Tyrosine-protein kinase membrane receptor 1 (ROR1) antibody-drug conjugates and uses thereof

Abstract

The present disclosure provides anti-tyrosine-protein kinase membrane receptor 1 antibody-drug conjugate structures. The antibody-drug conjugate structures include a branched linker, where two or more payloads per branched linker are attached to an antibody. In addition, the disclosure also encompasses compounds and methods for production of such conjugates. In addition, the disclosure also encompasses methods of using the conjugates.

Description

Attorney Docket No.14529-152-228 TYROSINE-PROTEIN KINASE MEMBRANE RECEPTOR 1 (ROR1) ANTIBODY- DRUG CONJUGATES AND USES THEREOF 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 514,784, filed July 20, 2023, the disclosure of which is incorporated by reference herein in its entirety. 2. SEQUENCE LISTING

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14529-152-228_SEQ_LISTING.xml”, was created on July 17, 2024, and is 159,402 bytes in size. 3. FIELD

[0003] The present disclosure relates generally to antibody-drug conjugates (ADCs) that bind to tyrosine-protein kinase membrane receptor 1 (ROR1, e.g., human ROR1) and methods of their use. 4. BACKGROUND

[0004] Various tumors can demonstrate cell-surface expression of tyrosine-protein kinase transmembrane receptor (ROR) antigens, as described in greater detail in Gentile, et al. (Cancer Res; 71(8) April 15, 2011), Rebagay, et al. (Front. Oncol., 18 April 2012), Zhang, et al. (American Journal of Pathology, Vol.181, No.6, December 2012), Henry, et al. (Oncotarget, Vol.6, No.372015), Zhang, et al. (PLoS ONE 7(3): e31127), and Bainbridge, et al. (PLoS ONE 9(7): e102695), each herein incorporated by reference in their entirety. In addition, ROR expression may not be expressed, or only demonstrate limited expression, in normal, e.g., non-cancerous, tissue as described in Balakrishnan et al. (Clin Cancer Res.2017 Jun 15; 23(12): 3061–3071), herein incorporated in its entirety. Thus, ROR antigens can be used as a tumor-specific marker in certain tumors. Examples of tumors and cancers with demonstrated ROR expression include, but are not limited to, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, and B-ALL, as described in Gohil et al. (Oncoimmunology.2017; 6(7): e1326437), herein incorporated in its entirety. Other cancers include, but are not limited to, hematological cancer, prostate cancer, colon cancer, renal cancer, and uterine cancer. Use of ROR multispecific antibodies, formatted in various 1 NAI-1540479824antibody platforms, to target tumors is described in Gohil, et al., international application WO 2017 / 053469, international application WO 2014 / 167022, U.S. Pub. No.2017 / 0198045, international application WO 2016 / 094873, international application WO 2017 / 127499, and international application WO 2016 / 142768, each of which is herein incorporated by reference in its entirety.

[0005] ROR antigen binding molecules thus have therapeutic potential in treatment of cancer. Multispecific ROR binding molecules that bind T cell surface antigens in addition to an ROR antigen have potential to provide T cell redirected killing of ROR-expressing cancer cells.

[0006] There remains a need in the art for ADCs that can target ROR to treat, prevent, or alleviate ROR-mediated diseases, disorders, or conditions, such as cancer. 5. SUMMARY

[0007] The present disclosure provides ADCs comprising an antibody that binds tyrosine- protein kinase membrane receptor 1 (“ROR1-ADC”). Such ROR1-ADCs, in some embodiments, bind to the same epitope of human ROR1 as an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) described herein.

[0008] The present disclosure also provides pharmaceutical compositions comprising an ROR1-ADC that comprises an antibody or fragment thereof that binds to ROR1 (“ROR1 antibody”) and a drug conjugated (directly or indirectly) thereto. Such pharmaceutical compositions, in some embodiments, include ROR1-ADCs comprising an antibody or fragment thereof that binds to essentially the same epitope of human ROR1 as an antibody comprising a VH and a VL described herein.

[0009] The present disclosure also provides methods of treating, preventing, or alleviating an ROR1-mediated disease, disorder, or condition, such as alleviating one or more symptoms of the ROR1-mediated disease, disorder, or condition with an ROR1-ADC.

[0010] More specifically, the present disclosure provides an ROR1-ADC comprising (a) an ROR1 antibody and (b) one or more pyridazine-pyrrolo coupling moieties comprising a drug conjugated to the pyridazine-pyrrolo coupling moiety through a linker, for example, using the Hydrazino-iso-Pictet-Spengler (HIPS) conjugation method.

[0011] Traditionally, the HIPS conjugation method has been used to produce conjugates carrying one payload per HIPS moiety per aldehyde tag, which produces antibody conjugates with DAR values of up to 4. In some embodiments, an ROR1-ADC as disclosed herein comprises branched HIPS linkers that carry two (or more) molecules of the same or different payload per one HIPS moiety and are therefore capable of conjugating two (or more) small 2 NAI-1540479824molecule payloads per one aldehyde group in a protein in a single conjugation step. Consequently, the usage of such branched linkers allows the generation of higher DAR site- specific conjugates (e.g., DAR up to 8) with controlled payload placement, which in the context of therapeutic ADCs would result in larger quantities of pharmaceutical agent delivered to the targeted tissue.

[0012] The present disclosure provides ROR1-ADC structures, each of which comprises (a) an ROR1 antibody, (b) a branched HIPS linker, and (c) a drug. The disclosure also encompasses compounds and methods for production of such conjugates, as well as methods of using the conjugates.

[0013] Aspects of the present disclosure include an ROR1-ADC comprising (a) an ROR1 antibody; and (b) one or more pyridazine-pyrrolo coupling moieties comprising one or more drugs conjugated to the pyridazine-pyrrolo coupling moiety via one or more linkers.

[0014] In some embodiments, an ROR1-ADC is represented by Formula (I), the ROR1- ADC comprising: a. an antibody that binds to tyrosine-protein kinase membrane receptor 1 (ROR1); and b. two or more drugs conjugated to a pyridazine-pyrrolo coupling moiety, each via a linkerwherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3, and R4are each selected from hydrogen and alkyl; LAis a first linker comprising: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, 3 NAI-1540479824wherein: a, b, c, d, e, and f are each independently 0 or 1, provided at least one of a, b, c, d, e, and f are 1; T1, T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LBis a second linker comprising: -(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-(T13-V13)m-, wherein: g, h, i, j, k, l, and m are each independently 0 or 1, provided that at least one of g, h, i, j, k, l, and m is 1; T7, T8, T9, T10, T11, T12, and T13are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para- 4 NAI-1540479824amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V7, V8, V9, V10, V11, V12,and V13are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; s is an integer from 1 to 10; W1is a first drug; and W2is a second drug.

[0015] In some embodiments, Z1is CR4.

[0016] In some embodiments, Z3is C-LB-W2.

[0017] In some embodiments, one or both of W1and W2are camptothecin analogues, for example, belotecan.

[0018] In some embodiments, LAcomprises: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein a, b, c, d, e, and f are each independently 0 or 1, provided at least one of a, b, c, d, e, and f is 1; T1, T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), 5 NAI-1540479824para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0019] In some embodiments of LA: T1is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:integer from 1 to 6 and r is 0 or 1; 6 NAI-15404798244-amino-piperidine each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; In further embodiments, a, b, c, and d are each 1; and e and f are 0.

[0020] In some embodiments, T1, T2, T3, T4, T5, and T6are each optionally substituted with a glycoside.

[0021] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0022] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0023] In some embodiments, LAis a linker wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)pand V3is absent (e.g., a covalent bond); T4is PABC and V4is absent (e.g., a covalent bond); p is an integer from 1 to 10; a, b, c, and d are each 1; and e and f are each 0.

[0024] In further embodiments, the PABC is substituted with a glycoside, for example, a hydrogen of PABC is replaced with a glycoside, such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0025] In some embodiments, LBcomprises: -(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-(T13-V13)m-, wherein g, h, i, j, k, l, and m are each independently 0 or 1, provided at least one of g, h, i, j, k, l, and m is 1; T7, T8, T9, T10, T11, T12, and T13are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy 7 NAI-1540479824(MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para- amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V7, V8, V9, V10, V11, V12, and V13are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0026] In some embodiments, T7, T8, T9, T10, T11, T12, and T13are each optionally substituted with a glycoside.

[0027] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0028] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0029] In some embodiments of LB: T7is a covalent bond; T8, T9, T10, T11, and T12are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V7, V8, V9, V10, V11, and V12are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, and -P(O)OH-; 8 NAI-1540479824wherein:integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; g, h, i, j, and k are each 1; and l and m are each 0.

[0030] In some embodiments, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12are each optionally substituted with a glycoside.

[0031] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0032] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0033] In some embodiments, LBis a linker wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is substituted (C1-C12)alkyl and V9is -CO-; T10is (AA)p and V10is absent (e.g., a covalent bond); T11is PABC and V11is absent (e.g., a covalent bond); p is an integer from 1 to 10; g, h, i, j, and k are each 1; and l and m are each 0.

[0034] In further embodiments, the PABC is substituted with a glycoside, for example, a hydrogen of PABC is replaced with a glycoside, such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0035] In some embodiments, an ROR1-ADC is represented by Formula (I): 9 NAI-1540479824wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3and R4are each selected from hydrogen and (C1-C12)alkyl; LAis a first linker wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)pwhere p is an integer from 1-20 and V3is a covalent bond; T4is PABC and V4is a covalent bond; a, b, c, and d are each 1; e and f are each 0; and LBis a second linker wherein: T7is a covalent bond and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is substituted (C1-C12)alkyl and V9is -CO-; T10is (AA)p where p is an integer from 1-20 and V10is a covalent bond; T11is PABC and V11is a covalent bond; and g, h, i, j, and k are each 1; and l and m are each 0; s is an integer from 1 to 10; W1is a first drug; and W2is a second drug.

[0036] In some embodiments, one or both of W1and W2are camptothecin analogues, for example, belotecan.

[0037] In some embodiments, an ROR1-ADC is represented by Formula (I): 10 NAI-1540479824wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3and R4are each selected from hydrogen and (C1-C12)alkyl; LAis a linker wherein: T1is (C1-C6)alkyl and V1is -CONH-; T2is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t isinteger from 2 to 10, optionally 8, and V2is -CO-; T3is (AA)2 and V3is a covalent bond; T4is PABC substituted with a glycoside and V4is a covalent bond; a, b, c, and d are each 1; and e and f are each 0; and LBis a linker wherein: T7is a covalent bond and V7is -NHCO-; T8is (C1-C6)alkyl and V8is -CONH-; T9is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t isinteger from 2 to 10, optionally 8, and V9is -CO-; T10is (AA)2 and V10is a covalent bond; T11is PABC substituted with a glycoside and V11is a covalent bond; g, h, i, j, and k are each 1; and l and m are each 0; s is an integer from 1 to 10; 11 NAI-1540479824W1is a first drug; and W2is a second drug.

[0038] In some embodiments, the PABC of one or both of T4and T11is substituted with a glucuronide. In some embodiments, one or both of T1and T8is ethyl. In some embodiments, one or both of T2and T9is C5 alkylene substituted with -NHCO(PEG)t, wherein (PEG)t isinteger from 5-10, optionally 8. In some embodiments, one or both of W1and W2are camptothecin analogues, for example, belotecan.

[0039] In some embodiments, an ROR1-ADC is represented by Formula (II):wherein: Ab represents the antibody that binds to ROR1; and s is an integer from 1 to 10.

[0040] In some embodiments, s is an integer from 1 to 8. In some embodiments, s is 2. In some embodiments, s is 4.

[0041] Formula (II) may be prepared by conjugating one or more linker-payloads of Formula (IIa), shown below, with an ROR1 antibody: 12 NAI-1540479824

[0042] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II), wherein Ab comprises: a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26.

[0043] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II), wherein Ab comprises: (i) a VH region comprising a VH CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, and 36, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 6, 7, 8, 9, and 10, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 11, 12, 13, 14, and 37; and (ii) a VL region comprising a VL CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 15, 16, 17, and 18, a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 19, 20, and 21, and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 22, 23, and 24.

[0044] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II), wherein the Ab competes with any one of the ROR1 antibodies as disclosed herein in binding to ROR1, for example human ROR1.

[0045] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a 13 NAI-1540479824framework 4 (FR4) sequence, for example, as set forth in any one of SEQ ID NOs: 25 and 26.

[0046] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises human framework sequences.

[0047] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises (i) a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0048] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:41 and a light chain comprising the amino acid sequence of SEQ ID NO:28. In further embodiments, s is 4. In some embodiments, an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:28. In further embodiments, wherein s is 2.

[0049] In some embodiments, an ROR1-ADC is represented by Formula (II), wherein s is 4 and wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:41 and a light chain comprising the amino acid sequence of SEQ ID NO:28. Accordingly, the drug-to-antibody ratio (DAR) of the ROR1-ADC is 8 and the ROR1-ADC is referred to herein as ADC-8. In some embodiments, an ROR1-ADC is represented by Formula (II), wherein s is 2 and wherein Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:28. Accordingly, the DAR of the ROR1-ADC is 4.

[0050] The present disclosure also provides a pharmaceutical composition comprising an ROR1-ADC, wherein the ROR1-ADC is represented by Formula (I) or Formula (II) and a pharmaceutically acceptable excipient, wherein the ROR1 antibody (ROR1 Ab or Ab) is as described in any embodiment described herein. In some embodiments, such a pharmaceutical composition exhibits a drug-to-antibody ratio (DAR) of the ROR1-ADC of about 1 to about 20, for example, a DAR of about 2 to about 8, about 1 to about 4, about 2 to about 4, about 3 to about 4, about 4, about 1 to about 8, about 2 to about 8, about 3 to about 8, about 4 to about 8, about 5 to about 8, about 6 to about 8, about 6.5 to about 8, about 6 to about 7, about 6.5 to about 7.5, about 7 to about 8, about 6.5, about 7, about 7.5, or about 8.

[0051] The present disclosure also provides a method for treating a cancer or a tumor in a subject comprising administering to the subject the ROR1-ADC, wherein the ROR1-ADC is represented by Formula (I) or (II) or the pharmaceutical composition comprising an ROR1- 14 NAI-1540479824ADC of Formula (I) or (II) and a pharmaceutically acceptable excipient, wherein the ROR1 antibody is as described in any embodiment herein.

[0052] Provided herein is a kit comprising the antibody-drug conjugate as disclosed herein or the pharmaceutical composition as disclosed herein, and instructions for use. 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG.1 provides exemplary in vitro cytotoxicity results as detailed in Example 5.

[0054] FIGs.2A-2B provide exemplary in vivo efficacy data in MDA-MB-231 triple- negative breast cancer (TNBC) xenograft model, as detailed in Example 6. FIG.2A plots tumor volumes, while FIG.2B plots tumor growth inhibition percentages (%TGI) over the isotype control on Day 29.

[0055] FIGs.3A-3B provide exemplary in vivo efficacy data in JEKO-1 mantle cell lymphoma (MCL) xenograft model, as detailed in Example 7. FIG.3A plots tumor volumes, while FIG.3B plots tumor growth inhibition percentages (%TGI) over the isotype control on Day 25.

[0056] FIG.4 provides exemplary pharmacokinetics (PK) results in rats as detailed in Example 9.

[0057] FIGs.5A-5B provide exemplary toxicokinetics (TK) results of ADC-8 (FIG.5A) and ADC-4 (FIG.5B) in rats as detailed in Example 10.

[0058] FIG.6 provides exemplary in vivo efficacy data in a non-small cell lung cancer (NSCLC) patient-derived xenograft (PDX) model, as detailed in Example 8. 7. DETAILED DESCRIPTION

[0059] The present disclosure provides antibody-drug conjugates (ADCs) that bind to ROR1 and a drug conjugated (directly or indirectly) thereto. Such ROR1-ADCs are useful in compositions and in methods of treating, preventing, or alleviating an ROR1-mediated disease, disorder, or condition, including one or more symptoms of the disease, disorder, or condition. ROR1-mediated diseases, disorders, and conditions include a variety of cancers, including, but not limited to, any cancer wherein the tumor cells express or overexpress an ROR1 antigen. In addition, ROR1-ADCs are useful for the killing and / or removal of tumor cells. ROR1-ADCs described herein are useful in compositions and in methods for treating cancer. 7.1 DEFINITIONS

[0060] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in 15 NAI-1540479824Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed.2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed.2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dübel eds., 2d ed.2010). Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0061] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art-recognized meanings.

[0062] “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-).

[0063] The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1carbon atom) have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is 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, -SO2-alkyl, -SO2-aryl, -SO2- heteroaryl, and -NRaRb, wherein R’and R”may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0064] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -O-, 16 NAI-1540479824-NR10-, -NR10C(O)-, -C(O)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like.

[0065] “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.

[0066] The term “alkane” refers to alkyl group and alkylene group, as defined herein.

[0067] 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.

[0068] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0069] “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 alkenyl-O-, cycloalkyl- O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0070] 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.

[0071] The term “alkoxyamino” refers to the group –NH-alkoxy, wherein alkoxy is defined herein.

[0072] The term “haloalkoxy” refers to 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.

[0073] 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.

[0074] 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. 17 NAI-1540479824

[0075] 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.

[0076] “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.

[0077] The term “substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0078] “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH2C≡CH).

[0079] The term “substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2- aryl, and -SO2-heteroaryl.

[0080] “Alkynyloxy” refers to the group –O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0081] “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)- 18 NAI-1540479824, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl- C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl- C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-

[0082] “Acylamino” refers to the groups –NR20C(O)alkyl, -NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, -NR20C(O)substituted cycloalkyl, - NR20C(O)cycloalkenyl, -NR20C(O)substituted cycloalkenyl, -NR20C(O)alkenyl, -NR20C(O)substituted alkenyl, -NR20C(O)alkynyl, -NR20C(O)substituted alkynyl, -NR20C(O)aryl, -NR20C(O)substituted aryl, -NR20C(O)heteroaryl, -NR20C(O)substituted heteroaryl, -NR20C(O)heterocyclic, and -NR20C(O)substituted heterocyclic, wherein R20is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0083] “Aminocarbonyl” or the term “aminoacyl” refers to the group -C(O)NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0084] “Aminocarbonylamino” refers to the group –NR21C(O)NR22R23where R21, R22, and R23are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.

