed to inhibit cancer cell growth is not more than about the amount that could otherwise be irreversibly toxic to the subject (i.e., maximum tolerated dose). In other cases the amount is around or even well below the toxic threshold, but still in an effective concentration range, or even as low as threshold dose.

[00403] Individual doses are typically not less than an amount required to produce a measurable effect on the subject, and may be determined based on the pharmacokinetics and pharmacology for absorption, distribution, metabolism, and excretion (“ADME”) of the antibody, and thus based on the disposition of the composition within the subject. This includes consideration of the route of administration as well as dosage amount, which can be adjusted for parenteral (applied by routes other than the digestive tract for systemic or local effects) applications, for example. For instance, administration of a subject antibody is typically via injection and often intravenous, intramuscular, intratumoral, intracranial, intraarterial, intraocular, intrathecal, or a combination thereof.

[00404] A uPAR ADC may be administered by infusion or by local injection. It also can be administered prior, at the time of, or after other therapeutic interventions, such as surgical intervention to remove cancerous cells. As noted above, a uPAR ADC can also be administered as part of a combination therapy, in which at least one of an immunotherapy, a cancer chemotherapy or a radiation therapy is administered to the subject (as described in detail above). [00405] Disposition of the agent and its corresponding biological activity within a subject is typically gauged against the fraction of agent present at a target of interest. For example, an ADC once administered can accumulate with uPAR or other biological target that concentrates the material in cancer cells and cancerous tissue. Thus dosing regimens in which the antibody is administered so as to accumulate in a target of interest over time can be part of a strategy to allow for lower individual doses. This can also mean that, for example, the dose of antibody that are cleared more slowly in vivo can be lowered relative to the effective concentration calculated from in vitro assays (e.g., effective amount in vitro approximates mM concentration, versus less than mM concentrations in vivo).

[00406] As an example, the effective amount of a dose or dosing regimen can be gauged from the IC₅₀ of a given antibody for inhibiting or binding uPAR. By“IC₅₀” is intended the concentration of a drug required for 50% inhibition in vitro. Alternatively, the effective amount can be gauged from the EC ₅₀ of a given antibody concentration. By“EC₅₀” is intended the plasma concentration required for obtaining 50% of a maximum effect in vivo.

[00407] In general, with respect to the uPAR ADCs of the present disclosure, an effective amount is usually not more than 200X the calculated IC₅₀. Typically, the amount of an antibody that is administered is less than about 200X, less than about 150X, less then about 100X and many embodiments less than about 75X, less than about 60X, 50X, 45X, 40X, 35X, 30X, 25X, 20X, 15X, 10X and even less than about 8X or 2X the calculated IC₅₀. In one embodiment, the effective amount is about IX to 50X of the calculated IC₅₀, and sometimes about 2X to 40X, about 3X to 30X or about 4X to 20X of the calculated IC₅₀. In other embodiments, the effective amount is the same as the calculated IC₅₀, and in certain embodiments the effective amount is an amount that is more than the calculated IC₅₀.

[00408] An effective amount may not be more than 100X the calculated EC50. For instance, the amount of antibody that is administered is less than about 100X, less than about 50X, less than about 40X, 35X, 30X, or 25X and many embodiments less than about 20X, less than about 15X and even less than about 10X, 9X, 9X, 7X, 6X, 5X, 4X, 3X, 2X or IX than the calculated EC50. In one embodiment, the effective amount is about IX to 30X of the calculated EC50, and sometimes about IX to 20X, or about IX to 10X of the calculated EC50. In other embodiments, the effective amount is the same as the calculated EC50, and in certain

embodiments the effective amount is an amount that is more than the calculated EC50. [00409] Effective amounts can readily be determined empirically from assays, from safety and escalation and dose range trials, individual clinician-patient relationships, as well as in vitro and in vivo assays such as those described herein and illustrated in the Experimental section, below.

[00410] The IC₅₀ may be calculated by inhibiting the agent binding to uPAR (e.g. uPAR alone or complexed uPAR, such as uPAR with integrins) in vitro. This aspect can be carried out by assessing the ability of the agent of interest to inhibit 3C6 antibody binding to uPAR. In general, the procedure is carried out by standard ELISA in which the plates are coated with uPAR as described in the examples at a concentration of about 1 pg/ml, and then processed and employed as described in the experimental examples to determine inhibition of antibody binding and the IC₅₀. These agents and others suitable for various aspects of this purpose can be employed.

Routes of administration

[00411] In practicing the methods, routes of administration (path by which a subject agent is brought into a subject in need of therapy or diagnosis) may vary, where representative routes of administration for a subject antibody are described in greater detail below. A subject agent alone or in combinations described above can be administered systemically (e.g., by parenteral administration, e.g., by an intravenous route) or locally (e.g., at a local tumor site, e.g., by intratumoral administration (e.g., into a solid tumor, into an involved lymph node in a lymphoma or leukemia), administration into a blood vessel supplying a solid tumor, etc.).

[00412] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. [00413] The formulations of the present disclosure can also be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as for use in a nebulizer or an atomizer.

[00414] Suppository formulations are also provided by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams.

[00415] Unit dosage forms for rectal administration such as syrups, elixirs, and

suspensions may be provided wherein each dosage unit contains a predetermined amount of the composition containing the antibody compositions. Similarly, unit dosage forms for injection or intravenous administration may comprise the antibody in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

The term“unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

KITS & SYSTEMS

[00416] Also provided are kits and systems that may find use in practicing the methods, as described above. For example, kits and systems may include one or more of the compositions described herein, such as a uPAR ADC (e.g., 3C6 or 2G10). Other optional components of the kit include: buffers, etc., for administering the uPAR ADC, and/or for performing a diagnostic assay. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.

[00417] The kits and systems for practicing the methods may include one or more pharmaceutical formulations that include the antibody compositions described herein. As such, the kits may include a single pharmaceutical composition present as one or more unit dosages. In yet other embodiments, the kits may include two or more separate pharmaceutical compositions. [00418] In addition to the above components, the kits may further include instructions for practicing the methods. These instructions may be present in the kits in a variety of forms, one or more of which may be present in or on the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in or on the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, DVD. Blu- Ray, flash drive, thumb drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

[00419] A kit may be provided for use in treating a host suffering from a cellular proliferative disease. This kit includes a pharmaceutical composition comprising antibody specific for uPAR, and instructions for the effective use of the pharmaceutical composition in a method of treating a host suffering from a cancerous condition by inhibiting the growth and/or metastasis of a cancer cell in a subject. Such instructions may include not only the appropriate handling properties, dosing regiment and method of administration, and the like, but can further include instructions to optionally screen the subject for uPAR associated with the disease. This aspect can assist the practitioner of the kit in gauging the potential responsiveness of the subject to treatment with an antibody of the present disclosure , including timing and duration of treatment relative to the type and growth stage of the cancer. Thus in another embodiment, the kit may further include an antibody or other reagent, such as an antibody having the CDRs of 3C6 or 2G10, for detecting uPAR on an extracellularly accessible surface of a cancer cell. The kit may also include an antibody that contains a conjugate with a detectable label, such as a fluorophore.

[00420] The term "system" as employed herein refers to a collection of antibodies described herein and one or more second therapeutic agents, present in single or disparate compositions that are brought together for the purpose of practicing the methods. For example, separately obtained antibody specific to uPAR and chemotherapy dosage forms brought together and co-administered to a subject are a system according to the present disclosure.

[00421] The following examples further illustrate the present invention and should not be construed as in any way limiting its scope. EXAMPLES

[00422] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

General Synthetic Procedures

[00423] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[00424] Compounds as described herein can be purified by any purification protocol known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J.

Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer- Verlag, New York, 1969.

[00425] During any of the processes for preparation of the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie,“Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wi_lts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in“The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie”, Houben-Weyl, 4^th edition, Vol. 15/1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit,“Aminosauren, Peptide, Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.

[00426] The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by

conventional synthetic methods. A variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.

EXAMPLE 1

[00427] FIG. 1 shows a schematic illustration of two types of anti-uPAR antibodies (2G10 and 3C6), which are antagonists and compete against uPAR interaction with urokinase plasminogen activator and b integrins.