[0085] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein. 19 NAI-1540479824

[0086] The term “acyloxy” refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0087] “Aminosulfonyl” refers to the group –SO2NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0088] “Sulfonylamino” refers to the group –NR21SO2R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0089] “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 that has 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 20 NAI-1540479824amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2- aryl, -SO2-heteroaryl and trihalomethyl.

[0090] “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.

[0091] “Amino” refers to the group –NH2.

[0092] 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.

[0093] The term “azido” refers to the group –N3.

[0094] “Carboxyl,” “carboxy” or “carboxylate” refers to –CO2H or salts thereof.

[0095] “Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0096] “(Carboxyl ester)oxy” or “carbonate” refers to the groups –O-C(O)O- alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O- C(O)O-alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O-C(O)O-cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O- substituted cycloalkenyl, -O-C(O)O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O- heterocyclic, and -O-C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, 21 NAI-1540479824cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0097] “Cyano” or “nitrile” refers to the group –CN.

[0098] “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.

[0099] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0100] “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.

[0101] The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO- alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0102] “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.

[0103] “Cycloalkoxy” refers to –O-cycloalkyl.

[0104] “Cycloalkenyloxy” refers to –O-cycloalkenyl. 22 NAI-1540479824

[0105] “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

[0106] “Hydroxy” or “hydroxyl” refers to the group –OH.

[0107] “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→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0108] 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.

[0109] “Heteroaryloxy” refers to –O-heteroaryl.

[0110] “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- 23 NAI-1540479824oxide, -S(O)-, or -SO2- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein.

[0111] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0112] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO- heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0113] “Heterocyclyloxy” refers to the group –O-heterocyclyl.

[0114] The term “heterocyclylthio” refers to the group heterocyclic-S-.

[0115] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.

[0116] The term “hydroxyamino” refers to the group -NHOH.

[0117] “Nitro” refers to the group –NO2.

[0118] “Oxo” refers to the atom (=O).

[0119] “Sulfonyl” refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cylcoalkyl, -SO2-cycloalkenyl, -SO2-substituted cylcoalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2- substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein alkyl, 24 NAI-1540479824substituted 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-SO2-, phenyl-SO2-, and 4- methylphenyl-SO2-.

[0120] “Sulfonyloxy” refers to the group -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2- alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cylcoalkyl, -OSO2- cycloalkenyl, -OSO2-substituted cylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2- heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, and -OSO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0121] “Sulfate” or “sulfate ester” refers the group -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2- O-substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O-SO2-O-substituted cylcoalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cylcoalkenyl, -O-SO2-O-aryl, -O-SO2-O-substituted aryl, -O-SO2-O-heteroaryl, -O-SO2-O- substituted heteroaryl, -O-SO2-O-heterocyclic, and -O-SO2-O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0122] The term “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0123] “Thiol” refers to the group -SH.

[0124] “Thioxo” or the term “thioketo” refers to the atom (=S).

[0125] “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.

[0126] The term “substituted thioalkoxy” refers to the group -S-substituted alkyl.

[0127] 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. 25 NAI-1540479824

[0128] 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.

[0129] 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.

[0130] In 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.

[0131] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2or =S) on saturated carbon atoms in the specified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O–M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)O-M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-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+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific 26 NAI-1540479824examples, -NR80R80is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl.

[0132] 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, -SO3M+, -SO3R70, -OSO2R70, -OSO3–M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2–M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2–M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+.

[0133] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0134] 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.

[0135] 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. 27 NAI-1540479824

[0136] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] “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, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate. 28 NAI-1540479824

[0141] “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.

[0142] “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.

[0143] 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.

[0144] “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.

[0145] A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease, disorder, or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition or associated symptom(s).

[0146] The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0147] “Patient” refers to human and non-human subjects, especially mammalian subjects.

[0148] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the 29 NAI-1540479824development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.

[0149] In some embodiments, the term “treating,” or “treatment” excludes a prophylactic treatment.

[0150] 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 polypeptide having the moiety of interest conjugated to the polypeptide through the fGly residue.

[0151] “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.

[0152] “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.

[0153] 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.

[0154] The term “subject” refers to human and non-human subjects, especially mammalian subjects.

[0155] The terms “native amino acid sequence” as used herein refers to the amino acid sequence of a polypeptide prior to modification to include a modified amino acid residue.

[0156] The terms “amino acid analog,” “unnatural amino acid,” and the like is 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 30 NAI-1540479824stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some embodiments, 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 can 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, and the like) or an atom (such as Cl or Br, and the like), deletion of a group, substitution of a covalent bond (single bond for double bond, and the like), or combinations thereof. For example, amino acid analogs can include α-hydroxy acids, and α- amino acids, and the like.

[0157] The term “amino acid side chain” is used to refer to the substituent attached to the α-carbon of an amino acid residue, including natural amino acids, unnatural amino acids, and amino acid analogs. An amino acid side chain can also include an amino acid side chain as described in the context of the modified amino acids and / or conjugates described herein.

[0158] The term “carbohydrate” is used to refer to monomer units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar is 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 can be adapted for use in the subject compounds and conjugates.

[0159] The term “glycoside” or “glycosyl” refers to a sugar molecule or group bound to a moiety via a glycosidic bond. For example, the moiety that the glycoside is bound to can be a cleavable linker as described herein. A glycosidic bond can link the glycoside to the other moiety through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O-glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). In some cases, glycosides can be cleaved from the moiety they are attached to, such as by chemically mediated hydrolysis or enzymatically mediated hydrolysis.

[0160] As used herein, “ROR1 antigens” refer to a member of the tyrosine-protein kinase transmembrane receptor (ROR) family. ROR2 is another member of the same family. Yet, in some embodiments, the ROR1-ADCs and ROR1 antibodies as disclosed herein do not bind to ROR2 (such as human ROR2). In some embodiments, the ROR1-ADCs and ROR1 antibodies as disclosed herein do not bind to human ROR2 or cyno ROR2. In other 31 NAI-1540479824embodiments, the ROR1-ADCs and ROR1 antibodies as disclosed herein bind to ROR1 (e.g., human ROR1) with higher affinity than to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to ROR1 (e.g., human ROR1) is at least 2 folds of that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to ROR1 (e.g., human ROR1) is at least 5 folds of that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to ROR1 (e.g., human ROR1) is at least 10 folds of that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to ROR1 (e.g., human ROR1) is at least 100 folds of that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to ROR1 (e.g., human ROR1) is at least 1000 folds of that to ROR2 (e.g., human ROR2).

[0161] Thus, in one embodiment, the term “ROR1” or “ROR1 antigen” as used herein refers to ROR1 (e.g., human ROR1).

[0162] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and are used in the broadest sense and specifically covers, for example polyclonal antibodies, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, recombinantly produced antibodies, single domain antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human versions of antibodies having full length heavy and / or light chains. Antibodies also include single antibody domains as well as antibody fragments (and / or polypeptides that comprise antibody fragments) that retain ROR1 binding characteristics. Non-limiting examples of antibody fragments include antigen- binding regions and / or effector regions of the antibody, e.g., Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single-chain antibody molecule, dual variable domain antibody, single variable domain antibody, linear antibody, V region, a multispecific antibody formed from antibody fragments, F(ab)2, Fd, Fc, diabody, di-diabody, disulfide-linked Fvs (dsFv), single-domain antibody (e.g., nanobody) or other fragments (e.g., fragments consisting of the variable regions of the heavy and light chains that are non-covalently coupled). In general terms, a variable (V) region can be any suitable arrangement of immunoglobulin heavy (VH) and / or light (VL) variable regions. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, 32 NAI-1540479824and an antibody heavy chain monomer. Thus, for example, the V region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers that bind ROR1. In any embodiment, a VH region and a VL region can be covalently coupled either directly or through a linker to form a single-chain Fv (scFv). For ease of reference, scFv proteins are referred to herein as included in the category “antibody fragments.” Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also termed “minimal recognition units” or “hypervariable regions”) can be obtained by constructing polynucleotides that encode one or more CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA of antibody-producing cells as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137, Wiley- Liss, Inc. (1995)). Antibody fragments can be incorporated, for example, into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, variable regions of new antigen receptors (v-NAR), and bis-single-chain Fv regions (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies comprising a VH and / or VL contain a light chain and / or a heavy chain constant region, such as one or more constant regions, including one or more IgG1, IgG2, IgG3 and / or IgG4 constant regions. In some embodiments, antibodies can include epitope-binding fragments of any of the above. The antibodies described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.

[0163] 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 33 NAI-1540479824(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No.5,565,332). In certain embodiments, framework substitutions are identified by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies contemplated for use in the present invention are described in U.S. Pat. Nos.5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and 4,816,567, and PCT publications WO 98 / 45331 and WO 98 / 45332. In particular embodiments, a subject rabbit antibody may be humanized according to the methods set forth in US20040086979 and US20050033031. Accordingly, the antibodies described above may be humanized using methods that are well known in the art.

[0164] 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.

[0165] The term “monospecific,” as used herein denotes an antibody that has one or more binding sites each of which binds to the same epitope of the same antigen.

[0166] The term “multispecific” when used in reference to an antibody means that the antibody is able to specifically bind to at least two distinct epitopes, for example two binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) or each formed by a pair of VHH domains binding to different antigens or to different epitopes on the same antigen. Such a bispecific antibody may have a 1+1 format (comprising one binding site for a first antigen or epitope and one binding site for a second antigen or epitope). Other bispecific antibody formats may be 2+1 or 1+2 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). When a bispecific antibody comprises two antigen-binding sites, each may bind to a different epitope. Such a 34 NAI-1540479824bispecific antibody may bind to two different epitopes on the same antigen (e.g., epitopes on ROR).

[0167] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, basic local alignment search tool (BLAST), ALIGN, MegAlign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about ten residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the full-length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about ten bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

[0168] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a side chain with similar chemical characteristics. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains 35 NAI-1540479824(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and / or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions that do not eliminate binding are well known in the art.

[0169] The term “polypeptide” refers to a polymer of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can include (e.g., be substituted with) non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as linkage to or conjugation with (directly or indirectly) a moiety such as a labeling component or a drug (e.g., toxin). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure can be based upon antibodies or other members of the immunoglobulin superfamily, in some embodiments, the polypeptides can occur as single chains or dimers of single chains.

[0170] As used herein, an “antigen” is a moiety or molecule that contains an epitope to which an antibody can bind. As such, an antigen can be bound by an antibody. In some embodiments, the antigen to which an antibody described herein binds is an ROR1 antigen (e.g., a human ROR1 antigen), or a fragment thereof.

[0171] As used herein, an “epitope” is a term in the art and refers to a localized region of an antigen to which an antibody can bind. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope), or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope), e.g., human ROR1. It will be appreciated by one of skill in the art that, in general, a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure. For example, in some embodiments, an antibody binds to a group of amino acids regardless of whether they are folded in a natural three-dimensional protein structure. In other embodiments, an antibody 36 NAI-1540479824requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.

[0172] An antibody binds “an epitope,” “essentially the same epitope,” or “the same epitope” as a reference antibody. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping, or adjacent epitopes in a three- dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent or enzyme labels.

[0173] As used herein, the terms “specifically binds,” “specifically recognizes,” “immunospecifically binds,” “selectively binds,” “immunospecifically recognizes” and “immunospecific” are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope) as understood by one skilled in the art.

[0174] In some embodiments, “specifically binds” means, for instance, that a polypeptide or molecule interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. For example, a molecule that specifically binds to an antigen can bind to other peptides or polypeptides, generally with lower affinity as determined by, e.g., immunoassays, BIACORE™, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), the OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, an antibody or antigen binding region binds to or specifically binds to an antigen when it binds to the antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIAs) and enzyme linked immunosorbent assays (ELISAs). Typically, a specific or selective reaction will be at least twice background signal or noise and can be more than ten times background. See, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed.1989) for a discussion regarding binding specificity. In some embodiments, the extent of binding of an antibody or antigen-binding region to a “non- target” protein is less than about 10% of the binding of the antibody or antigen-binding region to its target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIAs. In some embodiments, molecules that specifically bind to an antigen bind to the antigen with a KAthat is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen. In some embodiments, molecules 37 NAI-1540479824that specifically bind to an antigen do not cross react with other proteins. In another specific embodiment, molecules that specifically bind to an antigen do not cross react with other non- ROR1 antigens. In some embodiments, “specifically binds” means, for instance, that a polypeptide or molecule binds a protein or target with a KD of about 0.1 mM or less, but more usually less than about 1 µM. In some embodiments, “specifically binds” means that a polypeptide or molecule binds a target with a KDof at least about 0.1 µM or less, at least about 0.01 µM or less, or at least about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or molecule that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or molecule that recognizes more than one protein or target. It is understood that, in some embodiments, a polypeptide or molecule that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, e.g., binding to a single target. Thus, in some embodiments, a polypeptide or molecule can specifically bind more than one target. In some embodiments, multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule. For example, in some embodiments, an antibody can comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In alternative embodiments, an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities. Generally, but not necessarily, reference to “binding” means “specific binding.”

[0175] The term “binding affinity” generally refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., an antigen such as ROR). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity is known in the art, any of which can be used for purposes of the present disclosure. In one embodiment, the “KD” or “KDvalue” can be measured by biolayer interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA). Alternatively, the KD may also be 38 NAI-1540479824measured in a radiolabeled antigen binding assay (RIA), for example, performed with the Fab version of an antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865- 881) or using surface plasmon resonance (SPR) assays by BIACORE™, using, for example, a BIACORE™-2000 or a BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). An “on-rate” or “rate of association” or “association rate” or “kon,” as well as an “off-rate” or “rate of dissociation” or “dissociation rate” or “koff,” can also be determined with the same SPR or BLI techniques described herein using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA) or a BIACORE™-2000 or a BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ), respectively.

[0176] The term “compete,” when used in the context of an ROR1 antibody, describes an antibody that, in the presence of another antibody, is at least partially inhibited from binding to an epitope or binding site due to binding of the other antibody. Competition can be determined by an assay in which the antibody under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., ROR). Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference molecule for binding to ROR1 (e.g., human ROR1). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol.137:3614-3619 or Cheung et al., (1990) Virology 176:546-552); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using I-125 label (see, e.g., Morel et al., (1988) Molec. Immunol.25:7-15); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., ROR1, such as human ROR1) bound to a solid surface or cells bearing either of an unlabeled test antigen binding protein (e.g., test ROR1 antibody or ADC) or a labeled reference antigen binding protein (e.g., reference ROR1 antibody or ADC). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually, the test antigen binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and / or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to 39 NAI-1540479824occur (e.g., similar epitope or overlapping epitope). Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 20%, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. In some embodiments, binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.

[0177] The terms “constant region” and “constant domain” are used interchangeably herein, are well-known antibody terms of art, and refer to an antibody portion, for example, a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to an antigen, but which can exhibit various effector functions, such as interaction with an Fc receptor. The term includes the portion of an immunoglobulin molecule having a generally more conserved amino acid sequence relative to an immunoglobulin variable region.

[0178] Antibody “effector functions” refer to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0179] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226 (of the EU numbering system) or from Pro230 (of the EU numbering system) to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 of the EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. An exemplary Fc region sequence is provided below (CH2 domain = bold text; CH3 domain = underline text): CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:38). 40 NAI-1540479824

[0180] A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), and the like. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays as disclosed.

[0181] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and / or changed (e.g., isolated, purified, selected, including, or combining with other sequences such as variable region sequences) by a human. Native sequence human Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region; as well as naturally occurring variants thereof.

[0182] A “variant Fc region” comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, (e.g., substituting, addition, or deletion) preferably one or more amino acid substitution(s). In some embodiments, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region described herein can possess at least about 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith. The variant Fc region herein described herein can have a loss of effector function (e.g., silent Fc, (also referred to herein as “sFc”)).

[0183] In some embodiments, a sFc comprises an alanine (Ala, A) residue at position Leu234 (L234) according to the EU numbering system, an alanine (Ala, A) residue at position Leu235 (L235) according to the EU numbering system, and a lysine (Lys, K) residue at position Pro329 (P329) according to the EU numbering system (also referred to herein as “LALAPK” or “L234A / L235A / P329K”).

[0184] An exemplary variant Fc region (“silent Fc”) sequence is provided below (CH2 domain = bold text with amino acid changes underlined; CH3 domain = underline text): 41 NAI-1540479824CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:39).

[0185] Additionally or alternatively, a variant Fc region has a reduced potential immunogenicity. In further embodiments, a variant Fc region comprises a glutamic acid (Glu, E) residue at position Asp356 (D356) according to the EU numbering system, a glutamic acid (Glu, E) residue at position Glu357 (E357) according to the EU numbering system, and a methionine (Met, M) residue position Leu358 (L358) according to the EU numbering system (also referred to herein as “EEM” or “D356E / E357E / L358M”)

[0186] The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes one or more constant regions. The “heavy chain” can refer to any distinct types, e.g., for example, alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (µ), based on the amino acid sequence of the constant region, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4.

[0187] As used herein, the term “light chain” when used in reference to an antibody can refer to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, e.g., kappa (κ) or lambda (λ) based on the amino acid sequence of the constant regions. Light chain amino acid sequences are well known in the art. In one embodiment, a “chain” (e.g., a heavy chain or a light chain) is itself a molecule (e.g., a polypeptide). In another embodiment, a “chain” (e.g., a heavy chain or a light chain) is part of a molecule (e.g., a polypeptide), for example, is directly or indirectly conjugated to the remaining part of the molecule (such as polypeptide).

[0188] The terms “antigen binding fragment,” “antigen binding domain,” “antigen binding region,” and similar terms refer to a portion of an antibody that comprises amino acid residues that interact with an antigen and confer on the binding fragment or region its specificity and affinity for the antigen (e.g., the CDRs). “Antigen binding fragment” as used herein includes “antibody fragment,” which comprises a portion of an antibody including one or more CDRs, such as the antigen binding or variable region of the antibody. 42 NAI-1540479824

[0189] Antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, and the like), camelized antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0190] In some embodiments, antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain one or more antigen binding sites that bind to ROR1.