[00428] FIG. 2 shows a schematic illustration of an anti-uPAR antibody (e.g., 2G10 or 3C6) conjugated to a cytotoxic agent. FIG. 2 (panel A) shows the components of a site- specifically modified uPAR ADC. FIG. 2 (panel B) shows a schematic illustration of the production of an aldehyde-tagged antibody using formylglycine generating enzyme (FGE).

EXAMPLE 2

[00429] An in vitro triple-negative breast cancer (TNBC) model was used to test the efficacy of anti-uPAR antibodies 2G10 and 3C6.

[00430] FIG. 3 shows graphs demonstrating that anti-uPAR antibodies 2G10 and 3C6 are therapeutically effective in an in vitro triple-negative breast cancer (TNBC) model. FIG. 3 (panel A) shows a graph of % phosphorylated ERK (pERK) induction vs. inhibitor (antibody) used. As shown in FIG. 3 (panel A), the anti-uPAR antibodies 2G10 and 3C6 were effective for significantly reducing pERK levels. FIG. 3 (panel B) shows a graph of % invasion of MDA- MB-231 calls through a cross-linked matrix (Matrigel invasion assay) vs. antibody used. As shown in FIG. 3 (panel B), the anti-uPAR antibodies 2G10 and 3C6 significantly reduced invasion of MDA-MB-231 cells through a cross-linked matrix (Matrigel invasion assay) as compared to a control. FIG. 3 (panel C) shows a graph of % radioactivity internalized vs. antibody used. As shown in FIG. 3 (panel C), the anti-uPAR antibodies 2G10 and 3C6 induced uPAR internalization over time.

EXAMPLE 3

[00431] FIG. 4 shows a schematic illustration of the mechanism of action of an anti-uPAR ADC, according to embodiments of the present disclosure. As shown in FIG. 4, an anti-uPAR ADC is effective for delivering the drug of the ADC into the cell’s lysosome.

EXAMPLE 4

[00432] FIG. 5 shows images of intracellular trafficking of anti-uPAR Fabs, according to embodiments of the present disclosure. As shown in FIG. 5, the green color indicates lysosome, the red color indicates antibody, and the yellow color indicates an overlay between the antibody and the lysosome.

EXAMPLE 5

[00433] In vitro and in vivo triple-negative breast cancer (TNBC) models were used to test the efficacy of anti-uPAR ADCs, where an anti-uPAR antibody (e.g., 2G10 or 3C6) was conjugated to a drug, such as maytansine or MMAE.

[00434] FIG. 6 (panel A) shows a table of anti-uPAR ADCs that were tested in an in vivo TNBC model. The drug-antibody ratio (DAR) for each of the ADC tested was about 2. As shown in FIG. 6 (panel A), three different ADCs were tested: (1) 3C6-RED-l06-maytansine; (2) 3C6-RED-244-MMAE; and (3) 2Gl0-RED-l06-maytansine.

[00435] The structure of the RED-l06-maytansine linker-drug construct is shown below:

RED-l06-maytansine

[00436] The RED-l06-maytansine linker-drug construct was conjugated to a 3C6 anti- uPAR antibody at an aldehyde tag of the antibody as described herein.

[00437] The structure of the RED-244-MMAE linker-drug construct is shown below:

[00438] The RED-244-MMAE linker-drug construct was conjugated to a 3C6 anti-uPAR antibody at an aldehyde tag of the antibody as described herein.

[00439] FIG. 6 (panel B) shows graphs of % change in cell number relative to untreated cells after 110 hours of treatment with the ADC. The ADC 3C6-RED-244-MMAE demonstrated cell killing ability in vitro in MDA-MB-231 cells.

[00440] FIG. 7 shows a graph of tumor volume relative to initial implants (%) vs. time (days) for an in vivo TNBC model used to test the efficacy of anti-uPAR ADCs, where an anti- uPAR antibody (e.g., 2G10 or 3C6) was conjugated to a drug, e.g., maytansine or MMAE. As shown in FIG. 7, in the in vivo in mouse MDA-MB-231 xenograft model of TNBC, the ADCs 3C6-azaHIPS-4AP-MMAE (also referred to as 3C6-RED-244-MMAE) and 2G10-HIPS -4 AP- maytansine (also referred to as 2Gl0-RED-l06-maytansine) showed significant slowing or blocking of tumor growth without an effect on the mice body weight. See also FIG. 8. FIG. 8 shows a graph of body weight (g) vs. time (days) for the mice tested in the experimental example corresponding to FIG. 7.