[0191] An antibody, as described herein, can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In some embodiments, an ROR1 antibody, as described herein, is an IgG antibody (e.g., human IgG), or a class (e.g., human IgG1, IgG2, IgG3, or IgG4) or a subclass thereof.

[0192] In some embodiments, an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In further embodiments, the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical. In other embodiments, the amino acid sequences of the H chains are different from each other. Additionally or alternatively, the amino acid sequences of the L chains are different from each other. For example, an antibody comprises a first H / L chain pair and a second H / L chain pair, wherein the first H / L chain pair binds to an ROR1 antigen and the second H / L chain pair binds to another ROR1 antigen or a non-ROR1 antigen. In some embodiments, an antibody is a 2-chain antibody unit comprising a VHH-VHH pair. In further embodiments, the amino acid sequences of the VHH are identical. In other embodiments, the amino acid sequence of the VHH are different from each other. For example, an antibody comprises a first VHH and a second VHH, wherein the first VHH binds to an ROR1 antigen and the second VHH binds to another ROR1 antigen or a non-ROR1 antigen. In some embodiments, the H and / or L chains comprise constant regions, for example, human constant regions. In some embodiments, the L chain constant region of such antibodies is a kappa or lambda light chain constant region, for example, a human kappa or lambda light chain constant region. In some embodiments, the H chain constant region of such antibodies comprises a gamma heavy chain constant region, for example, a human gamma heavy chain constant region. In some embodiments, such antibodies comprise IgG constant regions, for example, human IgG constant regions (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions). 43 NAI-1540479824

[0193] As used herein, “ROR1 antibody” and “antibody that binds to ROR1” are used interchangeably and refer to an antibody that preferentially binds to ROR1. An antibody or fragment thereof can preferentially bind to ROR1, such as human ROR1, which means that the antibody or fragment thereof binds to ROR1, such as human ROR1, with greater affinity than it binds to an unrelated control protein. For example, the antibody or fragment thereof can specifically recognize and bind to ROR1 or a portion thereof. “Specific binding” means that the ROR1 antibody or fragment thereof binds to ROR1 with an affinity that is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times greater than the affinity for an unrelated control protein (e.g., hen egg white lysozyme). In some embodiments, the ROR1 antibody or fragment thereof can bind ROR1 substantially exclusively (e.g., is able to distinguish ROR1 from other known polypeptides, for example, by virtue of measurable differences in binding affinity). In some embodiments, an ROR1 antibody can react with ROR1 sequences other than human ROR1 sequences (e.g., cynomolgus ROR1 sequences).

[0194] The terms “variable region” and “variable domain” are used interchangeably to refer to a portion of the light and heavy chains of an antibody that are generally located at the amino-terminal of the light and heavy chain, has a length of about 120 to 130 amino acids in the heavy chain, about 100 to 110 amino acids in the light chain, and is used in the binding and specificity of each antibody for its antigen. The variable region of the heavy chain is referred to herein as “VH.” The variable region of the light chain is referred to herein as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” or alternatively called “complementarity determining regions.” The variable regions of heavy and light chains each comprise four frameworks (FR1, FR2, FR3 and FR4), largely adopting a β sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β sheet structure. The hypervariable regions in each chain are held together in proximity by the frameworks and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). The constant regions are not involved directly in 44 NAI-1540479824binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable region are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. In specific embodiments, the variable region is a human variable region.

[0195] The term “hypervariable region,” “HVR,” “HV,” “complementarity determining region, “and “CDR” when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions: three in the VH (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3), and three in the VL (L1 or VL CDR1, L2 or VL CDR2, and L3 or VL CDR3). Several hypervariable region delineations are in use and are encompassed herein. The Kabat CDRs are based on sequence variability and are the most used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol.2, Chapter 3, Springer Verlag). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

[0196] A universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT®) Information System (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT®is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable region is conserved between species and 45 NAI-1540479824present in structures called loops, by using numbering systems that align variable region sequences of structural features, CDR and framework residues and are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Plückthun, J. Mol. Biol. 309: 657-670 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT®unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) and is also illustrated below. Various systems known in the art or described herein represent different ways of delineating CDRs, and when they are used to define the same antibody, they are often considered equivalent. An exemplary system, shown herein, combines Kabat and Chothia.

[0197] Hypervariable regions can comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26- 35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. As used herein, the terms “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” are used interchangeably.

[0198] 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.

[0199] The terms “ROR1-mediated disease,” “ROR1-mediated disorder,” and “ROR1- mediated condition” are used interchangeably and refer to any disease, disorder or condition associated with or characterized by ROR1-expressing cells, such as ROR1-expressing tumor cells. An ROR1-mediated disease includes a cancer including, but not limited to, cancers that express or overexpress ROR1. 46 NAI-1540479824

[0200] The term “tumor,” in any embodiment herein, refers to any neoplastic cell growth or proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.

[0201] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0202] The term “ADC” refers to an antibody-drug conjugate, which in the context of the present invention refers to an ROR1 antibody, which is coupled to another moiety which includes a drug, as described herein.

[0203] As used herein, “drug” refers to a compound that has biological activity, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, and the like).

[0204] 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. Cancer chemotherapeutic agents include non-peptidic (e.g., 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.

[0205] Suitable cancer chemotherapeutic agents 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 ROR1-ADC of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD)).

[0206] Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, 47 NAI-1540479824cyclophosphamide (CYNOTAN™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0207] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U®), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6- thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10- propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0208] Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (TAXOL®), docetaxel (TAXOTERE®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine, and the like; podophyllotoxins, e.g., etoposide, teniposide, and the like; antibiotics, e.g., anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, and the like; phenoxizone biscyclopeptides, e.g., dactinomycin; basic glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mithramycin); anthracenediones, e.g., mitoxantrone; azirinopyrrolo indolediones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like; and the like.

[0209] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0210] Microtubule affecting agents that have antiproliferative activity are also suitable for use and include, but are not limited to, allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (TAXOL®), TAXOL®derivatives, docetaxel (TAXOTERE®), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0211] Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g., prednisone, dexamethasone, and the like; estrogens and pregestins, e.g., hydroxyprogesterone caproate, 48 NAI-1540479824medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; and the like; adrenocortical suppressants, e.g., aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (DROGENIL®), toremifene (FARESTON®), and goserelin (ZOLADEX®), and the like. Estrogens stimulate proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0212] Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis- DDP), carboplatin, and the like; ureas, e.g., hydroxyurea; hydrazines, e.g., N- methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; and the like Other anti-proliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); gefitinib (IRESSA®, ZD 1839, 4-(3- chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); and the like.

[0213] Taxanes are suitable for use. “Taxanes” include paclitaxel, as well as any active taxane derivative or pro-drug. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL®, TAXOTERE®(a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) can be readily prepared utilizing techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Pat. Nos.5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis). Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., TAXOTERE®docetaxel, as noted herein) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0214] Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazino and other derivatives described in WO 99 / 14209; 49 NAI-1540479824taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No.5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Patent No.5,821,263; and taxol derivative described in U.S. Patent No.5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No.5,824,701.

[0215] Biological response modifiers suitable for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that bind specifically to a tumor antigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.

[0216] An “effective amount” is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate the symptoms and / or underlying cause, prevent the occurrence of symptoms and / or their underlying cause, and / or improve or remediate the damage that results from or is associated with a disease, disorder, or condition. In some embodiments, the effective amount is a therapeutically effective amount.

[0217] The term “therapeutically effective amount” as used herein refers to the amount of an antibody or ADC described herein that is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder, or condition, and / or a symptom related thereto. A therapeutically effective amount of an agent, including a therapeutic agent, can be an amount necessary for (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and / or (iii) to improve or enhance the therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody or ADC described herein). A “therapeutically effective amount” of a substance / molecule / agent of the present disclosure (e.g., an ROR1 antibody or ADC) can vary based on a number of factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule / agent, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In some embodiments, the term “therapeutically effective amount” refers to an amount of an antibody or other agent (e.g., or drug) effective to “treat” a disease, disorder, or condition, in a subject or mammal.

[0218] In some embodiments, the drug is a microtubule affecting agent that has anti- proliferative activity, such as a maytansinoid. In some embodiments, the drug is an 50 NAI-1540479824antimitotic agent, such as an auristatin or an active auristatin analog or derivative thereof. In some embodiments, the drug is a DNA alkylating agent.

[0219] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0220] “Excipients” include carriers, excipients, preservatives, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed and can be included, for example, to affect stability, bulk up formulations, or to confer a therapeutic enhancement on the active ingredient in the final dosage form (e.g., facilitating absorption, reducing viscosity, enhancing solubility). An "excipient" can be an organic or inorganic ingredient, natural or synthetic with which the active ingredient is combined to facilitate the use of the active ingredient, e.g., the administration of the active ingredient to a subject. Examples of excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (e.g., less than about ten amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freund’s adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered. Such excipients can be sterile liquids, such as water and oils, such as those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, in any embodiment, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release 51 NAI-1540479824formulations, and the like. Oral compositions, such as formulations, can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable excipients are described in Remington: The Science and Practice of Pharmacy (2020) (Elsevier Science, Amsterdam, Netherlands), including pharmaceutical compounds, can contain an effective amount or therapeutically effective amount of an ROR1-ADC, for example, in isolated or purified form, together with a suitable amount of excipient to provide the form for proper administration to the subject. The formulation should suit the mode of administration.

[0221] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less variation of a given value or range.

[0222] As used herein, comparative terms as used herein, such as reduce, decrease, increase, or any grammatical variation thereof, can refer to certain variation from the reference. In some embodiments, such variation can refer to about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 1 fold, or about 2 fold, or about 3 fold, or about 4 fold, or about 5 fold, or about 10 fold, or about 20 fold, or about 30 fold, or about 40 fold, or about 100 fold or higher than the reference. In some embodiments, such variation can refer to about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% of the reference.

[0223] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0224] In some embodiments, the terms “first,” “second,” “third,” “fourth” and similar in a component name are used to distinguish and identify more than one component sharing certain identity in their names. For example, “first antibody” and “second antibody” are used to distinguish two antibodies.

[0225] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of” otherwise analogous embodiments described in terms of “consisting of” are also provided. 52 NAI-1540479824

[0226] The term “between” as used in a phrase as such “between A and B” or “between A- B” refers to a range including both A and B.

[0227] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0228] The term “optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances wherein the circumstance occurs, and the instances wherein the circumstance does not occur.

[0229] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0230] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0231] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (e.g., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing 53 NAI-1540479824such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0232] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0233] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0234] 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. 7.2 ROR1-ADCs

[0235] An antibody that binds to tyrosine-protein kinase membrane receptor 1 (ROR1) (also referred to herein as “ROR1 antibody,” “anti-ROR1 antibody,” “ROR1 Ab,” “Ab” or “antibody”) and a drug can be linked directly or indirectly to each other via a pyridazine- pyrrolo coupling moiety to form an ROR1-ADC as described herein. In certain embodiments, the ROR1 antibody and the two or more drugs or active agents are bound to each other through one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds can include a branched linker as described herein.

[0236] Moieties of interest (e.g., drugs or active agents) can be conjugated to the ROR1 antibody at any desired site of the antibody. Thus, the present disclosure provides, for example, an ROR1 antibody that has moieties conjugated at two or more sites on the antibody, such as a site at or near the C-terminus of the antibody, a position at or near the N- terminus of the antibody, and a position between the C-terminus and the N-terminus of the 54 NAI-1540479824antibody (e.g., at an internal site of the antibody). Combinations of the above conjugation sites are also possible.

[0237] In certain embodiments, a conjugate of the present disclosure includes two (or more) drugs or active agents conjugated to an amino acid residue of an ROR1 antibody at the α-carbon of an amino acid residue. Stated another way, a conjugate includes an ROR1 antibody where the side chain of an amino acid residue in the antibody has been modified and attached to two (or more) drugs or active agents (e.g., attached to two drugs or active agents through a branched linker as described herein). For example, a conjugate includes an ROR1 antibody where the α-carbon of an amino acid residue in the antibody has been modified and attached to two drugs or active agents (e.g., attached to two drugs or active agents through a branched linker as described herein).

[0238] Embodiments of the present disclosure include conjugates where an ROR1 antibody is conjugated to two or more moieties, such as 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, 10 moieties, 11 moieties, 12 moieties, 13 moieties, 14 moieties, 15 moieties, 16 moieties, 17 moieties, 18 moieties, 19 moieties, or 20 or more moieties. The moieties may be conjugated to the ROR1 antibody at multiple sites in the antibody. In some embodiments, two moieties may be conjugated to a single amino acid residue of the ROR1 antibody. For instance, two moieties may be conjugated to the same amino acid residue of the ROR1 antibody. In other embodiments, two moieties are conjugated to a first amino acid residue of the ROR1 antibody and two other moieties are conjugated to a second amino acid residue of the ROR1 antibody. For example, an ROR1 antibody can be conjugated to first and second moieties at a first amino acid residue and conjugated to third and fourth moieties at a second amino acid residue, etc. In some cases, two or more amino acid residues in the ROR1 antibody are each conjugated to a pair of moieties (e.g., two moieties), where each pair of moieties is conjugated to the ROR1 antibody through a branched linker as described herein. In some cases, 1 amino acid residue in the ROR1 antibody is conjugated to a pair of moieties through a branched linker as described herein. In other instances, 2 or more amino acid residues, such as 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid residues in the ROR1 antibody are each conjugated to a pair of moieties through a branched linker as described herein.

[0239] The one or more amino acid residues of the ROR1 antibody that are conjugated to the moieties of interest may be naturally occurring amino acids, unnatural amino acids, or combinations thereof. For instance, the conjugate may include moieties of interest (e.g., drugs or active agents) conjugated to a naturally occurring amino acid residue of the ROR1 55 NAI-1540479824antibody. In other instances, the conjugate may include moieties of interest conjugated to an unnatural amino acid residue of the ROR1 antibody. The moieties of interest may be conjugated to the ROR1 antibody at a single natural or unnatural amino acid residue as described above. One or more natural or unnatural amino acid residues in the ROR1 antibody may be conjugated to the moieties of interest as described herein. For example, two (or more) amino acid residues (e.g., natural or unnatural amino acid residues) in the ROR1 antibody may each be conjugated to two moieties through a branched linker, such that multiple sites in the ROR1 antibody are conjugated to the moieties of interest.

[0240] As described herein, an ROR1 antibody may be conjugated to two or more moieties of interest. In certain embodiments, the moiety of interest is a payload, for instance, a chemical entity, such as a drug, an active agent, or a detectable label. For example, drugs (or active agents, such as cytokines) may be conjugated to the ROR1 antibody, or in other embodiments, detectable labels may be conjugated to the ROR1 antibody. In other embodiments, combinations of different payloads may be conjugated to the ROR1 antibody. Thus, for instance, embodiments of the present disclosure include, but are not limited to, the following: a conjugate of an ROR1 antibody and two or more drugs; a conjugate of an ROR1 antibody and two or more active agents, such as cytokines; a conjugate of an ROR1 antibody and two or more detectable labels; and combinations thereof.

[0241] In certain embodiments, the ROR1 antibody and the moieties of interest (e.g., drugs or active agents) are conjugated through a conjugation moiety. For example, the ROR1 antibody and the moieties of interest may each be bound (e.g., covalently bonded) to the conjugation moiety, thus indirectly binding the ROR1 antibody and the moieties of interest together through the conjugation moiety. In some cases, the conjugation moiety includes a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound, or a derivative of a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl compound. For instance, a general scheme for coupling moieties of interest to an ROR1 antibody through a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety is shown in the general reaction scheme below. Hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also referred to herein as a hydrazino-iso-Pictet-Spengler (HIPS) conjugation moiety and an aza- hydrazino-iso-Pictet-Spengler (azaHIPS) conjugation moiety, respectively.56 NAI-1540479824

[0242] In the reaction scheme above, each R independently includes a moiety of interest (e.g., drug or active agent) that is conjugated to the ROR1 antibody (e.g., conjugated to the ROR1 antibody through a linker as described herein), where n is an integer from 1 to 4. As shown in the reaction scheme above, a conjugation moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety) is attached to two or more drugs or active agents, R. An ROR1 antibody that includes a 2-formylglycine residue (fGly) is reacted with the conjugation moiety to produce an ROR1 antibody conjugate, thus attaching the two or more drugs or active agents to the ROR1 antibody through the conjugation moiety.

[0243] As described herein, the moieties can be any of a variety of moieties such as, but not limited to, chemical entities, such as detectable labels, or drugs or active agents. R’ and R” may each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, 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 CR21, NR22, N, O or S, where R21and R22are each independently selected from any of the substituents described for R’ and R” above.

[0244] Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties may be attached (e.g., covalently attached) to two or more linkers. As such, embodiments of the present disclosure include a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety attached to two or more drugs or active agents each through a corresponding linker. Thus, conjugates of the present disclosure may include two or more linkers, where each linker attaches a corresponding drug or active agent to the hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety. Accordingly, the hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety and two or more linkers may be viewed overall as a “branched linker,” where the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety is attached to two of more “branches,” where each branch includes a linker attached to a drug or active agent.

[0245] Combinations of the same or different payloads may be conjugated to the ROR1 antibody through the branched linker. In certain embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are the same payload (e.g., 57 NAI-1540479824drug, active agent, or detectable label). For example, a first branch of a branched linker may be attached to a payload (e.g., drug, active agent, or detectable label) and a second branch of the branched linker may be attached to the same payload (e.g., drug, active agent, or detectable label) as the first branch.

[0246] In other embodiments, the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are different payloads (e.g., drugs, active agents, or detectable labels). For example, a first branch of a branched linker may be attached to a first payload (e.g., a first drug, active agent, or detectable label) and a second branch of the branched linker may be attached to a second payload (e.g., a second drug, active agent, or detectable label) different from the first payload (e.g., the first drug, active agent, or detectable label) attached to the first branch.

[0247] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. By “synergistic,” “synergism” or “synergy” is meant a therapeutic effect that is greater than the sum of the effects of the drugs or active agents taken separately. For example, in some instances, the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC.