EXAMPLE 6

[00441] FIG. 9 is a table showing different anti-uPAR 2G10 ADCs that were made and characterized according to the present disclosure. The structures of the RED-l06-maytansine and RED-244-MMAE linker-drug constructs are shown above. The structures of the RED-425 - maytansine, RED-432-maytansine, RED-412-MMAE, RED-388-MMAE, and RED-426-MMAE linker-drug constructs are shown below.

RED-388

RED-425

[00442] Each of the RED-l06-maytansine, RED-425-maytansine, RED-432-maytansine,

RED-412-MM AE, RED-388-MMAE, RED-244-MMAE, and RED-426-MMAE linker-drug constructs were conjugated to a 2G10 anti-uPAR antibody at an aldehyde tag of the antibody as described herein. [00443] FIG. 10 shows a graph of tumor volume relative to initial implants (%) vs time (days) for various anti-uPAR ADCs tested in an in vivo mouse MDA-MB-231 xenograft model of TNBC. As shown in FIG. 10, 2G10 anti-uPAR ADCs conjugated to maytansine slowed tumor growth. A greater effect was shown by 2G10 anti-uPAR ADCs conjugated to MMAE that blocked tumor growth and shrunk tumor volume in the mouse MDA-MB-231 xenograft model of TNBC, without an effect on the mice body weight. See also FIG. 11. FIG. 11 shows a graph of body weight (g) vs. time (days) for the mice tested in the experimental example corresponding to FIG. 10.

[00444] In summary, the anti-uPAR ADCs were able to recognize uPAR in TNBC cell lines. The ADC 3C6-azaHIPS-4AP-MMAE (also referred to as 3C6-RED-244-MMAE) exhibited the greatest therapeutic effect and reduced the tumor volume without toxicity in a mouse model of TNBC.

EXAMPLE 7

[00445] By comparing gene expression data in primary tumors and patient-derived cultured cell lines, elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) were identified in pancreatic adenocarcinoma (PD AC). It was found that antagonistic anti-uPAR antibodies recognize uPAR on PD AC lines, and on cells isolated from PD AC patient- derived xenografts (PDX) tumors. Next, a library of histologically and genetically validated PD AC PDX was developed by orthotopic implantation into NSG mice. Antibody-drug conjugates (ADC) based on 2G10 anti-uPAR conjugated to the cytotoxin, auristatin MMAE, were delivered in a dose of 10 mg/kgs to the three PD AC PDX mouse models that were developed. Conjugated antibodies had either 2 or 4 payload molecules. The 2G10-MMAE anti- uPAR ADC inhibited the growth of all three PDX lines without an effect on the mice body weight. Additionally, 3C6-MMAE anti-uPAR ADC was also delivered in a high dose of 18 mg/kg and similarly inhibited tumor growth without toxicity in vivo in a PD AC PDX.

[00446] FIG. 12 shows the development of an in vivo mouse PDX model and the work flow for testing anti-uPAR ADCs in vivo.

[00447] FIG. 13 shows characteristics of the grown patient-derived xenografts.

[00448] FIG. 14 shows that anti-uPAR antibodies recognize uPAR on epithelial cells isolated from patient-derived xenograft tumors. Panel A shows the morphology of confluent monolayers of the primary culture of cells isolated from the tumor of the PDX UCPDAC187. Panel B provides a histogram of cell count to log uPAR level and shows that 2G10 anti-uPAR igG bound uPAR on the cell surface of the isolated cells.

[00449] FIG. 15 shows a graph of tumor volume (%) vs. time (days) for an anti-uPAR 3C6-MMAE ADC tested in an in vivo mouse PDX model of pancreatic adenocarcinoma

(PDAC). As shown in FIG. 15, anti-uPAR 3C6-MMAE lst generation ADCs (l8mg/kg) blocked tumor growth in vivo in patient-derived xenografts (UCPDAC187). See also FIG. 16. FIG. 16 shows a graph of body weight (%) vs. time (days) for the mice tested in the experimental example corresponding to FIG. 15. The ADC 3C6-MMAE did not affect mice body weight.