[0248] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that provide an enhanced therapeutic benefit as compared to the use of the drugs or active agents separately, For example, the drugs or active agents may provide an increased effect on drug delivery of the ADC (e.g., some payloads, such as the iRGD peptide, can increase extravasation into tissues and augment tumor penetration).

[0249] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this may provide a decrease in tumor drug resistance by targeting multiple pathways. Examples of payload combinations can include, but are not limited to, cytotoxic drugs, immunomodulatory molecules to activate or inhibit immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, and the like.

[0250] In some embodiments, where two different payloads are attached to the branched linker, the payloads may be selected from combinations of drugs or active agents and detectable labels. For example, a first payload may be a detectable label that is used as an 58 NAI-1540479824imaging agent or tracer to detect the location of the ADC in vivo, while a second payload may be a drug or active agent that provides a therapeutic activity.

[0251] Various embodiments of the linkers that may couple the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the drugs or active agents are described in detail herein. For example, in some instances, the linker is a cleavable linker, such as a cleavable linker as described herein.

[0252] In certain embodiments, the ROR1 antibody may be conjugated to two or more moieties of interest, where one or more amino acids of the ROR1 antibody are modified before conjugation to the moieties of interest. Modification of one or more amino acids of the ROR1 antibody may produce an ROR1 antibody that contains one or more reactive groups suitable for conjugation to the moieties of interest. In some cases, the ROR1 antibody may include one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to the moieties of interest (e.g., where two or more moieties are attached to a conjugation moiety, such as a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety as described above). For example, an amino acid of the ROR1 antibody may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). A reactive aldehyde may be included in an “aldehyde tag” or “ald-tag”, which, as used herein, refers to an amino acid sequence derived from a sulfatase motif (e.g., L(C / S)TPSR, SEQ ID NO:99) 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 (e.g., a sulfatase motif in which a cysteine or serine residue has been converted to fGly by action of an FGE, e.g., L(fGly)TPSR, SEQ ID NO:123). A converted sulfatase motif may be produced from an amino acid sequence that includes an “unconverted” sulfatase motif (e.g., 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: LCTPSR, SEQ ID NO:100). 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. 59 NAI-1540479824

[0253] In some cases, to produce the conjugate, the ROR1 antibody containing the fGly residue may be conjugated to the moieties of interest by reaction of the fGly with a compound (e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety, as described above). For example, an fGly-containing ROR1 antibody may be contacted with a reactive partner under conditions suitable to provide for conjugation of two or more drugs to the ROR1 antibody. In some instances, the reactive partner may include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above. For example, two or more drugs or active agents may be attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. In some cases, the drugs or active agents are attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety, such as covalently attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl, where each drug or active agent is attached through a corresponding linker to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. Accordingly, the fGly residue conjugated to the moieties of interest after the reaction is referred to herein as fGly’.

[0254] In certain embodiments, a conjugate of the present disclosure includes an ROR1 antibody having at least one amino acid residue that has been attached to two or more moieties of interest (e.g., drugs or active agents). In order to make the conjugate, an amino acid residue of the ROR1 antibody may be modified and then coupled to two or more drugs or active agents attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above. In certain embodiments, an amino acid residue of the ROR1 antibody is a cysteine or serine residue that is modified to an fGly residue, as described above. In certain embodiments, the modified amino acid residue (e.g., fGly residue) is conjugated to two or more drugs or active agents containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above to provide a conjugate of the present disclosure where the two or more drugs or active agents are conjugated to the ROR1 antibody through the hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety. As used herein, the term fGly’ refers to the amino acid residue of the ROR1 antibody that is coupled to the moieties of interest (e.g., drugs or active agents).

[0255] In certain embodiments, the conjugate includes an ROR1 antibody having at least one amino acid residue attached to a branched linker as described herein, which in turn is attached to two or more drugs or active agents. For instance, the conjugate may include an ROR1 antibody having at least one amino acid residue (fGly’) that is conjugated to the moieties of interest (e.g., drugs or active agents) as described above. 60 NAI-1540479824

[0256] Aspects of the present disclosure include a conjugate of Formula (I):wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, Z3and Z4are each independently selected from CR4, N and C-LB-W2, wherein at least one Z1, Z2, Z3and Z4is C-LB-W2; R1is 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; R2and R3are 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 R2and R3are optionally cyclically linked to form a 5 or 6-membered heterocyclyl; each R4is 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; LAis a first linker; LBis a second linker; s is an integer from 1 to 10; W1is a first drug; and 61 NAI-1540479824W2is a second drug.

[0257] The substituents related to conjugates of Formula (I) are described in more detail below.

[0258] In certain embodiments, Z1, Z2, Z3and Z4are each independently selected from CR4, N and C-LB-W2, wherein at least one Z1, Z2, Z3and Z4is C-LB-W2. In certain embodiments, Z1is CR4. In certain embodiments, Z1is N. In certain embodiments, Z1is C- LB-W2. In certain embodiments, Z2is CR4. In certain embodiments, Z2is N. In certain embodiments, Z2is C-LB-W2. In certain embodiments, Z3is CR4. In certain embodiments, Z3is N. In certain embodiments, Z3is C-LB-W2. In certain embodiments, Z4is CR4. In certain embodiments, Z4is N. In certain embodiments, Z4is C-LB-W2. In some embodiments, each of Z1, Z3, and Z4is CR4. In some embodiments, Z3is C-LB-W2.

[0259] Combinations of various Z1, Z2, Z3and Z4are possible. For example, in some instances, Z1is C-LB-W2, Z2is CR4, Z3is CR4, and Z4is CR4. In some instances, Z1is CR4, Z2is C-LB-W2, Z3is CR4, and Z4is CR4. In some instances, Z1is CR4, Z2is CR4, Z3is C-LB- W2, and Z4is CR4. In some instances, Z1is CR4, Z2is CR4, Z3is CR4, and Z4is C-LB-W2.

[0260] In certain embodiments, R1is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl. In certain embodiments, R1is hydrogen. In certain embodiments, R1is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R1is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R1is alkynyl or substituted alkynyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R1is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R1is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R1is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R1is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl or C3-8substituted heterocyclyl, such as a 62 NAI-1540479824C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.

[0261] In certain embodiments, R2and R3are 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 R2and R3are optionally cyclically linked to form a 5 or 6-membered heterocyclyl.

[0262] In certain embodiments, R2is 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, R2is hydrogen. In certain embodiments, R2is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R2is methyl. In certain embodiments, R2is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R2is alkynyl or substituted alkynyl. In certain embodiments, R2is alkoxy or substituted alkoxy. In certain embodiments, R2is amino or substituted amino. In certain embodiments, R2is carboxyl or carboxyl ester. In certain embodiments, R2is acyl or acyloxy. In certain embodiments, R2is acyl amino or amino acyl. In certain embodiments, R2is alkylamide or substituted alkylamide. In certain embodiments, R2is sulfonyl. In certain embodiments, R2is thioalkoxy or substituted thioalkoxy. In certain embodiments, R2is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R2is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R2is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R2is heterocyclyl or substituted heterocyclyl, such as a C3-6heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl. 63 NAI-1540479824

[0263] In certain embodiments, R3is 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, R3is hydrogen. In certain embodiments, R3is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R3is methyl. In certain embodiments, R3is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4alkenyl or C2-4substituted alkenyl, or C2-3alkenyl or C2-3 substituted alkenyl. In certain embodiments, R3is alkynyl or substituted alkynyl. In certain embodiments, R3is alkoxy or substituted alkoxy. In certain embodiments, R3is amino or substituted amino. In certain embodiments, R3is carboxyl or carboxyl ester. In certain embodiments, R3is acyl or acyloxy. In certain embodiments, R3is acyl amino or amino acyl. In certain embodiments, R3is alkylamide or substituted alkylamide. In certain embodiments, R3is sulfonyl. In certain embodiments, R3is thioalkoxy or substituted thioalkoxy. In certain embodiments, R3is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R3is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6 substituted heteroaryl. In certain embodiments, R3is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R3is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5substituted heterocyclyl.

[0264] In certain embodiment, both R2and R3are methyl.

[0265] In certain embodiments, R2and R3are optionally cyclically linked to form a 5 or 6- membered heterocyclyl. In certain embodiments, R2and R3are cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R2and R3are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R2and R3are cyclically linked to form a 6-membered heterocyclyl.

[0266] In certain embodiments, each R4is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, 64 NAI-1540479824substituted 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.

[0267] The various possibilities for each R4are described in more detail as follows. In certain embodiments, R4is hydrogen. In certain embodiments, each R4is hydrogen. In certain embodiments, R4is halogen, such as F, Cl, Br, or I. In certain embodiments, R4is F. In certain embodiments, R4is Cl. In certain embodiments, R4is Br. In certain embodiments, R4is I. In certain embodiments, R4is alkyl or substituted alkyl, such as C1-6alkyl or C1-6substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R4is methyl. In certain embodiments, R4is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3alkenyl or C2-3substituted alkenyl. In certain embodiments, R4is alkynyl or substituted alkynyl. In certain embodiments, R4is alkoxy or substituted alkoxy. In certain embodiments, R4is amino or substituted amino. In certain embodiments, R4is carboxyl or carboxyl ester. In certain embodiments, R4is acyl or acyloxy. In certain embodiments, R4is acyl amino or amino acyl. In certain embodiments, R4is alkylamide or substituted alkylamide. In certain embodiments, R4is sulfonyl. In certain embodiments, R4is thioalkoxy or substituted thioalkoxy. In certain embodiments, R4is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, R4is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6substituted heteroaryl. In certain embodiments, R4is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3- 8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R4is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.

[0268] In certain embodiments, LAis a first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.

[0269] In certain embodiments, LBis a second linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below. 65 NAI-1540479824

[0270] In certain embodiments, W1is a first drug (or a first active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0271] In certain embodiments, W2is a second drug (or a second active agent). Examples of drugs and active agents that can be used in the conjugates of the present disclosure are described in more detail below.

[0272] In certain embodiments, Ab represents an antibody that binds to ROR1 (“ROR1 antibody”). In certain embodiments, the antibody Ab comprises one or more fGly’ residues as described herein. In certain embodiments, the ROR1 antibody is attached to the rest of the conjugate through an fGly’ residue as described herein. Examples of ROR1 antibodies that can be used in the conjugates of the present disclosure are described in more detail below.

[0273] In certain embodiments, the conjugate of Formula (I) includes a first linker, LA. The first linker, LA, may be utilized to bind a first moiety of interest (e.g., a first drug or active agent) to an ROR1 antibody through a conjugation moiety. The first linker, LA, may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein). For example, the first linker, LA, may attach a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to a first drug. The hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety may be used to conjugate the first linker, LA, (and thus the first drug) to an ROR1 antibody.

[0274] For example, as shown in Formula (I) above, LAis attached to the antibody Ab through a conjugation moiety, and thus the antibody Ab is indirectly bonded to the linker LAthrough the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As described above, the antibody Ab is an ROR1 antibody, and thus LAis attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the ROR1 antibody, e.g., the linker LAis indirectly bonded to the ROR1 antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0275] Any convenient linker may be utilized for the first linker LAin the subject conjugates and compounds. In certain embodiments, the first linker LAmay include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker LAmay include an alkyl or substituted alkyl group. In certain embodiments, the first linker LAmay include an alkenyl or substituted alkenyl group. 66 NAI-1540479824In certain embodiments, the first linker LAmay include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker LAmay include an alkoxy or substituted alkoxy group. In certain embodiments, the first linker LAmay include an amino or substituted amino group. In certain embodiments, the first linker LAmay include a carboxyl or carboxyl ester group. In certain embodiments, the first linker LAmay include an acyl amino group. In certain embodiments, the first linker LAmay include an alkylamide or substituted alkylamide group. In certain embodiments, the first linker LAmay include an aryl or substituted aryl group. In certain embodiments, the first linker LAmay include a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker LAmay include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker LAmay include a heterocyclyl or substituted heterocyclyl group.

[0276] In certain embodiments, the first linker LAmay include a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where 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.

[0277] In some embodiments, LAis a first linker described by the formula:wherein L1, L2, L3, L4, L5and L6are each independently a linker subunit, and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a, b, c, d, e, and f is 1.

[0278] In certain embodiments, the sum of a, b, c, d, e, and f is 1 to 6. In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1 and f is 0. In certain embodiments, a, b, c, and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e, and f are each 0. In certain embodiments, a is 1 and b, c, d, e, and f are each 0. 67 NAI-1540479824

[0279] In certain embodiments, the linker subunit L1is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). In certain embodiments, the linker subunit L2, if present, is attached to the first drug or active agent W1. In certain embodiments, the linker subunit L3, if present, is attached to the first drug or active agent W1. In certain embodiments, the linker subunit L4, if present, is attached to the first drug or active agent W1. In certain embodiments, the linker subunit L5, if present, is attached to the first drug or active agent W1. In certain embodiments, the linker subunit L6, if present, is attached to the first drug or active agent W1.

[0280] Any convenient linker subunits may be utilized in the first linker LA. Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, each of L1, L2, L3, L4, L5and L6(if present) comprise one or more groups independently selected from a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, and a diamine (e.g., a linking group that includes an alkylene diamine).

[0281] In some embodiments, L1(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L1comprises a polyethylene glycol. In some embodiments, L1comprises a modified polyethylene glycol. In some embodiments, L1comprises an amino acid residue. In some embodiments, L1comprises an alkyl group or a substituted alkyl. In some embodiments, L1comprises an aryl group or a substituted aryl group. In some embodiments, L1comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0282] In some embodiments, L2(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L2comprises a polyethylene glycol. In some embodiments, L2comprises a modified polyethylene glycol. In some embodiments, L2comprises an amino acid residue. In some embodiments, L2comprises an alkyl group or a substituted alkyl. In some embodiments, L2comprises an aryl group or a substituted aryl group. In some embodiments, L2comprises a diamine (e.g., a linking group comprising an alkylene diamine). 68 NAI-1540479824

[0283] In some embodiments, L3(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L3comprises a polyethylene glycol. In some embodiments, L3comprises a modified polyethylene glycol. In some embodiments, L3comprises an amino acid residue. In some embodiments, L3comprises an alkyl group or a substituted alkyl. In some embodiments, L3comprises an aryl group or a substituted aryl group. In some embodiments, L3comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0284] In some embodiments, L4(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L4comprises a polyethylene glycol. In some embodiments, L4comprises a modified polyethylene glycol. In some embodiments, L4comprises an amino acid residue. In some embodiments, L4comprises an alkyl group or a substituted alkyl. In some embodiments, L4comprises an aryl group or a substituted aryl group. In some embodiments, L4comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0285] In some embodiments, L5(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L5comprises a polyethylene glycol. In some embodiments, L5comprises a modified polyethylene glycol. In some embodiments, L5comprises an amino acid residue. In some embodiments, L5comprises an alkyl group or a substituted alkyl. In some embodiments, L5comprises an aryl group or a substituted aryl group. In some embodiments, L5comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0286] In some embodiments, L6(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L6comprises a polyethylene glycol. In some embodiments, L6comprises a modified polyethylene glycol. In some embodiments, L6comprises an amino acid residue. In some embodiments, L6comprises an alkyl group or a substituted alkyl. In some embodiments, L6comprises an aryl group or a substituted aryl group. In some embodiments, L6comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0287] In some embodiments, LAis a first linker comprising: -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-(L6)f-, 69 NAI-1540479824wherein:-(L4)d- is -(T4-V4)d-; -(L5)e- is -(T5-V5)e-; and -(L6)f- is -(T6-V6)f-, wherein: T1, T2, T3, T4, T5, and T6, if present, are tether groups; V1, V2, V3, V4, V5, and V6, if present, are covalent bonds or linking functional groups; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a, b, c, d, e, and f is 1.

[0288] In certain embodiments, the sum of a, b, c, d, e, and f is 1 to 6. In certain embodiments, the sum of a, b, c, d, e, and f is 1. In certain embodiments, the sum of a, b, c, d, e, and f is 2. In certain embodiments, the sum of a, b, c, d, e, and f is 3. In certain embodiments, the sum of a, b, c, d, e, and f is 4. In certain embodiments, the sum of a, b, c, d, e, and f is 5. In certain embodiments, the sum of a, b, c, d, e, and f is 6. In certain embodiments, a, b, c, d, e, and f are each 1. In certain embodiments, a, b, c, d, and e are each 1 and f is 0. In certain embodiments, a, b, c, and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e and f are each 0. In certain embodiments, a and b are each 1 and c, d, e, and f are each 0. In certain embodiments, a is 1 and b, c, d, e, and f are each 0.

[0289] As described above, in certain embodiments, L1is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). As such, in certain embodiments, T1is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). In certain embodiments, V1is attached to the first drug or active agent. In certain embodiments, L2, if present, is attached to the first drug or active agent. As such, in certain embodiments, T2, if present, is attached to the first drug or active agent, or V2, if present, is attached to the first drug or active agent. In certain embodiments, L3, if present, is attached to the first drug or active agent. As such, in certain embodiments, T3, if present, is attached to the first drug or active agent, or V3, if present, is attached to the first drug or active agent. In certain embodiments, L4, if present, is attached to the first drug or active agent. As such, in certain 70 NAI-1540479824embodiments, T4, if present, is attached to the first drug or active agent, or V4, if present, is attached to the first drug or active agent. In certain embodiments, L5, if present, is attached to the first drug or active agent. As such, in certain embodiments, T5, if present, is attached to the first drug or active agent, or V5, if present, is attached to the first drug or active agent. In certain embodiments, L6, if present, is attached to the first drug or active agent. As such, in certain embodiments, T6, if present, is attached to the first drug or active agent, or V6, if present, is attached to the first drug or active agent.

[0290] In certain embodiments, the conjugate of Formula (I) includes a second linker, LB. The second linker, LB, may be utilized to bind a second moiety of interest (e.g., a second drug or active agent) to an ROR1 antibody through a conjugation moiety. The second linker, LB, may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein). For example, the second linker, LB, may attach a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety to a second drug. The hydrazinyl-indolyl or hydrazinyl-pyrrolo- pyridinyl conjugation moiety may be used to conjugate the second linker, LB, (and thus the second drug) to an ROR1 antibody.