[00450] FIG. 17 shows a graph of tumor volume (%) vs. time (days) for anti-uPAR 2G10- MMAE ADCs tested in an in vivo mouse PDX model of PDAC. As shown in FIG. 17, 2G10- MMAE 2^nd generation ADCs blocked tumor growth in vivo in patient-derived xenografts (UCPDAC187). See also FIG. 18. FIG. 18 shows a graph of body weight (%) vs. time (days) for the mice tested in the experimental example corresponding to FIG. 17. The ADC 2G10-MMAE did not effect mice body weight.

[00451] In summary, the fully human anti-uPAR ADCs were able to recognize uPAR in cells isolated from PDXs of PDAC patients. 3C6-MMAE anti-uPAR ADC safely inhibits PDAC tumor growth in vivo at a high dose of 18 mg/kg. 2G10-MMAE anti-uPAR ADCs safely inhibit PDAC tumor growth in vivo at a dose of 10 mg/kg. Antagonist anti uPAR antibodies recognize uPAR on pancreatic cancer cell lines, and on cells isolated from PDAC PDX tumors.

EXAMPLE 8

Materials and Methods

[00452] Cloning, Expression, and Purification of Tagged IgG

[00453] Antagonistic anti-urokinase plasminogen activator receptor (uPAR) antibodies significantly inhibit uPAR-mediated cellular signaling and migration as disclosed in Duriseti et al. (2010) JBiol Chem. 285:26878.

[00454] Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes as disclosed in Drake et al. (2014) Bioconjug Chem. 25: 1331.

[00455] Bioconjugation, Purification, and HPLC Analytics [00456] Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes as disclosed in Drake et al. (2014) Bioconjug Chem. 25: 1331.

[00457] Flow Cytometry

[00458] The breast cancer cell lines were cultured in RPMI supplemented with 10% heat- inactivated FBS. Cells were washed with DPBS and harvested with TrypLE (Gibco). lxlO⁶ cells were incubated with 10 nM 2G10 IgG for 60 minutes at 4°C, followed by FITC-labeled anti-human IgG antibody (BD) for another 60 minutes at 4°C. Stained samples and controls were assayed on a BD FACSCalibur™. All experiments were performed in triplicate.

[00459] In vivo studies

[00460] PDX tumors were grown in NSG mice. When tumors reached an average of

100 mm , animals were randomized into groups of 10 mice and were dosed as described in the text. The animals were given four doses of 5 or lO-mg/kg of ADC, or vehicle alone. The animals were monitored twice weekly for body weight and tumor size. Tumors were measured twice weekly and tumor volume was estimated according to the

formula: tu mor volume (mm³) = where w is tumor width, h is tumor height and l is tumor

£ length. Animals were euthanized at the end of the study, or when tumors reached 2 cm .

[00461] Statistical analysis

[00462] All in vitro experiments were performed in triplicate. Statistical analysis was performed using GraphPad Prism software. In all cases, ANOVA followed by two-tailed, unpaired Student’s /-tests were performed to analyze statistical differences between groups. P values of <0.05 were considered statistically significant.

Claims

WHAT IS CLAIMED IS:

1. A conjugate comprising at least one modified amino acid residue with a side chain of formula (I):

wherein

Z is CR⁴ or N;

R¹ 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;

R 2 and R 3 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, acyl amino, amino acyl, 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;

each R⁴ 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, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker comprising -(T¹-V¹)_a-(T²-V²)_b-(T³-V³)_c-(T⁴-V⁴)_d-(T⁵-V⁵)_e- , wherein a, b, c, d and e are each independently 0 or 1, where the sum of a, b, c, d and e is 1 to 5;

T¹, T², T³, T⁴ and T⁵ are each independently selected from (Ci-Ci₂)alkyl, substituted (Ci- C_l2)alkyl, (EDA)_W, (PEG)_n, (AA)_P, -(CR^{l 3}OH)i_r, piperidin-4-amino (4AP), ), para-amino- benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol or a modified polyethylene glycol, and AA is an amino acid residue, 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;

V , V , V , V and V are each independently selected from the group consisting of a covalent bond, -CO-, -NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H4)-, -CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, - OC(O)-, -0-, -S-, -S(O)-, -SO2-, -SO2NR¹⁵-, -NR¹⁵S0₂- and -P(0)OH-, wherein q is an integer from 1 to 6;

each R is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl;

each R¹⁵ 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;

W¹ is a chemical entity; and

W is an anti-uP AR antibody.