[0291] For example, as shown in Formula (I) above, LBis attached to the antibody Ab through a conjugation moiety, and thus the antibody Ab is indirectly bonded to the second linker LBthrough the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As described above, the antibody Ab is an ROR1 antibody, and thus LBis attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the ROR1 antibody, e.g., the linker LBis indirectly bonded to the ROR1 antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0292] Any convenient linker may be utilized for the second linker LBin the subject conjugates and compounds. In certain embodiments, the second linker LBmay include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the second linker LBmay include an alkyl or substituted alkyl group. In certain embodiments, the second linker LBmay include an alkenyl or substituted alkenyl group. In certain embodiments, the second linker LBmay include an alkynyl or substituted alkynyl group. In certain embodiments, the second linker LBmay include an alkoxy or substituted alkoxy group. In certain embodiments, the second linker LBmay include an amino or substituted amino group. In certain embodiments, the second linker LBmay include a 71 NAI-1540479824carboxyl or carboxyl ester group. In certain embodiments, the second linker LBmay include an acyl amino group. In certain embodiments, the second linker LBmay include an alkylamide or substituted alkylamide group. In certain embodiments, the second linker LBmay include an aryl or substituted aryl group. In certain embodiments, the second linker LBmay include a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker LBmay include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker LBmay include a heterocyclyl or substituted heterocyclyl group.

[0293] In certain embodiments, the second linker LBmay include a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where 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.

[0294] In some embodiments, LBis a second linker described by the formula:wherein L7, L8, L9, L10, L11, L12and L13are each independently a linker subunit, and g, h, i, j, k, l, and m are each independently 0 or 1, provided at least one of g, h, i, j, k, l, and m is 1.

[0295] In certain embodiments, the sum of g, h, i, j, k, l, and m is 1 to 7. In certain embodiments, the sum of g, h, i, j, k, l, and m is 1. In certain embodiments, the sum of g, h, i, j, k, l, and m is 2. In certain embodiments, the sum of g, h, i, j, k, l, and m is 3. In certain embodiments, the sum of g, h, i, j, k, l, and m is 4. In certain embodiments, the sum of g, h, i, j, k, l, and m is 5. In certain embodiments, the sum of g, h, i, j, k, l, and m is 6. In certain embodiments, the sum of g, h, i, j, k, l, and m is 7. In certain embodiments, g, h, i, j, k, l, and m are each 1. In certain embodiments, g, h, i, j, k and l are each 1, and m is 0. In certain embodiments, g, h, i, j and k are each 1 and l and m are each 0. In certain embodiments, g, h, i and j are each 1 and k, l,and m are each 0. In certain embodiments, g, h, and i are each 1 and j, k, l, and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, l, and m are each 0. In certain embodiments, g is 1 and h, i, j, k, l, and m are each 0. In certain embodiments, g, h, i, j, k, l, and m are each 0.

[0296] In certain embodiments, the linker subunit L7is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). 72 NAI-1540479824In certain embodiments, the linker subunit L8, if present, is attached to the second drug or active agent W2. In certain embodiments, the linker subunit L9, if present, is attached to the second drug or active agent W2. In certain embodiments, the linker subunit L10, if present, is attached to the second drug or active agent W2. In certain embodiments, the linker subunit L11, if present, is attached to the second drug or active agent W2. In certain embodiments, the linker subunit L12, if present, is attached to the second drug or active agent W2. In certain embodiments, the linker subunit L13, if present, is attached to the second drug or active agent W2.

[0297] Any convenient linker subunits may be utilized in the second linker LB. Linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycols, polyethylenes and polyacrylates, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, each of L7, L8, L9, L10, L11, L12and L13(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).

[0298] In some embodiments, L7(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L7comprises a polyethylene glycol. In some embodiments, L7comprises a modified polyethylene glycol. In some embodiments, L7comprises an amino acid residue. In some embodiments, L7comprises an alkyl group or a substituted alkyl. In some embodiments, L7comprises an aryl group or a substituted aryl group. In some embodiments, L7comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0299] In some embodiments, L8(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L8comprises a polyethylene glycol. In some embodiments, L8comprises a modified polyethylene glycol. In some embodiments, L8comprises an amino acid residue. In some embodiments, L8comprises an alkyl group or a substituted alkyl. In some embodiments, L8comprises an aryl group or a substituted aryl group. In some embodiments, L8comprises a diamine (e.g., a linking group comprising an alkylene diamine). 73 NAI-1540479824

[0300] In some embodiments, L9(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L9comprises a polyethylene glycol. In some embodiments, L9comprises a modified polyethylene glycol. In some embodiments, L9comprises an amino acid residue. In some embodiments, L9comprises an alkyl group or a substituted alkyl. In some embodiments, L9comprises an aryl group or a substituted aryl group. In some embodiments, L9comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0301] In some embodiments, L10(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L10comprises a polyethylene glycol. In some embodiments, L10comprises a modified polyethylene glycol. In some embodiments, L10comprises an amino acid residue. In some embodiments, L10comprises an alkyl group or a substituted alkyl. In some embodiments, L10comprises an aryl group or a substituted aryl group. In some embodiments, L10comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0302] In some embodiments, L11(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L11comprises a polyethylene glycol. In some embodiments, L11comprises a modified polyethylene glycol. In some embodiments, L11comprises an amino acid residue. In some embodiments, L11comprises an alkyl group or a substituted alkyl. In some embodiments, L11comprises an aryl group or a substituted aryl group. In some embodiments, L11comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0303] In some embodiments, L12(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, a substituted aryl group, or a diamine. In some embodiments, L12comprises a polyethylene glycol. In some embodiments, L12comprises a modified polyethylene glycol. In some embodiments, L12comprises an amino acid residue. In some embodiments, L12comprises an alkyl group or a substituted alkyl. In some embodiments, L12comprises an aryl group or a substituted aryl group. In some embodiments, L12comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0304] In some embodiments, L13(if present) comprises a polyethylene glycol, a modified polyethylene glycol, an amino acid residue, an alkyl group, a substituted alkyl, an aryl group, 74 NAI-1540479824a substituted aryl group, or a diamine. In some embodiments, L13comprises a polyethylene glycol. In some embodiments, L13comprises a modified polyethylene glycol. In some embodiments, L13comprises an amino acid residue. In some embodiments, L13comprises an alkyl group or a substituted alkyl. In some embodiments, L13comprises an aryl group or a substituted aryl group. In some embodiments, L13comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0305] In some embodiments, LBis a second linker comprising:wherein: -(L7)g- is -(T7-V7)g-; -(L8)h- is -(T8-V8)h-; -(L9)i- is -(T9-V9)i-; -(L10)j- is -(T10-V10)j-;wherein: T7, T8, T9, T10, T11, T12, and T13, if present, are tether groups; V7, V8, V9, V10, V11, V12,and V13, if present, are covalent bonds or linking functional groups; and g, h, i, j, k, l, and m are each independently 0 or 1, provided at least one of g, h, i, j, k, l, and m is 1.

[0306] In certain embodiments, the sum of g, h, i, j, k, l, and m is 1 to 7. In certain embodiments, the sum of g, h, i, j, k, l, and m is 1. In certain embodiments, the sum of g, h, i, j, k, l, and m is 2. In certain embodiments, the sum of g, h, i, j, k, l, and m is 3. In certain embodiments, the sum of g, h, i, j, k, l, and m is 4. In certain embodiments, the sum of g, h, i, j, k, l, and m is 5. In certain embodiments, the sum of g, h, i, j, k, l, and m is 6. In certain embodiments, the sum of g, h, i, j, k, l, and m is 7. In certain embodiments, g, h, i, j, k, l, and m are each 1. In certain embodiments, g, h, i, j, k and l are each 1, and m is 0. In certain embodiments, g, h, i, j and k are each 1 and l and m are each 0. In certain embodiments, g, h, i and j are each 1 and k, l, and m are each 0. In certain embodiments, g, h, and i are each 1 and j, k, l, and m are each 0. In certain embodiments, g and h are each 1 and i, j, k, l, and m are each 0. In certain embodiments, g is 1 and h, i, j, k, l, and m are each 0. In certain embodiments, g, h, i, j, k, l, and m are each 0. 75 NAI-1540479824

[0307] As described above, in certain embodiments, L7is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). As such, in certain embodiments, T7is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). In certain embodiments, V7is attached to the second drug or active agent. In certain embodiments, L8, if present, is attached to the second drug or active agent. As such, in certain embodiments, T8, if present, is attached to the second drug or active agent, or V8, if present, is attached to the second drug or active agent. In certain embodiments, L9, if present, is attached to the second drug or active agent. As such, in certain embodiments, T9, if present, is attached to the second drug or active agent, or V9, if present, is attached to the second drug or active agent. In certain embodiments, L10, if present, is attached to the second drug or active agent. As such, in certain embodiments, T10, if present, is attached to the second drug or active agent, or V10, if present, is attached to the second drug or active agent. In certain embodiments, L11, if present, is attached to the second drug or active agent. As such, in certain embodiments, T11, if present, is attached to the second drug or active agent, or V11, if present, is attached to the second drug or active agent. In certain embodiments, L12, if present, is attached to the second drug or active agent. As such, in certain embodiments, T12, if present, is attached to the second drug or active agent, or V12, if present, is attached to the second drug or active agent. In certain embodiments, L13, if present, is attached to the second drug or active agent. As such, in certain embodiments, T13, if present, is attached to the second drug or active agent, or V13, if present, is attached to the second drug or active agent.

[0308] Regarding the tether groups, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and T13, any convenient tether groups may be utilized in the subject linkers. In some embodiments, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and T13each comprise one or more groups independently selected from a covalent bond, a (C1-C12)alkyl, a substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino- piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para- aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para- hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, where each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12. 76 NAI-1540479824

[0309] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes a (C1-C12)alkyl or a substituted (C1-C12)alkyl. In certain embodiments, (C1-C12)alkyl is a straight chain or branched alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, (C1-C12)alkyl may be an alkyl or substituted alkyl, such as C1-C12alkyl, or C1-C10alkyl, or C1-C6alkyl, or C1-C3alkyl. In some instances, (C1-C12)alkyl is a C2-alkyl. For example, (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C1-C12 alkylene, or C1-C10alkylene, or C1-C6alkylene, or C1-C3alkylene. In some instances, (C1-C12)alkyl is a C1-alkylene (e.g., CH2). In some instances, (C1-C12)alkyl is a C2-alkylene (e.g., CH2CH2). In some instances, (C1-C12)alkyl is a C3-alkylene (e.g., CH2CH2CH2).

[0310] In certain embodiments, substituted (C1-C12)alkyl is a straight chain or branched substituted alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, substituted (C1-C12)alkyl may be a substituted alkyl, such as substituted C1-C12alkyl, or substituted C1-C10alkyl, or substituted C1-C6alkyl, or substituted C1-C3alkyl. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkyl. For example, substituted (C1-C12)alkyl may be a substituted alkylene, such as substituted C1-C12alkylene, or substituted C1-C10alkylene, or substituted C1-C6alkylene, or substituted C1-C3alkylene. In some instances, substituted (C1-C12)alkyl is a substituted C1-alkylene (e.g., C1-alkylene substituted with -SO3H). In some instances, substituted (C1-C12)alkyl is a substituted C2-alkylene. In some instances, substituted (C1- C12)alkyl is a substituted C3-alkylene. For example, substituted (C1-C12)alkyl may include C1- C12 alkylene (e.g., C3-alkylene or C5-alkylene) substituted with a (PEG)k group as described herein (e.g.,-CONH(PEG)t, such as -CONH(PEG)3 or -CONH(PEG)5; or -NHCO(PEG)k, such as -NHCO(PEG)7, or may include C1-C12alkylene (e.g., C3-alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12 alkylene (e.g., C5-alkylene) substituted with a -NHCOCH2SO3H group.

[0311] In some embodiments, substituted (C1-C12)alkyl may include C1-C12alkylene (e.g., C3-alkylene or C5-alkylene) substituted with a (PEG)tgroup as described herein (e.g., -NHCO(PEG)t, whereinindicates the point of attachment 77 NAI-1540479824to carbonyl group of -NHCO-, and t is an integer), such as -NHCO(CH2CH2O)3CH3 or -NHCO(CH2CH2O)5CH3 or -NHCO(CH2CH2O)8CH3.

[0312] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12,and / or T13) includes an aryl or substituted aryl. For example, the aryl can be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl can be substituted with one or more substituents selected from (C1-C12)alkyl, a substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some instances, the substituted aryl is a substituted phenyl, where the substituent includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).

[0313] In some instances, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes a heteroaryl or substituted heteroaryl, such triazolyl (e.g., 1,2,3- triazolyl). In some instances, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12,and / or T13) includes a cycloalkyl or substituted cycloalkyl. In some instances, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes a heterocyclyl or substituted heterocyclyl. In some instances, the substituent on the substituted heteroaryl, substituted cycloalkyl or substituted heterocyclyl includes a cleavable moiety as described herein (e.g., an enzymatically cleavable moiety, such as a glycoside or glycoside derivative).

[0314] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12,and / or T13) includes an ethylene diamine (EDA) moiety, e.g., an EDA containing tether group. In certain embodiments, (EDA)w includes one or more EDA moieties, such as where w is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, or 6). The linked ethylene diamine (EDA) moieties may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, an acyl, a substituted acyl, an aryl, or a substituted aryl. In certain embodiments, the EDA moiety is described by the structure:, 78 NAI-1540479824where y is an integer from 1 to 6, r is 0 or 1, and each R12is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl. In certain embodiments, any two adjacent R12groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R12groups are an alkyl group, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R12groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

[0315] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12,and / or T13) 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 R12is 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, R12is a polyethylene glycol moiety. In certain embodiments, R12is a carboxy modified polyethylene glycol.

[0316] In certain embodiments, R12includes a polyethylene glycol moiety described by the formula: (PEG)k, which may be represented by the structure: 79 NAI-1540479824, where k is an integer from 1 to 20, such as from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 6, or from 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some instances, k is 2. In certain embodiments, R17is selected from OH, COOH, OR, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R17is COOH. In certain embodiments, R17is OH. In certain embodiments, R17is OCH3.

[0317] In certain embodiments, (PEG)k is (PEG)t having the following structure:, wherein t is an integer from 2 to 10. In certain embodiments, t is 8.

[0318] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes (PEG)n, where (PEG)n is a polyethylene glycol or a modified polyethylene glycol linking unit. In certain embodiments, (PEG)nis described by the structure:, wherein 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.

[0319] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) 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, 80 NAI-1540479824from 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.

[0320] In further embodiments, (AA)pcomprises a dipeptide of valine-alanine.

[0321] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes an amino acid analog. Amino acid analogs include compounds that are similar in structure and / or overall shape to one or more amino acids commonly found in naturally occurring proteins (e.g., Ala or A, Cys or C, Asp or D, Glu or E, Phe or F, Gly or G, His or H, Ile or I, Lys or K, Leu or L, Met or M, Asn or N, Pro or P, Gln or Q, Arg or R, Ser or S, Thr or T, Val or V, Trp or W, Tyr or Y). Amino acid analogs also include natural amino acids with modified side chains or backbones. Amino acid analogs also include amino acid analogs with the same stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs. In some instances, the amino acid analogs share backbone structures, and / or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids, and α- amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.

[0322] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12,and / or T13) includes a moiety described by the formula -(CR13OH)x-, where x is 0 or x 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, x is 1. In certain embodiments, x is 2. In certain embodiments, R13is 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, R13is hydrogen. In certain embodiments, R13is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R13is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 81 NAI-1540479824substituted alkenyl. In certain embodiments, R13is alkynyl or substituted alkynyl. In certain embodiments, R13is alkoxy or substituted alkoxy. In certain embodiments, R13is amino or substituted amino. In certain embodiments, R13is carboxyl or carboxyl ester. In certain embodiments, R13is acyl or acyloxy. In certain embodiments, R13is acyl amino or amino acyl. In certain embodiments, R13is alkylamide or substituted alkylamide. In certain embodiments, R13is sulfonyl. In certain embodiments, R13is thioalkoxy or substituted thioalkoxy. In certain embodiments, R13is aryl or substituted aryl, such as C5-8aryl or C5-8substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R13is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6 substituted heteroaryl. In certain embodiments, R13is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R13is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl.

[0323] In certain embodiments, R13is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13.

[0324] In certain embodiments, the tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13) includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.

[0325] In certain embodiments, a tether group (e.g., T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12,and / or T13) includes a meta-amino-benzyloxy (MABO), meta-amino- benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), or para-hydroxy-phenyl (PHP).

[0326] In some embodiments, a tether group includes a MABO group described by the following structure: 82 NAI-1540479824.

[0327] In some embodiments, a tether group includes a MABC group described by the following structure:.

[0328] In some embodiments, a tether group includes a PABO group described by the following structure:.

[0329] In some embodiments, a tether group includes a PABC group described by the following structure:.

[0330] In some embodiments, a tether group includes a PAB group described by the following structure: .

[0331] In some embodiments, a tether group includes a PABA group described by the following structure:

[0332] In some embodiments, a tether group includes a PAP group described by the following structure: 83 NAI-1540479824.

[0333] In some embodiments, a tether group includes a PHP group described by the following structure:.

[0334] In certain embodiments, each R14is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0335] In certain embodiments, R14is hydrogen. In certain embodiments, each R14is hydrogen. In certain embodiments, R14is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4alkyl or C1-4substituted alkyl, or C1-3alkyl or C1-3substituted alkyl. In certain embodiments, R14is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R14is alkynyl or substituted alkynyl. In certain embodiments, R14is alkoxy or substituted alkoxy. In certain embodiments, R14is amino or substituted amino. In certain embodiments, R14is carboxyl or carboxyl ester. In certain embodiments, R14is acyl or acyloxy. In certain embodiments, R14is acyl amino or amino acyl. In certain embodiments, R14is alkylamide or substituted alkylamide. In certain embodiments, R14is sulfonyl. In certain embodiments, R14is thioalkoxy or substituted thioalkoxy. In certain embodiments, R14is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5aryl or C5substituted aryl, or a C6aryl or C6substituted aryl. In certain embodiments, R14is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5- 8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6substituted heteroaryl. In certain embodiments, R14is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R14is heterocyclyl or substituted heterocyclyl, such as C3-8heterocyclyl 84 NAI-1540479824or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.

[0336] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0337] In certain embodiments, one or more of the tether groups T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and / or T13is each optionally substituted with a glycoside or glycoside derivative. For example, in some instances, T1, T2, T3, T4, T5, and T6are each optionally substituted with a glycoside. In some instances, T7, T8, T9, T10, T11, T12,and T13are each optionally substituted with a glycoside. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0338] In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. In some embodiments, the PABC is substituted with a glycoside, for example, a hydrogen of PABC is replaced with a glycoside, such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O- GalNAc.

[0339] For example, in some embodiments, the glycoside or glycoside derivative is selected from the following structures: 85 NAI-1540479824

[0340] Regarding the linking functional groups, V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, and V13any 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, V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, and V13are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, where q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2. In certain embodiments, q is 3. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6. In some embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0342] In certain embodiments, R15is hydrogen. In certain embodiments, each R15is hydrogen. In certain embodiments, R15is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R15is alkenyl or substituted alkenyl, such as C2-6alkenyl or C2-6substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R15is alkynyl or substituted alkynyl. In certain embodiments, R15is alkoxy or substituted alkoxy. In certain embodiments, R15is amino or substituted amino. In certain embodiments, R15is carboxyl or carboxyl ester. In certain 86 NAI-1540479824embodiments, R15is acyl or acyloxy. In certain embodiments, R15is acyl amino or amino acyl. In certain embodiments, R15is alkylamide or substituted alkylamide. In certain embodiments, R15is sulfonyl. In certain embodiments, R15is thioalkoxy or substituted thioalkoxy. In certain embodiments, R15is aryl or substituted aryl, such as C5-8 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, R15is heteroaryl or substituted heteroaryl, such as C5-8heteroaryl or C5-8substituted heteroaryl, such as a C5heteroaryl or C5substituted heteroaryl, or a C6heteroaryl or C6 substituted heteroaryl. In certain embodiments, R15is cycloalkyl or substituted cycloalkyl, such as C3-8cycloalkyl or C3-8substituted cycloalkyl, such as a C3-6cycloalkyl or C3-6substituted cycloalkyl, or a C3-5cycloalkyl or C3-5substituted cycloalkyl. In certain embodiments, R15is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5heterocyclyl or C3-5substituted heterocyclyl.

[0343] In certain embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15.

[0344] As described above, in some embodiments, LAis a first linker comprising -(T1-V1)a- (T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, where a, b, c, d, e and f are each independently 0 or 1, provided at least one of a, b, c, d, e, and f is 1.

[0345] In some embodiments, in the first linker LA: T1is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine, and an ester; and V1, V2, V3, V4, V5, and V6are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; 87 NAI-1540479824wherein: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-piperidineAA is an amino acid residue, where p is an integer from 1 to 20; and each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; each R13is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0346] In some embodiments, LAcomprises: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein: a, b, c, d, e, and f are each independently 0 or 1, provided at least one of a, b, c, d, e, and f is 1; T1, T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para- amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a 88 NAI-1540479824polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0347] In some embodiments of LA: T1is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein: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; 89 NAI-15404798244-amino-piperidine each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; a, b, c, and d are each 1; and e and f are 0.

[0348] In some embodiments, T1, T2, T3, T4, T5, and T6are each optionally substituted with a glycoside.

[0349] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0350] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0351] In certain embodiments, T1, T2, T3, T4, T5, and T6and V1, V2, V3, V4, V5, and V6are selected from the following: wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is (AA)p and V2is absent (e.g., a covalent bond); T3is PABC and V3is absent (e.g., a covalent bond); p is an integer from 1 to 10; and d, e, and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is (PEG)nand V2is -CO-; T3is (AA)p and V3is absent (e.g., a covalent bond); T4is PABC and V4is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is an amino acid analog and V2is -NH-; T3is (PEG)n and V3is -CO-; T4is (AA)pand V4is absent (e.g., a covalent bond); 90 NAI-1540479824T5is PABC and V5is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is (PEG)nand V2is -CO-; T3is (AA)pand V3is absent (e.g., a covalent bond); T4is PABC and V4is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)p and V3is absent (e.g., a covalent bond); T4is PABC and V4is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is (PEG)nand V2is -CO-; T3is (AA)p and V3is absent (e.g., a covalent bond); T4is PABA and V4is -CO-; T5is (C1-C12)alkyl and V5is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is 4AP and V2is -CO-; T3is (C1-C12)alkyl and V3is -CO-; T4is (AA)pand V4is absent (e.g., a covalent bond); T5is PABC and V5is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: 91 NAI-1540479824T1is (C1-C12)alkyl and V1is -CO-; T2is 4AP and V2is -CO-; T3is (C1-C12)alkyl and V3is -O-; T4is (C1-C12)alkyl and V4is -CO-; T5is (AA)p and V5is absent (e.g., a covalent bond); p is an integer from 1 to 10; and T6is PABC and V6is absent (e.g., a covalent bond); or wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is an amino acid analog and V2is absent (e.g., a covalent bond); T3is (AA)p and V3is absent (e.g., a covalent bond); T4is PABC and V4is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is (PEG)nand V2is -CONH-; T3is substituted (C1-C12)alkyl and V3is -CO-; T4is (AA)pand V4is absent (e.g., a covalent bond); T5is PABC and V5is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1is (C1-C12)alkyl and V1is -CO-; T2is an (AA)p and V2is -NH-; T3is (PEG)nand V3is -CO-; T4is (AA)p and V4is absent (e.g., a covalent bond); T5is PABC and V5is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is (PEG)nand V2is -CO-; T3is (AA)p and V3is absent (e.g., a covalent bond); 92 NAI-1540479824T4is PAP and V4is -C(O)O-; p is an integer from 1 to 10; and e and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)pand V3is absent (e.g., a covalent bond); T4is PABC and V4is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is PABC and V3is absent (e.g., a covalent bond); and d, e, and f are each 0.

[0352] In certain embodiments, the left-hand side of the above linker structure for the first linker LAis attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and the right-hand side of the above linker structure for the first linker LAis attached to the first drug or active agent.

[0353] As described above, in some embodiments, LBis a second linker comprising -(T7- V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-(T13-V13)m-, where g, h, i, j, k, l, and m are each independently 0 or 1, provided that at least one of g, h, i, j, k, l,and m is 1.

[0354] In some embodiments, in the second linker LB: T7is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T8, T9, T10, T11, T12, and T13are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine, and an ester; and V7, V8, V9, V10, V11, V12,and V13are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; wherein: 93 NAI-1540479824integer 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-piperidineAA is an amino acid residue, where p is an integer from 1 to 20; and each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; each R13is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0355] Any convenient tether groups may be utilized for T7, T8, T9, T10, T11, T12,and T13. For example, any of the tether groups described above in relation to T1, T2, T3, T4, T5, and T6may be used for the tether groups T7, T8, T9, T10, T11, T12, and T13.

[0356] Any convenient linking functional groups may be utilized for V7, V8, V9, V10, V11, V12, and V13. For example, any of the linking functional groups described above in relation to V1, V2, V3, V4, V5, and V6may be used for the linking functional groups V7, V8, V9, V10, V11, V12,and V13.

[0357] In certain embodiments, each R13is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13.

[0358] In certain embodiments, each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, 94 NAI-1540479824alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15. In these embodiments, various possible substituents are as described above for R15.

[0359] In certain embodiments of the second linker LB, one or more of the tether groups T7, T8, T9, T10, T11, T12,and T13is each optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0360] In certain embodiments of the second linker LB, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP tether structures shown above, the phenyl ring may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0361] In some embodiments, T7, T8, T9, T10, T11, T12,and T13are each optionally substituted with a glycoside.

[0362] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0363] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0364] In some embodiments of LB: g, h, i, j, and k are each 1; l and m are each 0; T7is a covalent bond; T8, T9, T10, T11, and T12are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V7, V8, V9, V10, V11, and V12are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, 95 NAI-1540479824-NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, and -P(O)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-piperidineeach R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring.

[0365] In some embodiments, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12are each optionally substituted with a glycoside.

[0366] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.

[0367] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

[0368] In certain embodiments, T7, T8, T9, T10, T11, T12, and T13and V7, V8, V9, V10, V11, V12, and V13are selected from the following: wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is (AA)pand V9is absent (e.g., a covalent bond); T10is PABC and V10is absent (e.g., a covalent bond); and k, l, and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is (PEG)n and V9is -CO-; 96 NAI-1540479824T10is (AA)p and V10is absent (e.g., a covalent bond); and T11is PABC and V11is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is an amino acid analog and V9is -NH-; T10is (PEG)n and V10is -CO-; T11is (AA)pand V11is absent (e.g., a covalent bond); T12is PABC and V12is absent (e.g., a covalent bond); and m is 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is (PEG)n and V9is -CO-; T10is (AA)pand V10is absent (e.g., a covalent bond); T11is PABC and V11is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is substituted (C1-C12)alkyl and V9is -CO-; T10is (AA)pand V10is absent (e.g., a covalent bond); T11is PABC and V11is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is (PEG)nand V9is -CO-; T10is (AA)pand V10is absent (e.g., a covalent bond); T11is PABA and V11is -CO-; T12is (C1-C12)alkyl and V12is absent (e.g., a covalent bond); and m is 0; or wherein: 97 NAI-1540479824T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is 4AP and V9is -CO-; T10is (C1-C12)alkyl and V10is -CO-; T11is (AA)p and V11is absent (e.g., a covalent bond); T12is PABC and V12is absent (e.g., a covalent bond); and m is 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is 4AP and V9is -CO-; T10is (C1-C12)alkyl and V10is -O-; T11is (C1-C12)alkyl and V11is -CO-; T12is (AA)p and V12is absent (e.g., a covalent bond); and T13PABC and V13is absent (e.g., a covalent bond); or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is an amino acid analog and V9is absent (e.g., a covalent bond); T10is (AA)pand V10is absent (e.g., a covalent bond); T11is PABC and V11is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is (PEG)nand V9is -CONH-; T10is substituted (C1-C12)alkyl and V10is -CO-; T11is (AA)p and V11is absent (e.g., a covalent bond); T12is PABC and V12is absent (e.g., a covalent bond); and m is 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is (AA)p and V9is -NH-; 98 NAI-1540479824T10is (PEG)n and V10is -CO-; T11is (AA)p and V11is absent (e.g., a covalent bond); T12is PABC and V12is absent (e.g., a covalent bond); and m is 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is (PEG)n and V9is -CO-; T10is (AA)pand V10is absent (e.g., a covalent bond); T11is PAP and V11is -C(O)O-; and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CO-; T9is (AA)p and V9is absent (e.g., a covalent bond); T10is PABC and V10is absent (e.g., a covalent bond); T11is PAP and V11is -C(O)O-; and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is substituted (C1-C12)alkyl and V9is -CO-; T10is PABC and V10is absent (e.g., a covalent bond); and k, l, and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is absent (e.g., a covalent bond); T9is heteroaryl and V9is absent (e.g., a covalent bond); T10is (C1-C12)alkyl and V10is -CONH-; T11is (PEG)nand V11is -CO-; and l and m are each 0; or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is absent (e.g., a covalent bond); 99 NAI-1540479824T9is heteroaryl and V9is absent (e.g., a covalent bond); T10is (C1-C12)alkyl and V10is -CONH-; T11is substituted (C1-C12)alkyl and V11is -CO-; T12is (AA)p and V12is absent (e.g., a covalent bond); and T13PAB and V13is absent (e.g., a covalent bond); or wherein: T7is absent (e.g., a covalent bond) and V7is -NHCO-; T8is (C1-C12)alkyl and V8is absent (e.g., a covalent bond); T9is heteroaryl and V9is absent (e.g., a covalent bond); T10is (C1-C12)alkyl and V10is -CONH-; T11is substituted (C1-C12)alkyl and V11is -CO-; T12is (AA)p and V12is absent (e.g., a covalent bond); and T13PABC and V13is absent (e.g., a covalent bond).

[0369] In certain embodiments, the left-hand side of the above linker structure for the second linker LBis attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, and the right-hand side of the above linker structure for the second linker LBis attached to the second drug or active agent.

[0370] In certain embodiments, the conjugate is an antibody-drug conjugate where the ROR1 antibody and the drugs are linked together by linkers as described above. In some instances, the linker m(e.g., LAand / or LB) is a cleavable linker. A cleavable linker is a linker that includes one or more cleavable moieties, where the cleavable moiety includes one or more bonds that can dissociate under certain conditions, thus separating the cleavable linker into two or more separable portions. For example, the cleavable moiety may include one or more covalent bonds, which under certain conditions, can dissociate or break apart to separate the cleavable linker into two or more portions. As such the linkers that are included in an antibody-drug conjugate can be cleavable linkers, such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at a desired target site of action for the drug.

[0371] In some instances, a cleavable linker includes two cleavable moieties, such as a first cleavable moiety and a second cleavable moiety. The cleavable moieties can be configured such that cleavage of both cleavable moieties is needed in order to separate or release the drug from the ROR1 antibody at a desired target site of action for the drug. For example, cleavage of a cleavable linker can be achieved by initially cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, a 100 NAI-1540479824cleavable linker includes a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety. By “hinders cleavage” is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood or substantially inhibits the cleavage of the first cleavable moiety, thus substantially reducing the amount or preventing the cleavage of the cleavable linker. For instance, the presence of uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety. The hinderance of cleavage of the first cleavable moiety by the presence of the second cleavable moiety, in turn, substantially reduces the amount or prevents the release of the drug from the antibody. For example, the premature release of the drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired target site of action for the drug.

[0372] In some cases, since the second cleavable moiety hinders cleavage of the first cleavable moiety, cleavage of the cleavable linker can be achieved by initially cleaving the second cleavable moiety and then cleaving the first cleavable moiety. Cleavage of the second cleavable moiety can reduce or eliminate the hinderance on the cleavage of the first cleavable moiety, thus allowing the first cleavable moiety to be cleaved. Cleavage of the first cleavable moiety can result in the cleavable linker dissociating or separating into two or more portions as described above to release the drug from the antibody-drug conjugate. In some instances, cleavage of the first cleavable moiety does not substantially occur in the presence of an uncleaved second cleavable moiety. By substantially is meant that about 10% or less cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety, such as about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less cleavage of the first cleavable moiety occurs in the presence of an uncleaved second cleavable moiety.

[0373] Stated another way, the second cleavable moiety can protect the first cleavable moiety from cleavage. For instance, the presence of uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. As such, cleavage of the second cleavable moiety exposes the first cleavable moiety (e.g., deprotects the first cleavable moiety), thus allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, which, in turn, separates or releases the drug from the antibody at a desired target site of action for the drug as described above. In certain instances, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of 101 NAI-1540479824the second cleavable moiety does not in and of itself result in cleavage of the cleavable linker (e.g., cleavage of the first cleavable moiety is still needed in order to cleave the cleavable linker).

[0374] The cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety. For example, the first cleavable moiety can be a first enzymatically cleavable moiety and the second cleavable moiety can be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more portions as described above through the enzymatic action of an enzyme. The enzymatically cleavable moiety can be any cleavable moiety that can be cleaved through the enzymatic action of an enzyme, such as, but not limited to, an ester, a peptide, a glycoside, and the like. In some instances, the enzyme that cleaves the enzymatically cleavable moiety is present at a desired target site of action, such as the desired target site of action of the drug that is to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in a significant amount in other areas, such as in whole blood, plasma, or serum. As such, the cleavage of an enzymatically cleavable moiety can be controlled such that substantial cleavage occurs at the desired site of action, whereas cleavage does not significantly occur in other areas or before the antibody-drug conjugate reaches the desired site of action.

[0375] For example, as described herein, antibody-drug conjugates of the present disclosure can be used for the treatment of cancer, such as for the delivery of a cancer therapeutic drug to a desired site of action where the cancer cells are present. In some cases, enzymes, such as an esterase that cleaves ester bonds or a glycosidase that cleaves glycosidic bonds, can be a biomarker for cancer that is overexpressed in cancer cells. The overexpression, and thus localization, of certain enzymes in cancer can be used in the context of the enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desired site of action (e.g., the site of the cancer (and overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester or a glycoside) that can be cleaved by an enzyme that is overexpressed in cancer cells. For instance, the enzyme can be an esterase. As such, in some instances, the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme. In some instances, the enzyme can be a glycosidase. As such, in some instances, the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that can be cleaved by a glycosidase enzyme. 102 NAI-1540479824

[0376] In certain embodiments, the enzymatically cleavable moiety is an ester bond. For example, the first cleavable moiety described above (e.g., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can include an ester. The presence of uncleaved second cleavable moiety can protect the first cleavable moiety (ester) from cleavage by an esterase enzyme, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action for the drug. In some instances, a portion of the linker adjacent to the first cleavable moiety is linked to or includes a substituent, where the substituent comprises the second cleavable moiety. In some instances, the second cleavable moiety includes a glycoside or glycoside derivative.

[0377] In some embodiments, the enzymatically cleavable moiety is sugar moiety, such as a glycoside (or glyosyl) or glycoside derivative. In some cases, the glycoside or glycoside derivative can facilitate an increase in the hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside or glycoside derivative. The glycoside or glycoside derivative can be any glycoside or glycoside derivative suitable for use in the cleavable linker and that can be cleaved through the enzymatic action of an enzyme. For example, the second cleavable moiety (e.g., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside or glycoside derivative. For instance, in some embodiments, the first cleavable moiety includes an ester, and the second cleavable moiety includes a glycoside or glycoside derivative. In certain embodiments, the second cleavable moiety is a glycoside or glycoside derivative selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O- GalNAc. In some instances, the second cleavable moiety is a glucuronide. In some instances, the second cleavable moiety is a galactoside. In some instances, the second cleavable moiety is a glucoside. In some instances, the second cleavable moiety is a mannoside. In some instances, the second cleavable moiety is a fucoside. In some instances, the second cleavable moiety is O-GlcNAc. In some instances, the second cleavable moiety is O-GalNAc.

[0378] The glycoside or glycoside derivative can be attached (covalently bonded) to the cleavable linker through a glycosidic bond. The glycosidic bond can link the glycoside or glycoside derivative to the cleavable linker through various types of bonds, such as, but not limited to, an O-glycosidic bond (an O-glycoside), an N-glycosidic bond (a glycosylamine), an S-glycosidic bond (a thioglycoside), or C-glycosidic bond (a C-glycoside or C-glycosyl). In some instances, the glycosidic bond is an O-glycosidic bond (an O-glycoside). In some cases, the glycoside or glycoside derivative can be cleaved from the cleavable linker it is 103 NAI-1540479824attached to by an enzyme (e.g., through enzymatically mediated hydrolysis of the glycosidic bond). A glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme that is able to carry out the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. An example of an enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is a glycosidase, such as a glucuronidase, a galactosidase, a glucosidase, a mannosidase, a fucosidase, and the like. Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug of the antibody-drug conjugate. For instance, the enzyme can be a lysosomal enzyme, such as a lysosomal glycosidase, found in cells at or near the desired site of action for the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the target site where the enzyme that mediates cleavage of the first cleavable moiety is found.

[0379] In some embodiments, an ROR1-ADC is represented by Formula (I):wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3and R4are each selected from hydrogen and (C1-C12)alkyl; LAis a first linker wherein: T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)p where p is an integer from 1-20 and V3is a covalent bond; T4is PABC and V4is a covalent bond; 104 NAI-1540479824a, b, c, and d are each 1; e and f are each 0; and LBis a second linker wherein: T7is a covalent bond and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is substituted (C1-C12)alkyl and V9is -CO-; T10is (AA)pwhere p is an integer from 1-20 and V10is a covalent bond; T11is PABC and V11is a covalent bond; and h, i, j, and k are each 1; and l and m are each 0; s is an integer from 1 to 10; W1is a first drug; and W2is a second drug.

[0380] In some embodiments, one or both of W1and W2are camptothecin analogues, for example, belotecan.

[0381] In some embodiments, an ROR1-ADC is represented by Formula (I):wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3and R4are each selected from hydrogen and (C1-C12)alkyl; LAis a linker wherein: T1is (C1-C6)alkyl and V1is -CONH-; T2is (C1-C6)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10, and V2is -CO-; T3is (AA)2 and V3is a covalent bond; T4is PABC substituted with a glycoside and V4is a covalent bond; 105 NAI-1540479824a, b, c, and d are each 1; and e and f are each 0; and LBis a linker wherein: T7is a covalent bond and V7is -NHCO-; T8is (C1-C6)alkyl and V8is -CONH-; T9is (C1-C6)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10, and V9is -CO-; T10is (AA)2 and V10is a covalent bond; T11is PABC substituted with a glycoside and V11is a covalent bond; h, i, j, and k are each 1; and l and m are each 0; s is an integer from 1 to 10; W1is a first drug; and W2is a second drug.

[0382] In some embodiments, an ROR1-ADC is represented by Formula (I):wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3and R4are each selected from hydrogen and (C1-C12)alkyl; LAis a linker wherein: T1is (C1-C6)alkyl and V1is -CONH-; T2is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)tisinteger from 2 to 10, optionally 8, and V2is -CO-; T3is (AA)2 and V3is a covalent bond; 106 NAI-1540479824T4is PABC substituted with a glycoside and V4is a covalent bond; a, b, c, and d are each 1; and e and f are each 0; and LBis a linker wherein: T7is a covalent bond and V7is -NHCO-; T8is (C1-C6)alkyl and V8is -CONH-; T9is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)tisinteger from 2 to 10, optionally 8, and V9is -CO-; T10is (AA)2and V10is a covalent bond; T11is PABC substituted with a glycoside and V11is a covalent bond; h, i, j, and k are each 1; and l and m are each 0; s is an integer from 1 to 10; W1is a first drug; and W2is a second drug.

[0383] In some embodiments, the PABC of one or both of T4and T11is substituted with a glucuronide. In some embodiments, one or both of T1and T8is ethyl. In some embodiments, one or both of T2and T9is C5 alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 5 to 10. In some embodiments, one or both of W1and W2are camptothecin analogues, for example, belotecan.

[0384] In some embodiments, the PABC of one or both of T4and T11is substituted with a glucuronide. In some embodiments, one or both of T1and T8is ethyl. In some embodiments, one or both of T2and T9is C5alkylene substituted with -NHCO(PEG)t, wherein (PEG)tisinteger from 5 to 10. In some embodiments, one or both of W1and W2are camptothecin analogues, for example, belotecan.

[0385] In some embodiments, s is an integer from 1 to 4. In further embodiments, s is 4.

[0386] In some embodiments, an ROR1-ADC is represented by Formula (II): 107 NAI-1540479824wherein: Ab represents the antibody that binds to ROR1; and s is an integer from 1 to 10.

[0387] In some embodiments, s is an integer from 1 to 4.

[0388] Any of the chemical entities, linkers and conjugation moieties set forth in the structures above may be adapted for use in the subject compounds and conjugates.

[0389] Additional disclosure related to hydrazinyl-indolyl and hydrazinyl-pyrrolo-pyridinyl compounds and methods for producing a conjugate is found in U.S. Patent No.9,310,374, U.S. Patent No.9,493,413, U.S. Patent NO.11,564,989, and International Publication Number WO 2022 / 187370, the disclosures of each of which are incorporated herein by reference. 7.3 TYROSINE-PROTEIN KINASE MEMBRANE RECEPTOR 1 (ROR1) ANTIBODIES

[0390] As noted above, a subject conjugate comprises an antibody (Ab) that binds to ROR1. The amino acid sequence of the antibody can be modified to include a 2- formylglycine (fGly) residue. As used herein, amino acids may be referred to by their standard name, their standard three letter abbreviation and / or their standard one letter 108 NAI-1540479824abbreviation, such as: Alanine or Ala or A; Cysteine or Cys or C; Aspartic acid or Asp or D; Glutamic acid or Glu or E; Phenylalanine or Phe or F; Glycine or Gly or G; Histidine or His or H; Isoleucine or Ile or I; Lysine or Lys or K; Leucine or Leu or L; Methionine or Met or M; Asparagine or Asn or N; Proline or Pro or P; Glutamine or Gln or Q; Arginine or Arg or R; Serine or Ser or S; Threonine or Thr or T; Valine or Val or V; Tryptophan or Trp or W; and Tyrosine or Tyr or Y.

[0391] In some embodiments, the present disclosure provides tyrosine-protein kinase membrane receptor 1 (ROR1) antibodies that can be used herein as therapeutic agents for treatment of cancer. Such agents include antibodies (e.g., monospecific or multispecific, including bispecific) that bind to ROR1. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0392] In some embodiments, described herein are ROR1 antibodies that bind to ROR1, including an ROR1 polypeptide, an ROR1 polypeptide fragment, an ROR1 peptide or an ROR1 epitope. In some embodiments, the ROR1 antibodies are human or humanized antibodies (e.g., comprising human constant regions) that bind ROR1, including an ROR1 polypeptide, an ROR1 polypeptide fragment, an ROR1 peptide or an ROR1 epitope. In some embodiments, an ROR1 antibody, such as a human ROR1 antibody, can bind to ROR1 expressed on the surface of a mammalian (e.g., human) cell, including an ROR1 expressing cancer cell. In some embodiments, an ROR1 antibody, such as a human ROR1 antibody, can bind to ROR1 expressed on the surface of a mammalian (e.g., human) cell, including an ROR1 overexpressing cancer cell. In some embodiments, an ROR1 antibody binds an ROR1 extracellular epitope exposed on a cell such as a cancer cell. In some embodiments, described herein is an ROR1 antibody that binds to ROR1, such as human ROR1 or a portion thereof. In some embodiments, ROR1 is a human ROR1. In some embodiments, an ROR1 antibody is a human ROR1 antibody (e.g., an antibody that binds to human ROR1). In some embodiments, ROR1 antibodies bind to both human and cyno ROR1. In other embodiments, ROR1 antibodies bind to human ROR1 but not to cyno ROR1.

[0393] In some embodiments, the ROR1 antibody provided herein binds to ROR1 (e.g., human ROR1, cyno ROR1, mouse ROR1, and / or rat ROR1) with a dissociation constant (KD) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including by RIA, for example, performed with the Fab version of an 109 NAI-1540479824antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by OCTET®, using, for example, an OCTET®Red96 system, or by BIACORE®, using, for example, a BIACORE®TM-2000 or a BIACORE®TM-3000. An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above using, for example, the OCTET®Red96, the BIACORE®TM-2000, the BIACORE®TM-3000 system, the BIACORE®TM-8K, or the BIACORE®TM-8K+ system.

[0394] In some embodiments, the ROR1 antibody provided herein does not bind to ROR2 (e.g., human ROR2, cyno ROR2, mouse ROR2, and / or rat ROR2). In some embodiments, the ROR1 antibody provided herein does not bind to human ROR2. In some embodiments, the ROR1 antibody provided herein does not bind to human ROR2, cyno ROR2, mouse ROR2, and / or rat ROR2. In other embodiments, the ROR1 antibody provided herein binds to ROR1 with higher affinity than to ROR2 (e.g., human ROR2, cyno ROR2, mouse ROR2, and / or rat ROR2). In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 2-fold of that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 5-fold of that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 10-fold of that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 100-fold of that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 1000 fold of that to ROR2.

[0395] In some embodiments, the ROR1 antibodies described herein comprise a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 of any one of the antibodies described herein, such as an amino acid sequence of a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and / or VL CDR3 depicted in Table 1. Accordingly, in some embodiments, an ROR1 antibody described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from the antibody designated A27 as shown in Table 1. In some embodiments, an ROR1 antibody described herein comprises any one, any two, and / or all three heavy chain CDRs and any one, any two, and / or all three light chain CDRs from the antibody designated A27 as shown in Table 1. In some embodiments, CDRs of an ROR1 antibody as used herein are disclosed in US Patent Application Publication No. US20210155692A1, which is incorporated by reference in its entirety. In some embodiments, 110 NAI-1540479824an ROR1 antibody as used herein is disclosed in US Patent Application Publication No. US20210155692A1, which is incorporated by reference in its entirety.

[0396] In some embodiments, an ROR1 antibody comprises a VH region, which comprises a VH CDR1, a VH CDR2, and / or a VH CDR3, and / or a VL region, which comprises a VL CDR1, a VL CDR2, and / or a VL CDR3, of any one of the antibodies described herein (see, e.g., any one of Tables 1-2). Accordingly, in some embodiments, an ROR1 antibody described herein comprises any one, any two, and / or all three heavy chain CDRs and / or any one, any two, and / or all three light chain CDRs from Table 1.

[0397] In some embodiments, the ROR1 antibody provided herein comprises (i) a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25, and / or (ii) a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26.

[0398] In some embodiments, the ROR1 antibody provided herein comprises a VH CDR1, a VH CDR2, and / or a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and / or a VL CDR1, a VL CDR2, and / or a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein comprises a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. CDR sequences can be determined according to well-known numbering systems or a combination thereof. In some embodiments, the CDRs are according to exemplary numbering. In some embodiments, the CDRs are according to IMGT numbering. In some embodiments, the CDRs are according to Kabat numbering. In some embodiments, the CDRs are according to AbM numbering. In other embodiments, the CDRs are according to Chothia numbering. In other embodiments, the CDRs are according to Contact numbering. In some embodiments, the CDR sequences are determined according to a combination of any two or more of the above-mentioned numbering systems, for example, a combination of Kabat and Chothia. Various exemplary CDR numbering systems are described and illustrated above in Section 7.1.

[0399] In some embodiments, the ROR1 antibody provided herein comprises (a) a VH region comprising a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5; and 36 a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, and 10; and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 111 NAI-154047982413, 14, and 37; and / or (b) a VL region comprising a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18; a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21; and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24.

[0400] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0401] In some embodiments, the ROR1 binding agent provided herein (e.g., an antibody) comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:36, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:37; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0402] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:12; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0403] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0404] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:13; and a VL region comprising a VL CDR1 comprising 112 NAI-1540479824the amino acid sequence of SEQ ID NO:17, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0405] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:5, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:14; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:21, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:24.

[0406] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a VL region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22.

[0407] In some embodiments, the antibody further comprises one or more framework regions of SEQ ID NOs: 25 and / or 26. In some embodiments, the antibody or fragment thereof further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence as set forth in any one of SEQ ID NOs: 25 and 26. In some embodiments, the antibody provided herein is a humanized antibody. Framework regions described herein are determined based upon the boundaries of the CDR numbering system. In other words, if the CDRs are determined by, e.g., Kabat, IMGT, or Chothia, then the framework regions are the amino acid residues surrounding the CDRs in the variable region in the format, from the N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR2 is defined as the amino acid residues between CDR1 and CDR2 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, FR3 is defined as the amino acid residues between CDR2 and CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system, and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined by, e.g., the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system. 113 NAI-1540479824

[0408] In some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise a VH region or VH domain. Additionally or alternatively, in some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise a VL region or VL domain. In some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein have a combination of (i) a VH domain or VH region; and (ii) a VL domain or VL region.

[0409] In some embodiments, the ROR1 antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:25. In some embodiments, the ROR1 antibody provided herein comprises a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein comprises a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0410] In certain embodiments, the ROR1 provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a CDR, VH or VL in Table 1, or any full-length antibody chain as disclosed herein. In some embodiments, the ROR1 antibody provided herein comprises CDRs of any antibody or fragment thereof provided herein, for example in Table 1. In further embodiments, the ROR1 antibody provided herein comprises amino acid sequences with certain percent identity (such as at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or as at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or higher) relative to any antibody or fragment thereof provided herein, for example, a VH or VL in Table 1, or any full-length antibody chain as disclosed herein. 114 NAI-1540479824

[0411] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A.87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A.90:58735877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol.215:403 (1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, word length=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25:33893402 (1997). In some embodiments, the percent identity between two sequences is calculated by dividing the number of residue(s) varied (excluding or including conservative amino acid substitution(s) or degenerate nucleotide substitution(s)) between the two sequences in the alignment with the residue number of any one of the following: (i) full length of the shorter sequence, (ii) full length of the longer sequence, (iii) mean length of the two sequences, (iv) total length of the non-gap portion of the alignment, (v) length of the alignment excluding overhangs, or (vi) length of the alignment including overhangs. Overhangs as used herein with respect to a sequence alignment refer to either or both ends of the alignment where residues of one sequence are considered as aligning to no residues (e.g., gap) in the other sequence. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or 115 NAI-1540479824without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0412] In some embodiments, the antibody provided herein contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody comprising that sequence retains the ability to bind to ROR1. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in a reference amino acid sequence. In some embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs and / or constant regions).

[0413] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of an ROR1 antibody, including a human ROR1 antibody, described herein may vary by one, two, three, four, five, or six amino acid positions so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, the position defining a CDR of any of Table 1 may vary by shifting the N-terminal and / or C-terminal boundary of the CDR by one, two, three, four, five, or six amino acids, relative to the current CDR position, so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) region of an ROR1 antibody, including a human ROR1 antibody, described herein may vary (e.g., be shorter or longer) by one, two, three, four, five, or more amino acids, so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). For example, in some embodiments, a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids shorter than one or more of the CDRs described by SEQ ID NOS:1-24, 36, and 37, so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In other embodiments, a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be one, two, three, four, five or more amino acids longer than one or more of the CDRs described by SEQ ID NOS: 1- 24, 36, and 37, so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). In some embodiments, the amino terminus of a VH and / or VL 116 NAI-1540479824CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 1-24, 36, and 37, so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Additionally or alternatively, in some embodiments, the carboxy terminus of a VH and / or VL CDR1, CDR2, and / or CDR3 described herein may be extended or shortened by one, two, three, four, five or more amino acids compared to one or more of the CDRs described by SEQ ID NOS: 1-24, 36, and 37, so long as binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). Any method known in the art can be used to ascertain whether binding to ROR1 (e.g., human ROR1) is maintained, for example, the binding assays and conditions described in the “Examples” section described herein.

[0414] In other embodiments, the ROR1 antibodies, including human ROR1 antibodies, presented herein that bind to ROR1, further comprise conservative sequence modifications. With respect to polypeptides that are ROR1 antibodies, such as human ROR1 antibodies, conservative sequence modifications include conservative amino acid substitutions that include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Thus, in some embodiments, a predicted nonessential amino acid residue in an ROR1 is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding and nucleotides encoding thereof are well-known in the art (see, e.g., Brummell et al., Biochem.32:1180-1187 (1993); Kobayashi et al. Protein Eng.12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, the conservative sequence modifications described herein modify the amino acid sequences of the ROR1 antibodies, including human ROR1 antibodies, by 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 98%, or 99%. In some embodiments, the amino acid sequence modifications refer to at most 1, 2, 3, 4, 5, or 6 amino acid substitutions to the CDRs, such as those described in any one of Table 1. Thus, for example, each such CDR may contain up to 5 conservative amino acid substitutions, for example up to (not more than) 4 conservative amino acid substitutions, for example up to (not more than) 3 conservative amino acid substitutions, for example up to (not more than) 2 conservative amino acid substitutions, or no more than 1 conservative amino acid substitution. In some 117 NAI-1540479824embodiments, an ROR1 antibody, including a human ROR1 antibody, contains one or more, including six, CDRs having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the CDRs of A27 (see, e.g., Tables 1-2).

[0415] In some embodiments, an ROR1 antibody, including a human ROR1 antibody, contains a VH and a VL comprising CDRs identical to those of A27 (see, e.g., Tables 1-2). In some embodiments, the amino acid sequence modifications do not include any modification within an SDR. In some embodiments, the amino acid sequence modifications do not include any modification within a CDR (such as CDR1, CDR2, CDR3, or any combination thereof). Additionally or alternatively, the amino acid sequence modifications are in the framework, constant region, and / or fragment crystallizable region (Fc).

[0416] In some embodiments, the antibody or fragment provided herein comprises a VH domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:25, and / or a VL domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:26, and the binding of the antibody or fragment thereof to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0417] In some embodiments, functional epitopes can be mapped, e.g., by combinatorial alanine scanning, to identify amino acids in the ROR1 protein that are necessary for interaction with ROR1 antibodies provided herein. In some embodiments, conformational and crystal structure of ROR1 antibodies bound to ROR1 may be employed to identify the epitopes. In some embodiments, the present disclosure provides an antibody that specifically binds to the same epitope as any of the ROR1 antibodies provided herein.

[0418] For example, in some embodiments, the ROR1 antibody provided herein (e.g., an antibody) binds to the same epitope as an anti-ROR1 antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein binds to the same epitope as an anti-ROR1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:25, and a VL comprising the amino acid sequence of SEQ ID NO:26. 118 NAI-1540479824

[0419] In some embodiments, the ROR1 antibody provided herein further comprises an Fc or a variant thereof. In some embodiments, the Fc comprises an amino acid sequence as set forth in SEQ ID NO: 38. In other embodiments, the Fc variant is a silent Fc (sFc). In further embodiments, the silent Fc comprises an alanine (Ala, A) residue at position Leu234 (L234) according to the EU numbering system and an alanine (Ala, A) residue at position Leu235 (L235) according to the EU numbering system. In some embodiments, the silent Fc comprises an alanine (Ala, A) residue at position Leu234 (L234) according to the EU numbering system and an alanine (Ala, A) residue at position Leu235 (L235) according to the EU numbering system, and a lysine (Lys, K) residue at position Pro329 (P329) according to the EU numbering system (also referred to herein as “LALAPK” or “L234A / L235A / P329K”). In some embodiments, the silent Fc comprises an amino acid sequence as set forth in SEQ ID NO: 39. Additionally or alternatively, a variant Fc region has a reduced potential immunogenicity. In further embodiments, a variant Fc region comprises a glutamic acid (Glu, E) residue at position Asp356 (D356) according to the EU numbering system, a glutamic acid (Glu, E) residue at position Glu357 (E357) according to the EU numbering system, and a methionine (Met, M) residue position Leu358 (L358) according to the EU numbering system (also referred to herein as “EEM” or “D356E / E357E / L358M”). Other suitable Fc can be found, for example in US20230071196 and US20220389055, each of which is incorporated herein by reference in its entirety.

[0420] In some embodiments, the ROR1 antibody provided herein specifically binds to ROR1 competitively with any one of the anti-ROR1 antibodies or fragments thereof described herein.

[0421] In some embodiments, the ROR1 antibody provided herein specifically binds to ROR1 competitively with an anti-ROR1 antibody comprising a VH CDR1, a VH CDR2, and a VH CDR3 as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein specifically binds to ROR1 competitively with an anti-ROR1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO:25, and a VL comprising the amino acid sequence of SEQ ID NO:26.

[0422] In some embodiments, the ROR1 antibody comprises six CDRs of the antibody designated A27. In yet further embodiments, the ROR1 antibody comprises six CDRs as listed in one column of Table 1. In some embodiments, the ROR1 antibody comprises three CDRs of the heavy chain variable region as set forth in SEQ ID NO:25 and three CDRs of the 119 NAI-1540479824light chain variable regions as set forth in SEQ ID NO:26. In some embodiments, the ROR1 antibody comprises the heavy chain variable region as set forth in SEQ ID NO:25 and the light chain variable regions as set forth in SEQ ID NO:26.

[0423] In some embodiments, the ROR1 antibody is an IgG, for example IgG1, IgG2, IgG3, or IgG4. In further embodiments, the ROR1 antibody is an IgG1. Additionally or alternatively, the ROR1 antibody comprises a kappa (κ) light chain (e.g., a kappa (κ) antibody). In other embodiments, the ROR1 antibody comprises a lambda (λ) light chain (e.g., a lambda (λ) antibody). In some embodiments, the ROR1 antibody is an IgG1 kappa antibody.

[0424] In some embodiments, the antibodies are superior developability based on a known assay in the art, for example, various chromatographic methods, including size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and standup monolayer adsorption chromatography (SMAC). In some embodiments, the antibodies are superior developability based on measurement of monomer percentage, solubility, and / or antibody aggregation or precipitation.

[0425] In some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise a heavy chain having a combination of (i) a VH described herein, such as in Table 1; and (ii) one or more heavy chain constant domains (e.g., CH1, Hinge, CH2, and CH3). An exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, Hinge, CH2, and CH3 amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:31)

[0426] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include unconverted sulfatase motifs. In some embodiments, the unconverted sulfatase motif comprises the amino acid sequence of LCTPSR (SEQ ID NO:100).

[0427] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include unconverted sulfatase motifs in the CH1 region and in 120 NAI-1540479824the CT region. An exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, Hinge, CH2, and CH3 amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALCTPSRGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCTPSRGS (SEQ ID NO:32)

[0428] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include unconverted sulfatase motif in the CH1 region. An exemplary IgG heavy chain comprises any VH sequence as described herein and the following CH1, Hinge, CH2, and CH3 amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSLCTPSRNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:33)

[0429] In further embodiments, the carboxyl terminus (C terminus) of the VH is conjugated directly or indirectly to the amino terminus (N terminus) of the one or more heavy chain constant domains.

[0430] In some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise a light chain having a combination of (i) a VL domain described herein, such as in any one of Table 1; and (ii) a light chain constant domain (CL). An exemplary light chain (e.g., for pairing with an IgG heavy chain) comprises any VL sequence described herein and the following CL amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:34)

[0431] In further embodiments, the C terminus of the VL is conjugated directly or indirectly to the N terminus of the CL. 121 NAI-1540479824

[0432] In some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise (a) a heavy chain having a combination of (i) a VH described herein, such as in Table 1, and (ii) one or more heavy chain constant domains (e.g., CH1, Hinge, CH2, and CH3); and (b) a light chain having a combination of (i) a VL described herein, such as in Table 1, and (ii) a light chain constant domain in an IgG format (CL or CL1). In some embodiments, the ROR1 antibody comprises an IgG heavy chain comprising any VH sequence as described herein and the amino acid sequence of SEQ ID NO:31, and a light chain comprising any VL sequence as described herein and the amino acid sequence of SEQ ID NO:34. In some embodiments, the ROR1 antibody comprises an IgG heavy chain comprising any VH sequence as described herein and the amino acid sequence of SEQ ID NO:32, and a light chain comprising any VL sequence as described herein and the amino acid sequence of SEQ ID NO:34. In some embodiments, the ROR1 antibody comprises an IgG heavy chain comprising any VH sequence as described herein and the amino acid sequence of SEQ ID NO:33, and a light chain comprising any VL sequence as described herein and the amino acid sequence of SEQ ID NO:34.

[0433] In some embodiments, ROR1 antibodies (e.g., antibodies such as monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise (a) a heavy chain described herein, such as in Table 2, and (b) a light chain described herein, such as in Table 2. In some embodiments, ROR1 antibodies (e.g., monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise: (a) a heavy chain having the amino acid sequence of SEQ ID NO:27, and (b) a light chain having the amino acid sequence of SEQ ID NO:28. In some embodiments, ROR1 antibodies (e.g., monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise: (a) a heavy chain having the amino acid sequence of SEQ ID NO:29, and (b) a light chain having the amino acid sequence of SEQ ID NO:28. In some embodiments, ROR1 antibodies (e.g., monospecific or bispecific antibodies), including human ROR1 antibodies, described herein comprise: (a) a heavy chain having the amino acid sequence of SEQ ID NO:30, and (b) a light chain having the amino acid sequence of SEQ ID NO:28.

[0434] In some embodiments, ROR1 antibodies (e.g., monospecific or bispecific antibodies), including huma...

Claims

CLAIMS What is claimed is:

1. An antibody-drug conjugate (ADC) of Formula (I) comprising: a. an antibody that binds to tyrosine-protein kinase membrane receptor 1 (ROR1); and b. two or more drugs conjugated to a pyridazine-pyrrolo coupling moiety, each via a linkerwherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4are each independently CR4; Z3is C-LB-W2; R1, R2, R3, and R4are each selected from hydrogen and alkyl; LAis a first linker comprising: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, wherein: a, b, c, d, e, and f are each independently 0 or 1, provided at least one of a, b, c, d, e, and f are 1; T1, T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino- benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene 206 NAI-1540479824glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LBis a second linker comprising: -(T7-V7)g-(T8-V8)h-(T9-V9)i-(T10-V10)j-(T11-V11)k-(T12-V12)l-(T13-V13)m-, wherein: g, h, i, j, k, l,and m are each independently 0 or 1, provided that at least one of g, h, i, j, k, l, and m is 1; T7, T8, T9, T10, T11, T12,and T13are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para- amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), an acetal group, a hydrazine, a disulfide, and an ester, wherein EDA is an ethylene diamine moiety, PEG is a polyethylene glycol, and AA is an amino acid residue or an amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V7, V8, V9, V10, V11, V12,and V13are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein each q is an integer from 1 to 6; 207 NAI-1540479824each R13is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; s is an integer from 1 to 10; W1is a first drug; and W2 is a second drug.

2. The ADC of claim 1, wherein:T1is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V1, V2, V3, V4, V5, and V6are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, and -P(O)OH-;4-amino-piperidineeach R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; q is an integer from 1 to 6; 208 NAI-1540479824r is 0 or 1; and yis an integer from 1 to 6.

3. The ADC of claims 1 or 2, wherein:T1is (C1-C12)alkyl and V1is -CONH-; T2is substituted (C1-C12)alkyl and V2is -CO-; T3is (AA)p and V3is absent; T4is PABC and V4is absent; p is an integer from 1 to 10; and a, b, c, and d are each 1; and e and f are each 0.

4. The ADC of any one of claims 1-3, wherein: T7is a covalent bond; T8, T9, T10, T11, and T12are each independently selected from a covalent bond, (C1- C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V7, V8, V9, V10, V11, and V12are each independently selected from the group consisting of a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, and -P(O)OH-; wherein:integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:209 NAI-1540479824each R12is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12groups may be cyclically linked to form a piperazinyl ring; g, h, i, j, and k are each 1; and l and m are each 0.

5. The ADC of any one of claims 1-4, wherein: T7is absent and V7is -NHCO-; T8is (C1-C12)alkyl and V8is -CONH-; T9is substituted (C1-C12)alkyl and V9is -CO-; T10is (AA)pand V10is absent; T11is PABC and V11is absent; p is an integer from 1 to 10; and g, h, i, j, and k are each 1; and l and m are each 0.

6. The ADC of any one of claims 1-5, wherein one or both of T2and T9is (C1- C6)alkylene substituted with -NHCO(PEG)t, whereinand t is an integer from 2 to 10, optionally 8.

7. The ADC of any one of claims 1-6, wherein one or both T3and T10has p of 2.

8. The ADC of any one of claims 1-7, wherein s is 2 or 4.

9. The ADC of any one of claims 1-8, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside, optionally wherein the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.

10. The ADC of any one of claims 1-9, wherein one or both of W1and W2is a camptothecin analog, optionally wherein the camptothecin analog is belotecan.

11. The ADC of any one of claim 1-10, wherein each of W1and W2is belotecan. 210 NAI-154047982412. An ADC represented by Formula (II):wherein: Ab represents the antibody that binds to ROR1; and s is an integer from 1 to 10.

13. The ADC of any one of claims 1-12, wherein s is 2 or 4.

14. The ADC of any one of claims 1-12, wherein s is 2.

15. The ADC of any one of claims 1-12, wherein s is 4.

16. The ADC of any one of claims 1-15, wherein the antibody Ab comprises: a heavy chain variable region (VH) complementarity determining region 1 (CDR1), a VH complementarity determining region 2 (CDR2), and a VH complementarity determining region 3 (CDR3) as set forth in a VH comprising the amino acid sequence of SEQ ID NO:25 and a light chain variable region (VL) CDR1, a VL CDR2, and a VL CDR3 as set forth in a VL comprising the amino acid sequence of SEQ ID NO:

26. 211 NAI-154047982417. The ADC of any one of claims 1-16, wherein the antibody Ab comprises: (a) a VH comprising: (1) a VH CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, and 36; (2) a VH CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, and 10; and (3) a VH CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, and 37; and (b) a VL comprising: (1) a VL CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, and 18; (2) a VL CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, and 21; and (3) a VL CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24.

18. The ADC of any one of claims 1-17, wherein the antibody Ab comprises any one or more of (i)-(vii): (i) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:6, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22; (ii) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:7, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:12; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:16, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22; (iii) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:6, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a VL 212 NAI-1540479824comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22; (iv) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:13; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:17, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:23; (v) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:5, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:9, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:14; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:21, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:24; (vi) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:22; or (vii) a VH comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:36, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:37; and a VL comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

23.

19. The ADC of any one of claims 1-18, wherein the antibody Ab further comprises a framework 1 (FR1), a framework 2 (FR2), a framework 3 (FR3) and / or a framework 4 (FR4) sequence. 213 NAI-154047982420. The ADC of any one of claims 1-19, wherein the antibody Ab further comprises human framework sequences, optionally an FR1, an FR2, an FR3 and / or an (FR4 sequence as set forth in any one of SEQ ID NOs: 25 and 26.

21. The ADC of any one of claims 1-20, wherein the antibody Ab comprises: a VH comprising the amino acid sequence of SEQ ID NO:25 and a VL comprising the amino acid sequence of SEQ ID NO:

26.

22. The ADC of any one of claims 1-21, wherein the antibody Ab comprises a sequence of Formula (VIII) X1(fGly’)X2Z20X3Z30(VIII) wherein: fGly’ is the amino acid residue coupled to the drug through a linker; Z20is either a proline (P) or alanine (A) residue; Z30is a basic amino acid residue optionally selected from the group consisting of: arginine (R), lysine (K), and histidine (H), or an aliphatic amino acid residue optionally selected from the group consisting of: alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), and proline (P); X1is present or absent and, when present, can be any amino acid residue, with the proviso that when the sequence of Formula (VIII) is at the N-terminus of the antibody Ab, X1is present; and X2and X3independently can be any amino acid residue, optionally wherein the sequence of Formula (VIII) is selected from the group consisting of: L(fGly’)TPSR (SEQ ID NO:146), M(fGly’)TPSR (SEQ ID NO:147), V(fGly’)TPSR (SEQ ID NO:148), L(fGly’)SPSR (SEQ ID NO:149), L(fGly’)APSR (SEQ ID NO:150), L(fGly’)VPSR (SEQ ID NO:151), L(fGly’)GPSR (SEQ ID NO:152), I(fGly’)TPAR (SEQ ID NO:153), L(fGly’)TPSK (SEQ ID NO:154), M(fGly’)TPSK (SEQ ID NO:155), V(fGly’)TPSK (SEQ ID NO:156), L(fGly’)SPSK (SEQ ID NO:157), L(fGly’)APSK (SEQ ID NO:158), L(fGly’)VPSK (SEQ ID NO:159), L(fGly’)GPSK (SEQ ID NO:160), L(fGly’)TPSA (SEQ ID NO:161), I(fGly’)TPAA (SEQ ID NO:162), M(fGly’)TPSA (SEQ ID NO:163), V(fGly’)TPSA (SEQ ID NO:164), L(fGly’)SPSA (SEQ ID NO:165), L(fGly’)APSA (SEQ ID NO:166), L(fGly’)VPSA (SEQ ID NO:167), and L(fGly’)GPSA (SEQ ID NO:168); 214 NAI-1540479824further optionally wherein the sequence of Formula (VIII) comprises L(fGly’)TPSR (SEQ ID NO:146).

23. The ADC of any one of claims 1-22, wherein the antibody Ab is an IgG1 antibody, optionally an IgG1 kappa antibody.

24. The ADC of any one of claims 1-23, wherein the antibody Ab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:27 or a variant thereof further comprising one or more sequences of Formula (VIII) and a light chain comprising the amino acid sequence of SEQ ID NO:

28.

25. The ADC of any one of claims 1-24, wherein the antibody Ab comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO:41 and a light chain comprising the amino acid sequence of SEQ ID NO:28; or (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO:44 and a light chain comprising the amino acid sequence of SEQ ID NO:

28.

26. The ADC of any one of claims 1-25, wherein the antibody Ab is a monoclonal antibody.

27. The ADC of any one of claims 1-26, wherein the antibody Ab is a humanized, human, or chimeric antibody.

28. The ADC of any one of claims 1-22, wherein the antibody Ab is a Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single chain antibody molecule, dual variable region antibody, single variable region antibody, linear antibody, V region, or a multispecific antibody formed from antibody fragments.

29. The ADC of any one of claims 1-28, wherein the antibody Ab is conjugated or recombinantly fused to a diagnostic agent, detectable agent, or therapeutic agent, optionally wherein the therapeutic agent is a chemotherapeutic agent, cytotoxin, or drug.

30. The ADC of any one of claims 1-27, wherein the antibody Ab is a multispecific antibody, optionally a bispecific antibody. 215 NAI-154047982431. A pharmaceutical composition comprising the ADC of any one of claims 1-30 and a pharmaceutically acceptable excipient.

32. The pharmaceutical composition of claim 31, characterized by an ADC drug-to- antibody ratio (DAR) of about 1 to about 20.

33. The pharmaceutical composition of claim 32, wherein the DAR is about 2 to about 8.

34. The pharmaceutical composition of claim 32, wherein the DAR is about 4 to about 8.

35. The pharmaceutical composition of claim 32, wherein the DAR is about 4.

36. The pharmaceutical composition of claim 32, wherein the DAR is about 8.

37. A method for treating a subject with cancer, the method comprising administering a therapeutically effective amount of the ADC of any one of claims 1-30 or the pharmaceutical composition of any one of claims 31-36 to the subject.

38. The method of claim 37, wherein the cancer is an ROR1 antigen expressing cancer.

39. The method of any of claim 37 or claim 38, wherein the cancer is selected from the group consisting of: pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematological cancer, prostate cancer, colon cancer, renal cancer, thyroid cancer, liver cancer, urothelial carcinoma, melanoma, endometrial cancer, clear cell renal cell carcinoma, clear cell carcinoma, and uterine cancer, optionally wherein the cancer is triple negative breast cancer, non-small cell lung cancer, or mantle cell lymphoma. 216 NAI-1540479824

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