2. The conjugate of Claim 1, wherein:

T¹ is selected from a (Ci-Ci2)alkyl and a substituted (Ci-Ci2)alkyl;

T², T³, T⁴ and T⁵ are each independently selected from (EDA)_W, (PEG)_n, (Ci-Ci2)alkyl, substituted (Ci-Ci2)alkyl, (AA)_P , -(CR OH)_h-, 4-amino-piperidine (4AP), ), para-amino- benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl (PAB), an acetal group, a hydrazine, and an ester; and

V , V , V , V and V are each independently selected from the group consisting of a covalent bond, -CO-, -NR¹⁵-, -NR¹⁵(CH₂)_q-, -NR¹⁵(C₆H4)-, -CONR¹⁵-, -NR¹⁵CO-, -C(0)0-, - OC(O)-, -0-, -S-, -S(O)-, -SO2- , -SO2NR¹⁵-, -NR¹⁵S0₂-, and -P(0)OH-;

wherein:

integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

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

4-amino-piperidine

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

R 13 is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl.

3. The conjugate of Claim 1, wherein the chemical entity is a drug or a detectable label.

4. The conjugate of Claim 3, wherein the drug is selected from the group consisting of a maytansinoid and an auristatin.

5. The conjugate of Claim 4, wherein the maytansinoid is of the formula:

where indicates the point of attachment between the maytansinoid and L.

6. The conjugate of Claim 4, wherein the auristatin is monomethyl auristatin E (MMAE).

7. The conjugate of Claim 1, wherein the anti-uPAR antibody comprises a sequence of the formula (II):

X ¹ (FGly’ )X²Z²⁰X³Z³⁰ (II)

wherein

FGly’ is the modified amino acid residue of formula (I);

Z²⁰ is either a proline or alanine residue;

Z³⁰ is a basic amino acid or an aliphatic amino acid;

X¹ may be present or absent and, when present, can be any amino acid, with the proviso that when the sequence is at the N-terminus of the conjugate, X¹ is present; and

X and X are each independently any amino acid.

8. The conjugate of Claim 7, wherein

Z³⁰ is selected from R, K, H, A, G, L, V, I, and P;

X^I is selected from L, M, S, and V; and

X and X are each independently selected from S, T, A, V, G, and C.

9. The conjugate of Claim 7, wherein the sequence is L(FGly’)TPSR.

10. The conjugate of Claim 1, wherein the modified amino acid residue is positioned at a C- terminus of a heavy chain constant region of the anti-uPAR antibody.

11. The conjugate of Claim 10, wherein the heavy chain constant region comprises the sequence S LS LS LGS L(F Gly’ )TPS RGS .

12. The conjugate of Claim 1, wherein the modified amino acid residue is positioned in a CH1 region of the anti-uPAR antibody.

13. The conjugate of Claim 12, wherein the CH1 region comprises the sequence

WNS G AL(F Gly’ )TPS RG VHTFP A .

14. The conjugate of Claim 1, wherein the modified amino acid residue is positioned in a light chain constant region of the anti-uPAR antibody.

15. The conjugate of Claim 1, wherein the modified amino acid residue is positioned in a heavy chain CH2 region of the anti-uPAR antibody.

16. The conjugate of Claim 1, wherein the modified amino acid residue is positioned in a heavy chain CH3 region of the anti-uPAR antibody.

17. A pharmaceutical composition comprising:

a conjugate of any of Claims 1 to 16; and

a pharmaceutically acceptable excipient.

18. A method comprising:

administering to a subject an effective amount of a conjugate of any of Claims 1 to 16.

19. A method of treating cancer in a subject, the method comprising:

administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate of any of Claims 1 to 16, wherein the administering is effective to treat cancer in the subject.

20. A method of delivering a drug to a target site in a subject, the method comprising:

administering to the subject a pharmaceutical composition comprising a conjugate of any of Claims 1 to 16, wherein the administering is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject.