WO2025019787A1 - Tyrosine-protein kinase membrane receptor 1 (ror1) antibody-drug conjugates and uses thereof - Google Patents

Tyrosine-protein kinase membrane receptor 1 (ror1) antibody-drug conjugates and uses thereof Download PDF

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WO2025019787A1
WO2025019787A1 PCT/US2024/038778 US2024038778W WO2025019787A1 WO 2025019787 A1 WO2025019787 A1 WO 2025019787A1 US 2024038778 W US2024038778 W US 2024038778W WO 2025019787 A1 WO2025019787 A1 WO 2025019787A1
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amino acid
alkyl
antibody
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Kathleen GOGAS
Seema Kantak
Brian Alan MENDELSOHN
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Exelixis Inc
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Exelixis Inc
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Priority to CN202480052974.6A priority patent/CN121752598A/en
Publication of WO2025019787A1 publication Critical patent/WO2025019787A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • 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.
  • ADCs antibody-drug conjugates
  • ROR1 tyrosine-protein kinase membrane receptor 1
  • BACKGROUND Various tumors can demonstrate cell-surface expression of tyrosine-protein kinase transmembrane receptor (ROR) antigens, as described in greater detail in Gentile, et al.
  • ROR expression may not be expressed, or only demonstrate limited expression, in normal, e.g., non-cancerous, tissue as described in Balakrishnan et al.
  • ROR antigens can be used as a tumor-specific marker in certain tumors.
  • 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.
  • ROR multispecific antibodies formatted in various 1 NAI-1540479824 antibody 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. [00005] 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.
  • ADCs comprising an antibody that binds tyrosine- protein kinase membrane receptor 1 (“ROR1-ADC”).
  • ROR1-ADCs 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.
  • 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.
  • ROR1 antibody an antibody or fragment thereof that binds to ROR1
  • Such pharmaceutical compositions 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.
  • 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.
  • 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.
  • HIPS Hydrazino-iso-Pictet-Spengler
  • 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.
  • 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-1540479824 molecule 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.
  • DAR site-specific conjugates e.g., DAR up to 8
  • 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.
  • 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.
  • 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.
  • L A is a first linker comprising: -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) f -, 3 NAI-1540479824 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
  • Z 1 is CR 4 .
  • Z 3 is C-L B -W 2 .
  • W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • L A comprises: -(T 1 -V 1 )a-(T 2 -V 2 )b-(T 3 -V 3 )c-(T 4 -V 4 )d-(T 5 -V 5 )e-(T 6 -V 6 )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; T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl
  • T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from a covalent bond, (C1- C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG)n, (AA)p, -(CR 13 OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V 1 , V 2 , V 3 , V 4
  • T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L A is a linker wherein: T 1 is (C1-C12)alkyl and V 1 is -CONH-; T 2 is substituted (C 1 -C 12 )alkyl and V 2 is -CO-; T 3 is (AA) p and V 3 is absent (e.g., a covalent bond); T 4 is PABC and V 4 is 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.
  • 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.
  • a glycoside such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L B comprises: -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V 10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l -(T 13 -V 13 ) 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; T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each independently selected from a covalent bond, (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 7 is a covalent bond
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L B is a linker wherein: T 7 is absent (e.g., a covalent bond) and V 7 is -NHCO-; T 8 is (C 1 -C 12 )alkyl and V 8 is -CONH-; T 9 is substituted (C1-C12)alkyl and V 9 is -CO-; T 10 is (AA)p and V 10 is absent (e.g., a covalent bond); T 11 is PABC and V 11 is 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.
  • 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.
  • a glycoside such as a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • an ROR1-ADC is represented by Formula (I): 9 NAI-1540479824 wherein: Ab represents the antibody that binds to ROR1; Z 1 , Z 2 , and Z 4 are each independently CR 4 ; Z 3 is C-L B -W 2 ; R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (C1-C12)alkyl; L A is a first linker wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-; T 2 is substituted (C1-C12)alkyl and V 2 is -CO-; T 3 is (AA) p where p is an integer from 1-20 and V 3 is a covalent bond; T 4 is PABC and V 4 is a covalent bond; a, b, c, and d are each 1; e and f are each 0; and L B is a second linker wherein: T 7 is a
  • an ROR1-ADC is represented by Formula (I): 10 NAI-1540479824 wherein: Ab represents the antibody that binds to ROR1; Z 1 , Z 2 , and Z 4 are each independently CR 4 ; Z 3 is C-L B -W 2 ; R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (C1-C12)alkyl; L A is a linker wherein: T 1 is (C 1 -C 6 )alkyl and V 1 is -CONH-; T 2 is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is integer from 2 to 10, optionally 8, and V 2 is -CO-; T 3 is (AA)2 and V 3 is a covalent bond; T 4 is PABC substitute
  • the PABC of one or both of T 4 and T 11 is substituted with a glucuronide.
  • one or both of T 1 and T 8 is ethyl.
  • one or both of T 2 and T 9 is C5 alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is integer from 5-10, optionally 8.
  • one or both of W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • 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.
  • Formula (II) may be prepared by conjugating one or more linker-payloads of Formula (IIa), shown below, with an ROR1 antibody: 12 NAI-1540479824
  • 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.
  • 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.
  • 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.
  • 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-1540479824 framework 4 (FR4) sequence, for example, as set forth in any one of SEQ ID NOs: 25 and 26.
  • an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises human framework sequences.
  • 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.
  • 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.
  • s is 4.
  • 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. [00049] 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.
  • DAR drug-to-antibody ratio
  • 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.
  • 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.
  • 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.
  • DAR drug-to-antibody ratio
  • 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-1540479824 ADC of Formula (I) or (II) and a pharmaceutically acceptable excipient, wherein the ROR1 antibody is as described in any embodiment herein.
  • a kit comprising the antibody-drug conjugate as disclosed herein or the pharmaceutical composition as disclosed herein, and instructions for use. 6.
  • FIG.1 provides exemplary in vitro cytotoxicity results as detailed in Example 5.
  • 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.
  • FIGs.3A-3B provide exemplary in vivo efficacy data in JEKO-1 mantle cell lymphoma (MCL) xenograft model, as detailed in Example 7.
  • MCL mantle cell lymphoma
  • FIG.3A plots tumor volumes, while FIG.3B plots tumor growth inhibition percentages (%TGI) over the isotype control on Day 25.
  • FIG.4 provides exemplary pharmacokinetics (PK) results in rats as detailed in Example 9.
  • 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.
  • 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.
  • NSCLC non-small cell lung cancer
  • PDX patient-derived xenograft
  • the present disclosure provides antibody-drug conjugates (ADCs) that bind to ROR1 and a drug conjugated (directly or indirectly) thereto.
  • ADCs antibody-drug conjugates
  • 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.
  • 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.
  • 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 (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).
  • substituted alkyl refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C 1 carbon 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, thioheter
  • 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 -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 - and the like.
  • This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH 2 CH(CH 3 )-), (-C(CH 3 ) 2 CH 2 CH 2 -), (-C(CH 3 ) 2 CH 2 C(O)-), (-C(CH 3 ) 2 CH 2 C(O)NH-), (-CH(CH3)CH2-), and the like.
  • “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below.
  • alkane refers to alkyl group and alkylene group, as defined herein.
  • alkylaminoalkyl 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.
  • alkaryl or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.
  • 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.
  • alkoxy also refers to alkenyl-O-, cycloalkyl- O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
  • 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.
  • alkoxyamino refers to the group –NH-alkoxy, wherein alkoxy is defined herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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).
  • 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, al
  • Alkynyloxy refers to the group –O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.
  • 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, substituted alkenyl
  • acyl includes the “acetyl” group CH 3 C(O)- [00082]
  • “Acylamino” refers to the groups –NR 20 C(O)alkyl, -NR 20 C(O)substituted alkyl, N R 20 C(O)cycloalkyl, -NR 20 C(O)substituted cycloalkyl, - NR 20 C(O)cycloalkenyl, -NR 20 C(O)substituted cycloalkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O)substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O)substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O)substituted aryl, -NR 20 C(O)heteroary
  • Aminocarbonyl or the term “aminoacyl” refers to the group -C(O)NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted
  • Aminocarbonylamino refers to the group –NR 21 C(O)NR 22 R 23 where R 21 , R 22 , and R 23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.
  • 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.
  • acyloxy refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.
  • Aminosulfonyl refers to the group –SO2NR 21 R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R 21 and R 22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted
  • “Sulfonylamino” refers to the group –NR 21 SO2R 22 , wherein R 21 and R 22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 21 and R 22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, substituted
  • 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.
  • 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-1540479824 amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, substituted thioalkoxy,
  • 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.
  • Amino refers to the group –NH 2 .
  • 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.
  • azido refers to the group –N3.
  • Carboxyl,” “carboxy” or “carboxylate” refers to –CO 2 H or salts thereof.
  • 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(C(O)O
  • (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, -
  • Cyano or “nitrile” refers to the group –CN.
  • 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.
  • 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,
  • 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.
  • 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,
  • 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.
  • Cycloalkoxy refers to –O-cycloalkyl.
  • Cycloalkenyloxy refers to –O-cycloalkenyl. 22 NAI-1540479824
  • Halo or “halogen” refers to fluoro, chloro, bromo, and iodo.
  • “Hydroxy” or “hydroxyl” refers to the group –OH.
  • 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.
  • 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.
  • 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.
  • N ⁇ O N-oxide
  • sulfinyl sulfonyl moieties.
  • This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.
  • 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, thio
  • heteroarylkyl 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.
  • Heteroaryloxy refers to –O-heteroaryl.
  • Heterocycle refers 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.
  • the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N- 23 NAI-1540479824 oxide, -S(O)-, or -SO2- moieties.
  • 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.
  • 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,
  • 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,
  • Heterocyclyloxy refers to the group –O-heterocyclyl.
  • heterocyclylthio refers to the group heterocyclic-S-.
  • heterocyclene refers to the diradical group formed from a heterocycle, as defined herein.
  • hydroxyamino refers to the group -NHOH.
  • Niro refers to the group –NO 2 .
  • “Sulfonyl” refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO 2 -substituted alkenyl, -SO 2 -cycloalkyl, -SO 2 -substituted cylcoalkyl, -SO 2 -cycloalkenyl, -SO 2 -substituted cylcoalkenyl, -SO 2 -aryl, -SO 2 -substituted aryl, -SO 2 -heteroaryl, -SO 2 - substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein alkyl, 24 NAI-1540479824 substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitute
  • Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4- methylphenyl-SO2-.
  • “Sulfonyloxy” refers to the group -OSO 2 -alkyl, -OSO 2 -substituted alkyl, -OSO 2 - alkenyl, -OSO 2 -substituted alkenyl, -OSO 2 -cycloalkyl, -OSO 2 -substituted cylcoalkyl, -OSO 2 - cycloalkenyl, -OSO2-substituted cylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2- heteroaryl, -OSO 2 -substituted heteroaryl, -OSO 2 -heterocyclic, and -OSO 2 -substituted hetero
  • “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-SO 2 -O-substituted cylcoalkyl, -O-SO 2 -O-cycloalkenyl, -O-SO 2 -O-substituted cylcoalkenyl, -O-SO 2 -O-aryl, -O-SO 2 -O-substituted aryl, -O-SO 2 -O-heteroaryl, -O-SO 2 -O- substituted heteroaryl, -O-SO2-O-heterocyclic, and -O-SO-SO2-
  • 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.
  • Thiol refers to the group -SH.
  • Alkylthio or the term “thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein.
  • sulfur may be oxidized to -S(O)-.
  • the sulfoxide may exist as one or more stereoisomers.
  • substituted thioalkoxy refers to the group -S-substituted alkyl.
  • 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 [00128]
  • thioheteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.
  • heterocyclooxy refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.
  • 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.
  • Each M + may independently be, for example, an alkali ion, such as K + , Na + , Li + ; an ammonium ion, such as + N(R 60 ) 4 ; or an alkaline earth ion, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 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).
  • an alkali ion such as K + , Na + , Li +
  • an ammonium ion such as + N(R 60 ) 4
  • -NR 80 R 80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl.
  • substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R 60 , halo, -O-M + , -OR 70 , -SR 70 , -S – M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , -N 3 , -SO 2 R 70 , -SO 3 M + , -SO 3 R 70 , -OSO 2 R 70 , -OSO 3 – M + , -OSO 3 R 70 , -PO 3 -2 (M + ) 2 , -P(O)(OR 70 )O – M + , -P(O)(OR 70 ) 2 , -C(O)R 70 ,
  • substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R 60 , -O-M + , -OR 70 , -SR 70 , -S-M + , -NR 80 R 80 , trihalomethyl, -CF 3 , -CN, -NO, -NO 2 , -S(O) 2 R 70 , -S(O) 2 O-M + , -S(O) 2 OR 70 , -OS(O)2R 70 , -OS(O)2O-M + , -OS(O)2OR 70 , -P(O)(O-)2(M + )2, -P(O)(OR 70 )O-M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70
  • a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
  • 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.
  • the maximum number of such substitutions is three.
  • 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.
  • the subject compounds include all stereochemical isomers arising from the substitution of these compounds.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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 [00141] “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.
  • pyrazoles imidazoles, benzimidazoles, triazoles, and tetrazoles.
  • a pharmaceutically or therapeutically effective amount refers 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.
  • 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.
  • 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).
  • 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.
  • “Patient” refers to human and non-human subjects, especially mammalian subjects.
  • treating 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-1540479824 development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.
  • the term “treating,” or “treatment” excludes a prophylactic treatment.
  • 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.
  • FGE Formylglycine Generating Enzyme
  • 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.
  • “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.
  • 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.
  • 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.
  • subject refers to human and non-human subjects, especially mammalian subjects.
  • 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.
  • 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-1540479824 stereochemistry as in the naturally occurring D-form, as well as the L-form of amino acid analogs.
  • 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.
  • 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.
  • amino acid analogs can include ⁇ -hydroxy acids, and ⁇ - amino acids, and the like.
  • amino acid side chain is used to refer to the substituent attached to the ⁇ -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.
  • carbohydrate is used to refer to monomer units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
  • sugar is be used to refer to the smaller carbohydrates, such as monosaccharides, disaccharides.
  • 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.
  • glycoside or “glycosyl” refers to a sugar molecule or group bound to a moiety via a glycosidic bond.
  • 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).
  • O-glycosidic bond an O-glycoside
  • N-glycosidic bond a glycosylamine
  • S-glycosidic bond a thioglycoside
  • C-glycosidic bond a C-glycoside or C-glycosyl
  • ROR1 antigens refer to a member of the tyrosine-protein kinase transmembrane receptor (ROR) family.
  • ROR2 is another member of the same family.
  • the ROR1-ADCs and ROR1 antibodies as disclosed herein do not bind to ROR2 (such as human ROR2).
  • the ROR1-ADCs and ROR1 antibodies as disclosed herein do not bind to human ROR2 or cyno ROR2.
  • 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).
  • ROR1 e.g., human ROR1
  • the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to ROR1 is at least 2 folds of that to ROR2 (e.g., human ROR2).
  • 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).
  • the binding affinity of the ROR1-ADCs and ROR1 antibodies as disclosed herein to 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).
  • ROR1 refers to ROR1 (e.g., human ROR1).
  • ROR1 e.g., human ROR1.
  • antibody immunoglobulin
  • Ig immunoglobulin
  • 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.
  • 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).
  • variable (V) region can be any suitable arrangement of immunoglobulin heavy (VH) and/or light (VL) variable regions.
  • antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, 32 NAI-1540479824 and an antibody heavy chain monomer.
  • the V region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers that bind ROR1.
  • a VH region and a VL region can be covalently coupled either directly or through a linker to form a single-chain Fv (scFv).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • 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).
  • 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).
  • a bispecific antibody comprises two antigen-binding sites, each may bind to a different epitope.
  • Such a 34 NAI-1540479824 bispecific antibody may bind to two different epitopes on the same antigen (e.g., epitopes on ROR).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains
  • 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).
  • a moiety such as a labeling component or a drug (e.g., toxin).
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • an “antigen” is a moiety or molecule that contains an epitope to which an antibody can bind.
  • an antigen can be bound by an antibody.
  • the antigen to which an antibody described herein binds is an ROR1 antigen (e.g., a human ROR1 antigen), or a fragment thereof.
  • 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.
  • 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.
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • an antibody binds to a group of amino acids regardless of whether they are folded in a natural three-dimensional protein structure.
  • an antibody 36 NAI-1540479824 requires 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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, BIACORETM, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), the OctetQK384 system (ForteBio, Menlo Park, CA), or other assays known in the art.
  • 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).
  • 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.
  • 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.
  • FACS fluorescence activated cell sorting
  • molecules that specifically bind to an antigen bind to the antigen with a K A that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen.
  • molecules 37 NAI-1540479824 that specifically bind to an antigen do not cross react with other proteins.
  • molecules that specifically bind to an antigen do not cross react with other non- ROR1 antigens.
  • “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 ⁇ M.
  • “specifically binds” means that a polypeptide or molecule binds a target with a K D of at least about 0.1 ⁇ M or less, at least about 0.01 ⁇ M 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.
  • a polypeptide or molecule can specifically bind more than one target.
  • multiple targets can be bound by the same antigen-binding site on the polypeptide or molecule.
  • an antibody can comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins.
  • an antibody can be bispecific and comprise at least two antigen-binding sites with differing specificities.
  • 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.
  • the “K D ” or “K D value” can be measured by biolayer interferometry (BLI) using, for example, the OctetQK384 system (ForteBio, Menlo Park, CA).
  • the KD may also be 38 NAI-1540479824 measured 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.
  • BIACORETM surface plasmon resonance (SPR) assays by BIACORETM, using, for example, a BIACORETM-2000 or a BIACORETM-3000 (BIAcore, Inc., Piscataway, NJ).
  • SPR surface plasmon resonance
  • 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 “k off ,” 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 BIACORETM-2000 or a BIACORETM-3000 (BIACORETM, Inc., Piscataway, NJ), respectively.
  • 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).
  • a reference molecule e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody
  • ROR1 e.g., human ROR1
  • assays examples 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.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA enzyme immunoassay
  • 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.
  • 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).
  • ROR1 purified antigen
  • ADC unlabeled test antigen binding protein
  • 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.
  • the test antigen binding protein is present in excess.
  • Antibodies identified by competition assay 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-1540479824 occur (e.g., similar epitope or overlapping epitope).
  • competing antibody when it 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%.
  • binding is inhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.
  • the terms “constant region” and “constant domain” 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.
  • 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.
  • 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.
  • 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.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • 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.
  • 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.
  • 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).
  • 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”)).
  • 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”).
  • a variant Fc region has a reduced potential immunogenicity.
  • 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”) [00186]
  • 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 ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), 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.
  • 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.
  • Light chain amino acid sequences are well known in the art.
  • a “chain” e.g., a heavy chain or a light chain
  • 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).
  • 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.
  • 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.
  • synthetic antibodies e.g., 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’) fragment
  • 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.
  • 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.
  • 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.
  • an antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs.
  • the amino acid sequences of the H chains are identical and the amino acid sequences of the L chains are identical.
  • 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.
  • 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.
  • an antibody is a 2-chain antibody unit comprising a VHH-VHH pair.
  • the amino acid sequences of the VHH are identical. In other embodiments, the amino acid sequence of the VHH are different from each other.
  • 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.
  • the H and/or L chains comprise constant regions, for example, human constant regions.
  • 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.
  • the H chain constant region of such antibodies comprises a gamma heavy chain constant region, for example, a human gamma heavy chain constant region.
  • such antibodies comprise IgG constant regions, for example, human IgG constant regions (e.g., IgG1, IgG2, IgG3, and/or IgG4 constant regions).
  • IgG constant regions for example, human IgG constant regions (e.g., IgG1, IgG2, IgG3, and/or IgG4 constant regions).
  • 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.
  • 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).
  • 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).
  • an ROR1 antibody can react with ROR1 sequences other than human ROR1 sequences (e.g., cynomolgus ROR1 sequences).
  • 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.”
  • 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.
  • variable 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 ⁇ sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ 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-1540479824 binding 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.
  • variable region is a human variable region.
  • HVR hypervariable region
  • HV complementarity determining region
  • CDR when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops.
  • 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).
  • VH H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3
  • VL three in the VL
  • 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.
  • IMGT ® ImMunoGeneTics
  • IMGT ® is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates.
  • CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain.
  • location of the CDRs within the structure of the immunoglobulin variable region is conserved between species and 45 NAI-1540479824 present 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.
  • 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.
  • the terms “hypervariable region,” “HVR,” “HV,” “complementarity determining region,” or “CDR” are used interchangeably.
  • isolated is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs.
  • ROR1-mediated disease 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.
  • ROR1-mediated disorder is 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.
  • 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.
  • cancer and cancerous refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • 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.
  • 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).
  • cytotoxic compound e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, and the like.
  • examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent.
  • 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.
  • 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.
  • 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)).
  • PBD pyrrolobenzodiazepine
  • 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-1540479824 cyclophosphamide (CYNOTANTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
  • alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates
  • 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.
  • CYTOSAR-U ® cytarabine
  • cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6- thio
  • Suitable natural products and their derivatives 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
  • anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • 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.
  • NSC 406042 Halichondrin B
  • Hormone modulators and steroids 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-1540479824 medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; and the like; adrenocortical suppressants, e.g., aminoglutethimide; 17 ⁇ -ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, amino
  • 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.
  • 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-(
  • 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.
  • 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).
  • paclitaxel-PEG paclitaxel-dextran
  • paclitaxel-xylose paclitaxel-xylose
  • 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-1540479824 taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No.5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No.5,821,263; and taxol derivative described in U.S. Patent No.5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No.5,824,701.
  • 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- ⁇ ; (7) IFN- ⁇ ; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.
  • RTK tyrosine kinase
  • 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.
  • 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 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).
  • 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 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.
  • 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.
  • the drug is a microtubule affecting agent that has anti- proliferative activity, such as a maytansinoid.
  • the drug is an 50 NAI-1540479824 antimitotic agent, such as an auristatin or an active auristatin analog or derivative thereof.
  • the drug is a DNA alkylating agent.
  • pharmaceutically acceptable 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.
  • 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.
  • 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 TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants such as ascorbic acid
  • 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.
  • 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 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-1540479824 formulations, and the like.
  • Oral compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • 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.
  • 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.
  • 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.
  • the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.
  • 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.
  • first antibody and “second antibody” are used to distinguish two antibodies.
  • 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).
  • 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.
  • ROR1-ADCs 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.
  • 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.
  • the one or more functional groups and covalent bonds can include a branched linker as described herein.
  • Moieties of interest can be conjugated to the ROR1 antibody at any desired site of the antibody.
  • 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-1540479824 antibody (e.g., at an internal site of the antibody). Combinations of the above conjugation sites are also possible.
  • 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 ⁇ -carbon of an amino acid residue.
  • 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).
  • a conjugate includes an ROR1 antibody where the ⁇ -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).
  • 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.
  • two moieties may be conjugated to the same amino acid residue of the ROR1 antibody.
  • 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.
  • 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.
  • 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.
  • 1 amino acid residue in the ROR1 antibody is conjugated to a pair of moieties through a branched linker as described herein.
  • 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.
  • 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.
  • 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-1540479824 antibody.
  • 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.
  • 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.
  • an ROR1 antibody may be conjugated to two or more moieties of interest.
  • the moiety of interest is a payload, for instance, a chemical entity, such as a drug, an active agent, or a detectable label.
  • drugs or active agents, such as cytokines
  • detectable labels may be conjugated to the ROR1 antibody.
  • combinations of different payloads may be conjugated to the ROR1 antibody.
  • 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.
  • the ROR1 antibody and the moieties of interest are conjugated through a conjugation moiety.
  • 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.
  • 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.
  • 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.
  • HIPS hydrazino-iso-Pictet-Spengler
  • azaHIPS aza- hydrazino-iso-Pictet-Spengler
  • 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.
  • a conjugation moiety e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety
  • R 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.
  • 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.
  • substituent such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino
  • Z may be CR 21 , NR 22 , N, O or S, where R 21 and R 22 are each independently selected from any of the substituents described for R’ and R” above.
  • R 21 and R 22 are each independently selected from any of the substituents described for R’ and R” above.
  • Other hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties are also possible, as shown in the conjugates and compounds described herein.
  • the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moieties may be attached (e.g., covalently attached) to two or more linkers.
  • 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.
  • 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.
  • 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.
  • Combinations of the same or different payloads may be conjugated to the ROR1 antibody through the branched linker.
  • the two payloads (e.g., drugs, active agents, or detectable labels) attached to the branched linker are the same payload (e.g., 57 NAI-1540479824 drug, active agent, or detectable label).
  • 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.
  • 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).
  • 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.
  • the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect.
  • “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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the payloads may be selected from combinations of drugs or active agents and detectable labels.
  • a first payload may be a detectable label that is used as an 58 NAI-1540479824 imaging agent or tracer to detect the location of the ADC in vivo
  • a second payload may be a drug or active agent that provides a therapeutic activity.
  • linkers that may couple the hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the drugs or active agents are described in detail herein.
  • the linker is a cleavable linker, such as a cleavable linker as described herein.
  • 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.
  • 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).
  • 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”).
  • FGE formylglycine generating enzyme
  • the fGly residue generated by an FGE may also be referred to as a “formylglycine.”
  • 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).
  • 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.
  • 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.
  • 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).
  • a compound e.g., a compound containing a hydrazinyl-indolyl or a hydrazinyl-pyrrolo- pyridinyl conjugation moiety, as described above.
  • 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.
  • the reactive partner may include a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety as described above.
  • two or more drugs or active agents may be attached to a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • 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.
  • fGly residue conjugated to the moieties of interest after the reaction is referred to herein as fGly’.
  • 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).
  • 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.
  • an amino acid residue of the ROR1 antibody is a cysteine or serine residue that is modified to an fGly residue, as described above.
  • 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.
  • 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).
  • 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.
  • 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.
  • aspects of the present disclosure include a conjugate of Formula (I): wherein: Ab represents the antibody that binds to ROR1; Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 4 , N and C-L B -W 2 , wherein at least one Z 1 , Z 2 , Z 3 and Z 4 is C-L B -W 2 ; R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitute
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 4 , N and C-L B -W 2 , wherein at least one Z 1 , Z 2 , Z 3 and Z 4 is C-L B -W 2 .
  • Z 1 is CR 4 .
  • Z 1 is N.
  • Z 1 is C- L B -W 2 .
  • Z 2 is CR 4 .
  • Z 2 is N.
  • Z 2 is C-L B -W 2 .
  • Z 3 is CR 4 . In certain embodiments, Z 3 is N. In certain embodiments, Z 3 is C-L B -W 2 . In certain embodiments, Z 4 is CR 4 . In certain embodiments, Z 4 is N. In certain embodiments, Z 4 is C-L B -W 2 . In some embodiments, each of Z 1 , Z 3 , and Z 4 is CR 4 . In some embodiments, Z 3 is C-L B -W 2 . [00259] Combinations of various Z 1 , Z 2 , Z 3 and Z 4 are possible.
  • Z 1 is C-L B -W 2
  • Z 2 is CR 4
  • Z 3 is CR 4
  • Z 4 is CR 4
  • Z 1 is CR 4
  • Z 2 is C-L B -W 2
  • Z 3 is CR 4
  • Z 4 is CR 4
  • Z 1 is CR 4
  • Z 2 is CR 4
  • Z 3 is C-L B - W 2
  • Z 4 is CR 4
  • Z 1 is CR 4
  • Z 2 is CR 4
  • Z 3 is C-L B - W 2
  • Z 4 is CR 4
  • Z 1 is CR 4
  • Z 2 is CR 4
  • Z 3 is CR 4
  • Z 4 is C-L B -W 2 .
  • R 1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl.
  • R 1 is hydrogen.
  • R 1 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl.
  • R 1 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl.
  • R 1 is alkynyl or substituted alkynyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl.
  • R 1 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C6 aryl or C6 substituted aryl.
  • R 1 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 1 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3- 8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 1 is heterocyclyl or substituted heterocyclyl, such as C 3-8 heterocyclyl or C 3-8 substituted heterocyclyl, such as a 62 NAI-1540479824 C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.
  • R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl.
  • R 2 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 2 is hydrogen. In certain embodiments, R 2 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 2 is methyl. In certain embodiments, R 2 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl. In certain embodiments, R 2 is alkynyl or substituted alkynyl.
  • R 2 is alkoxy or substituted alkoxy. In certain embodiments, R 2 is amino or substituted amino. In certain embodiments, R 2 is carboxyl or carboxyl ester. In certain embodiments, R 2 is acyl or acyloxy. In certain embodiments, R 2 is acyl amino or amino acyl. In certain embodiments, R 2 is alkylamide or substituted alkylamide. In certain embodiments, R 2 is sulfonyl. In certain embodiments, R 2 is thioalkoxy or substituted thioalkoxy.
  • R 2 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl.
  • R 2 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 2 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 2 is heterocyclyl or substituted heterocyclyl, such as a C 3-6 heterocyclyl or C 3- 6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl.
  • R 3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 3 is hydrogen. In certain embodiments, R 3 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C1-4 alkyl or C1-4 substituted alkyl, or C1-3 alkyl or C1-3 substituted alkyl. In certain embodiments, R 3 is methyl. In certain embodiments, R 3 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C 2-4 alkenyl or C 2-4 substituted alkenyl, or C 2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R 3 is alkynyl or substituted alkynyl.
  • R 3 is alkoxy or substituted alkoxy. In certain embodiments, R 3 is amino or substituted amino. In certain embodiments, R 3 is carboxyl or carboxyl ester. In certain embodiments, R 3 is acyl or acyloxy. In certain embodiments, R 3 is acyl amino or amino acyl. In certain embodiments, R 3 is alkylamide or substituted alkylamide. In certain embodiments, R 3 is sulfonyl. In certain embodiments, R 3 is thioalkoxy or substituted thioalkoxy.
  • R 3 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl.
  • R 3 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C6 substituted heteroaryl.
  • R 3 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 3 is 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 C 3-5 substituted heterocyclyl.
  • both R 2 and R 3 are methyl.
  • R 2 and R 3 are optionally cyclically linked to form a 5 or 6- membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R 2 and R 3 are cyclically linked to form a 6-membered heterocyclyl.
  • each R 4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, 64 NAI-1540479824 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.
  • R 4 is hydrogen. In certain embodiments, each R 4 is hydrogen. In certain embodiments, R 4 is halogen, such as F, Cl, Br, or I. In certain embodiments, R 4 is F. In certain embodiments, R 4 is Cl. In certain embodiments, R 4 is Br. In certain embodiments, R 4 is I. In certain embodiments, R 4 is alkyl or substituted alkyl, such as C 1-6 alkyl or C 1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 4 is methyl.
  • R 4 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C 2-3 alkenyl or C 2-3 substituted alkenyl.
  • R 4 is alkynyl or substituted alkynyl.
  • R 4 is alkoxy or substituted alkoxy.
  • R 4 is amino or substituted amino.
  • R 4 is carboxyl or carboxyl ester.
  • R 4 is acyl or acyloxy.
  • R 4 is acyl amino or amino acyl.
  • R 4 is alkylamide or substituted alkylamide. In certain embodiments, R 4 is sulfonyl. In certain embodiments, R 4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R 4 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C 6 aryl or C 6 substituted aryl (e.g., phenyl or substituted phenyl).
  • R 4 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C 6 substituted heteroaryl.
  • R 4 is 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 C 3-5 substituted cycloalkyl.
  • R 4 is 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.
  • L A is a first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.
  • L B is a second linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below.
  • W 1 is 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.
  • W 2 is 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.
  • Ab represents an antibody that binds to ROR1 (“ROR1 antibody”).
  • the antibody Ab comprises one or more fGly’ residues as described herein.
  • the ROR1 antibody is attached to the rest of the conjugate through an fGly’ residue as described herein.
  • the conjugate of Formula (I) includes a first linker, L A .
  • the first linker, L A 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, L A may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein).
  • the first linker, L A 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, L A , (and thus the first drug) to an ROR1 antibody.
  • L A is attached to the antibody Ab through a conjugation moiety, and thus the antibody Ab is indirectly bonded to the linker L A through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • the antibody Ab is an ROR1 antibody, and thus L A is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the ROR1 antibody, e.g., the linker L A is indirectly bonded to the ROR1 antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • Any convenient linker may be utilized for the first linker L A in the subject conjugates and compounds.
  • the first linker L A may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • the first linker L A may include an alkyl or substituted alkyl group.
  • the first linker L A may include an alkenyl or substituted alkenyl group. 66 NAI-1540479824 In certain embodiments, the first linker L A may include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker L A may include an alkoxy or substituted alkoxy group. In certain embodiments, the first linker L A may include an amino or substituted amino group. In certain embodiments, the first linker L A may include a carboxyl or carboxyl ester group. In certain embodiments, the first linker L A may include an acyl amino group. In certain embodiments, the first linker L A may include an alkylamide or substituted alkylamide group.
  • the first linker L A may include an aryl or substituted aryl group. In certain embodiments, the first linker L A may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker L A may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker L A may include a heterocyclyl or substituted heterocyclyl group. [00276] In certain embodiments, the first linker L A may include a polymer.
  • 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.
  • the polymer is a polyalkylene glycol.
  • the polymer is a polyethylene glycol.
  • L A is a first linker described by the formula: wherein L 1 , L 2 , L 3 , L 4 , L 5 and L 6 are 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.
  • 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.
  • 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.
  • 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.
  • the linker subunit L 1 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • the linker subunit L 2 if present, is attached to the first drug or active agent W 1 .
  • the linker subunit L 3 if present, is attached to the first drug or active agent W 1 .
  • the linker subunit L 4 if present, is attached to the first drug or active agent W 1 .
  • linker subunit L 5 if present, is attached to the first drug or active agent W 1 .
  • linker subunit L 6 if present, is attached to the first drug or active agent W 1 .
  • Any convenient linker subunits may be utilized in the first linker L A .
  • 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.
  • each of L 1 , L 2 , L 3 , L 4 , L 5 and L 6 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).
  • L 1 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.
  • L 1 comprises a polyethylene glycol. In some embodiments, L 1 comprises a modified polyethylene glycol. In some embodiments, L 1 comprises an amino acid residue. In some embodiments, L 1 comprises an alkyl group or a substituted alkyl. In some embodiments, L 1 comprises an aryl group or a substituted aryl group. In some embodiments, L 1 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00282] In some embodiments, L 2 (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.
  • L 2 comprises a polyethylene glycol. In some embodiments, L 2 comprises a modified polyethylene glycol. In some embodiments, L 2 comprises an amino acid residue. In some embodiments, L 2 comprises an alkyl group or a substituted alkyl. In some embodiments, L 2 comprises an aryl group or a substituted aryl group. In some embodiments, L 2 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • a diamine e.g., a linking group comprising an alkylene diamine
  • L 3 (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, L 3 comprises a polyethylene glycol. In some embodiments, L 3 comprises a modified polyethylene glycol. In some embodiments, L 3 comprises an amino acid residue. In some embodiments, L 3 comprises an alkyl group or a substituted alkyl. In some embodiments, L 3 comprises an aryl group or a substituted aryl group.
  • L 3 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 4 (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.
  • L 4 comprises a polyethylene glycol.
  • L 4 comprises a modified polyethylene glycol.
  • L 4 comprises an amino acid residue.
  • L 4 comprises an alkyl group or a substituted alkyl.
  • L 4 comprises an aryl group or a substituted aryl group.
  • L 4 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 5 (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.
  • L 5 comprises a polyethylene glycol.
  • L 5 comprises a modified polyethylene glycol.
  • L 5 comprises an amino acid residue.
  • L 5 comprises an alkyl group or a substituted alkyl.
  • L 5 comprises an aryl group or a substituted aryl group.
  • L 5 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 6 (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.
  • L 6 comprises a polyethylene glycol.
  • L 6 comprises a modified polyethylene glycol.
  • L 6 comprises an amino acid residue.
  • L 6 comprises an alkyl group or a substituted alkyl.
  • L 6 comprises an aryl group or a substituted aryl group.
  • L 6 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L A is a first linker comprising: -(L 1 )a-(L 2 )b-(L 3 )c-(L 4 )d-(L 5 )e-(L 6 )f-, 69 NAI-1540479824 wherein: -(L 4 )d- is -(T 4 -V 4 )d-; -(L 5 ) e - is -(T 5 -V 5 ) e -; and -(L 6 ) f - is -(T 6 -V 6 ) f -, wherein: T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , if present, are tether groups; V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 , if present, are tether groups; V 1 ,
  • 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.
  • the sum of a, b, c, d, e, and f is 6.
  • a, b, c, d, e, and f are each 1.
  • a, b, c, d, and e are each 1 and f is 0.
  • a, b, c, and d are each 1 and e and f are each 0.
  • a, b, and c are each 1 and d, e and f are each 0.
  • a and b are each 1 and c, d, e, and f are each 0.
  • a is 1 and b, c, d, e, and f are each 0.
  • L 1 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • T 1 is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • V 1 is attached to the first drug or active agent.
  • L 2 if present, is attached to the first drug or active agent.
  • T 2 if present, is attached to the first drug or active agent, or V 2 , if present, is attached to the first drug or active agent.
  • L 3 if present, is attached to the first drug or active agent.
  • T 3 if present, is attached to the first drug or active agent, or V 3 , if present, is attached to the first drug or active agent.
  • L 4 if present, is attached to the first drug or active agent.
  • T 4 is attached to the first drug or active agent, or V 4 , if present, is attached to the first drug or active agent.
  • L 5 is attached to the first drug or active agent.
  • T 5 is attached to the first drug or active agent, or V 5 , if present, is attached to the first drug or active agent.
  • L 6 is attached to the first drug or active agent.
  • T 6 is attached to the first drug or active agent, or V 6 , if present, is attached to the first drug or active agent.
  • the conjugate of Formula (I) includes a second linker, L B .
  • the second linker, L B 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, L B may be bound (e.g., covalently bonded) to the conjugation moiety (e.g., as described herein).
  • the second linker, L B 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, L B , (and thus the second drug) to an ROR1 antibody.
  • L B is attached to the antibody Ab through a conjugation moiety, and thus the antibody Ab is indirectly bonded to the second linker L B through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • the antibody Ab is an ROR1 antibody, and thus L B is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the ROR1 antibody, e.g., the linker L B is indirectly bonded to the ROR1 antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety.
  • Any convenient linker may be utilized for the second linker L B in the subject conjugates and compounds.
  • the second linker L B may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • the second linker L B may include an alkyl or substituted alkyl group.
  • the second linker L B may include an alkenyl or substituted alkenyl group. In certain embodiments, the second linker L B may include an alkynyl or substituted alkynyl group. In certain embodiments, the second linker L B may include an alkoxy or substituted alkoxy group. In certain embodiments, the second linker L B may include an amino or substituted amino group. In certain embodiments, the second linker L B may include a 71 NAI-1540479824 carboxyl or carboxyl ester group. In certain embodiments, the second linker L B may include an acyl amino group. In certain embodiments, the second linker L B may include an alkylamide or substituted alkylamide group.
  • the second linker L B may include an aryl or substituted aryl group. In certain embodiments, the second linker L B may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker L B may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker L B may include a heterocyclyl or substituted heterocyclyl group. [00293] In certain embodiments, the second linker L B may include a polymer.
  • 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.
  • the polymer is a polyalkylene glycol.
  • the polymer is a polyethylene glycol.
  • L B is a second linker described by the formula: wherein L 7 , L 8 , L 9 , L 10 , L 11 , L 12 and L 13 are 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.
  • the sum of g, h, i, j, k, l, and m is 1 to 7.
  • 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.
  • 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, h, and i are each 1 and j, k, l, and m are each 0.
  • 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. [00296] In certain embodiments, the linker subunit L 7 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • the linker subunit L 8 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 9 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 10 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 11 if present, is attached to the second drug or active agent W 2 .
  • the linker subunit L 12 if present, is attached to the second drug or active agent W 2 .
  • linker subunit L 13 if present, is attached to the second drug or active agent W 2 .
  • Any convenient linker subunits may be utilized in the second linker L B .
  • 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.
  • each of L 7 , L 8 , L 9 , L 10 , L 11 , L 12 and L 13 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).
  • L 7 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.
  • L 7 comprises a polyethylene glycol. In some embodiments, L 7 comprises a modified polyethylene glycol. In some embodiments, L 7 comprises an amino acid residue. In some embodiments, L 7 comprises an alkyl group or a substituted alkyl. In some embodiments, L 7 comprises an aryl group or a substituted aryl group. In some embodiments, L 7 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00299] In some embodiments, L 8 (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.
  • L 8 comprises a polyethylene glycol. In some embodiments, L 8 comprises a modified polyethylene glycol. In some embodiments, L 8 comprises an amino acid residue. In some embodiments, L 8 comprises an alkyl group or a substituted alkyl. In some embodiments, L 8 comprises an aryl group or a substituted aryl group. In some embodiments, L 8 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 9 (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, L 9 comprises a polyethylene glycol. In some embodiments, L 9 comprises a modified polyethylene glycol. In some embodiments, L 9 comprises an amino acid residue. In some embodiments, L 9 comprises an alkyl group or a substituted alkyl. In some embodiments, L 9 comprises an aryl group or a substituted aryl group.
  • L 9 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 10 (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.
  • L 10 comprises a polyethylene glycol.
  • L 10 comprises a modified polyethylene glycol.
  • L 10 comprises an amino acid residue.
  • L 10 comprises an alkyl group or a substituted alkyl.
  • L 10 comprises an aryl group or a substituted aryl group.
  • L 10 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 11 (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.
  • L 11 comprises a polyethylene glycol.
  • L 11 comprises a modified polyethylene glycol.
  • L 11 comprises an amino acid residue.
  • L 11 comprises an alkyl group or a substituted alkyl.
  • L 11 comprises an aryl group or a substituted aryl group.
  • L 11 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 12 (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.
  • L 12 comprises a polyethylene glycol.
  • L 12 comprises a modified polyethylene glycol.
  • L 12 comprises an amino acid residue.
  • L 12 comprises an alkyl group or a substituted alkyl.
  • L 12 comprises an aryl group or a substituted aryl group.
  • L 12 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L 13 (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-1540479824 a substituted aryl group, or a diamine.
  • L 13 comprises a polyethylene glycol.
  • L 13 comprises a modified polyethylene glycol.
  • L 13 comprises an amino acid residue.
  • L 13 comprises an alkyl group or a substituted alkyl.
  • L 13 comprises an aryl group or a substituted aryl group.
  • L 13 comprises a diamine (e.g., a linking group comprising an alkylene diamine).
  • L B is a second linker comprising: wherein: -(L 7 ) g - is -(T 7 -V 7 ) g -; -(L 8 )h- is -(T 8 -V 8 )h-; -(L 9 )i- is -(T 9 -V 9 )i-; -(L 10 ) j - is -(T 10 -V 10 ) j -; wherein: T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 , if present, are tether groups; V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 , if present, are tether groups; V 7 , V 8
  • 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.
  • 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.
  • 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.
  • L 7 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • T 7 is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above).
  • V 7 is attached to the second drug or active agent.
  • L 8 if present, is attached to the second drug or active agent.
  • T 8 is attached to the second drug or active agent, or V 8 , if present, is attached to the second drug or active agent.
  • L 9 is attached to the second drug or active agent.
  • T 9 is attached to the second drug or active agent, or V 9 , if present, is attached to the second drug or active agent.
  • L 10 is attached to the second drug or active agent.
  • T 10 is attached to the second drug or active agent, or V 10 , if present, is attached to the second drug or active agent.
  • L 11 if present, is attached to the second drug or active agent.
  • T 11 if present, is attached to the second drug or active agent, or V 11 , if present, is attached to the second drug or active agent.
  • L 12 if present, is attached to the second drug or active agent.
  • T 12 if present, is attached to the second drug or active agent, or V 12 , if present, is attached to the second drug or active agent.
  • L 13 if present, is attached to the second drug or active agent.
  • T 13 is attached to the second drug or active agent
  • V 13 if present, is attached to the second drug or active agent.
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 any convenient tether groups may be utilized in the subject linkers.
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 each 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, -(CR 13 OH)x-, 4-amino- piperidine (4AP), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para- aminobenzyl
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes a (C1-C12)alkyl or a substituted (C1-C12)alkyl.
  • (C 1 -C 12 )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.
  • (C 1 -C 12 )alkyl may be an alkyl or substituted alkyl, such as C 1 -C 12 alkyl, or C 1 -C 10 alkyl, or C 1 -C 6 alkyl, or C 1 -C 3 alkyl.
  • (C 1 -C 12 )alkyl is a C 2 -alkyl.
  • (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C1-C12 alkylene, or C 1 -C 10 alkylene, or C 1 -C 6 alkylene, or C 1 -C 3 alkylene.
  • (C 1 -C 12 )alkyl is a C 1 -alkylene (e.g., CH 2 ).
  • (C 1 -C 12 )alkyl is a C 2 -alkylene (e.g., CH 2 CH 2 ).
  • (C1-C12)alkyl is a C3-alkylene (e.g., CH2CH2CH2).
  • 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.
  • substituted (C1-C12)alkyl may be a substituted alkyl, such as substituted C 1 -C 12 alkyl, or substituted C 1 -C 10 alkyl, or substituted C 1 -C 6 alkyl, or substituted C 1 -C 3 alkyl.
  • substituted (C 1 -C 12 )alkyl is a substituted C2-alkyl.
  • substituted (C1-C12)alkyl may be a substituted alkylene, such as substituted C 1 -C 12 alkylene, or substituted C 1 -C 10 alkylene, or substituted C 1 -C 6 alkylene, or substituted C 1 -C 3 alkylene.
  • substituted (C 1 -C 12 )alkyl is a substituted C1-alkylene (e.g., C1-alkylene substituted with -SO3H). In some instances, substituted (C 1 -C 12 )alkyl is a substituted C 2 -alkylene. In some instances, substituted (C 1 - C 12 )alkyl is a substituted C 3 -alkylene.
  • substituted (C 1 -C 12 )alkyl may include C 1 - 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 C 1 -C 12 alkylene (e.g., C 3 -alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12 alkylene (e.g., C5-alkylene) substituted with a -NHCOCH2SO3H group.
  • substituted (C 1 -C 12 )alkyl may include C 1 -C 12 alkylene (e.g., C 3 -alkylene or C 5 -alkylene) substituted with a (PEG) t group as described herein (e.g., -NHCO(PEG)t, wherein indicates the point of attachment 77 NAI-1540479824 to carbonyl group of -NHCO-, and t is an integer), such as -NHCO(CH2CH2O)3CH3 or -NHCO(CH2CH2O)5CH3 or -NHCO(CH2CH2O)8CH3.
  • a (PEG) t group as described herein (e.g., -NHCO(PEG)t, wherein indicates the point of attachment 77 NAI-1540479824 to carbonyl group of -NHCO-, and t is an integer), such as -NHCO(CH2CH2O)3CH3 or -NHCO(CH2CH2O)
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes an aryl or substituted aryl.
  • the aryl can be phenyl.
  • the substituted aryl is a substituted phenyl.
  • the substituted phenyl can be substituted with one or more substituents selected from (C 1 -C 12 )alkyl, a substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • 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).
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes a heteroaryl or substituted heteroaryl, such triazolyl (e.g., 1,2,3- triazolyl).
  • triazolyl e.g., 1,2,3- triazolyl
  • the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes a cycloalkyl or substituted cycloalkyl. In some instances, the tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes a heterocyclyl or substituted heterocyclyl.
  • 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).
  • the tether group e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13
  • EDA ethylene diamine
  • (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.
  • the EDA moiety is described by the structure: , 78 NAI-1540479824 where y is an integer from 1 to 6, r is 0 or 1, and each R 12 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl. In certain embodiments, any two adjacent R 12 groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R 12 groups are an alkyl group, cyclically linked to form a piperazinyl ring.
  • y is 1 and the adjacent R 12 groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).
  • the tether group e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13
  • the tether group includes a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidin-4-amino, P4A).
  • the 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituents, e.g., with an alkyl, a substituted alkyl, a polyethylene glycol moiety, an acyl, a substituted acyl, an aryl, or a substituted aryl.
  • the 4AP moiety is described by the structure: where R 12 is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., a polyethylene glycol or a modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 12 is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., a polyethylene glycol or
  • R 12 is a polyethylene glycol moiety. In certain embodiments, R 12 is a carboxy modified polyethylene glycol. [00316] In certain embodiments, R 12 includes 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.
  • PEG 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
  • R 17 is 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.
  • R 17 is COOH.
  • R 17 is OH.
  • R 17 is OCH 3 .
  • (PEG)k is (PEG)t having the following structure: , wherein t is an integer from 2 to 10.
  • t is 8.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes (PEG)n, where (PEG)n is a polyethylene glycol or a modified polyethylene glycol linking unit.
  • (PEG) n is 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.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) 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.
  • p is an integer from 1 to 50, such as from 1 to 40, from 1 to 30, 80 NAI-1540479824 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.
  • p is 1.
  • p is 2.
  • (AA) p comprises a dipeptide of valine-alanine.
  • a tether group e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) 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.
  • 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.
  • 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.
  • amino acid analogs may include ⁇ -hydroxy acids, and ⁇ - amino acids, and the like. Examples of amino acid analogs include, but are not limited to, sulfoalanine, and the like.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) includes a moiety described by the formula -(CR 13 OH) 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.
  • R 13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 13 is hydrogen.
  • R 13 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl.
  • R 13 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 81 NAI-1540479824 substituted alkenyl.
  • R 13 is alkynyl or substituted alkynyl.
  • R 13 is alkoxy or substituted alkoxy. In certain embodiments, R 13 is amino or substituted amino. In certain embodiments, R 13 is carboxyl or carboxyl ester. In certain embodiments, R 13 is acyl or acyloxy. In certain embodiments, R 13 is acyl amino or amino acyl. In certain embodiments, R 13 is alkylamide or substituted alkylamide. In certain embodiments, R 13 is sulfonyl. In certain embodiments, R 13 is thioalkoxy or substituted thioalkoxy.
  • R 13 is aryl or substituted aryl, such as C 5-8 aryl or C 5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl.
  • R 13 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5- 8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C6 substituted heteroaryl.
  • R 13 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 13 is 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 C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
  • R 13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .
  • the tether group e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13
  • the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group.
  • a tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.
  • a tether group (e.g., T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 ) 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).
  • MABO meta-amino-benzyloxy
  • MABC
  • a tether group includes a MABO group described by the following structure: 82 NAI-1540479824 .
  • a tether group includes a MABC group described by the following structure: .
  • a tether group includes a PABO group described by the following structure: .
  • a tether group includes a PABC group described by the following structure: .
  • a tether group includes a PAB group described by the following structure: .
  • a tether group includes a PABA group described by the following structure: [00332] In some embodiments, a tether group includes a PAP group described by the following structure: 83 NAI-1540479824 . [00333] In some embodiments, a tether group includes a PHP group described by the following structure: .
  • each R 14 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 14 is hydrogen. In certain embodiments, each R 14 is hydrogen. In certain embodiments, R 14 is alkyl or substituted alkyl, such as C1-6 alkyl or C1-6 substituted alkyl, or C 1-4 alkyl or C 1-4 substituted alkyl, or C 1-3 alkyl or C 1-3 substituted alkyl. In certain embodiments, R 14 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R 14 is alkynyl or substituted alkynyl.
  • R 14 is alkoxy or substituted alkoxy. In certain embodiments, R 14 is amino or substituted amino. In certain embodiments, R 14 is carboxyl or carboxyl ester. In certain embodiments, R 14 is acyl or acyloxy. In certain embodiments, R 14 is acyl amino or amino acyl. In certain embodiments, R 14 is alkylamide or substituted alkylamide. In certain embodiments, R 14 is sulfonyl. In certain embodiments, R 14 is thioalkoxy or substituted thioalkoxy.
  • R 14 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C 5 aryl or C 5 substituted aryl, or a C 6 aryl or C 6 substituted aryl.
  • R 14 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5- 8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C 6 substituted heteroaryl.
  • R 14 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl.
  • R 14 is heterocyclyl or substituted heterocyclyl, such as C 3-8 heterocyclyl 84 NAI-1540479824 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.
  • 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.
  • one or more of the tether groups T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and/or T 13 is each optionally substituted with a glycoside or glycoside derivative.
  • T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside.
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each optionally substituted with a glycoside.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • 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.
  • the phenyl ring may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • the PABC is substituted with a glycoside
  • 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.
  • the glycoside or glycoside derivative is selected from the following structures: 85 NAI-1540479824 [00340] Regarding the linking functional groups, V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 any convenient linking functional groups may be utilized in the subject linkers.
  • Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like.
  • V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 are each independently selected from a covalent bond, -CO-, -NR 15 -, -NR 15 (CH2)q-, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO 2 -, -SO 2 NR 15 -, -NR 15 SO 2 - and -P(O)OH-, where q is an integer from 1 to 6.
  • 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.
  • each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • R 15 is hydrogen. In certain embodiments, each R 15 is hydrogen. In certain embodiments, R 15 is 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, R 15 is alkenyl or substituted alkenyl, such as C 2-6 alkenyl or C 2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R 15 is alkynyl or substituted alkynyl.
  • R 15 is alkoxy or substituted alkoxy. In certain embodiments, R 15 is amino or substituted amino. In certain embodiments, R 15 is carboxyl or carboxyl ester. In certain 86 NAI-1540479824 embodiments, R 15 is acyl or acyloxy. In certain embodiments, R 15 is acyl amino or amino acyl. In certain embodiments, R 15 is alkylamide or substituted alkylamide. In certain embodiments, R 15 is sulfonyl. In certain embodiments, R 15 is thioalkoxy or substituted thioalkoxy.
  • R 15 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl.
  • R 15 is heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5- 8 substituted heteroaryl, such as a C 5 heteroaryl or C 5 substituted heteroaryl, or a C 6 heteroaryl or C6 substituted heteroaryl.
  • R 15 is cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 substituted cycloalkyl, such as a C 3-6 cycloalkyl or C 3-6 substituted cycloalkyl, or a C 3-5 cycloalkyl or C 3-5 substituted cycloalkyl.
  • R 15 is 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 C 3-5 heterocyclyl or C 3-5 substituted heterocyclyl.
  • each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • 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 R 15 .
  • L A is a first linker comprising -(T 1 -V 1 ) a - (T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -(T 6 -V 6 ) 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.
  • T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl
  • T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine, and an ester; and V 1 , V
  • L A comprises: -(T 1 -V 1 )a-(T 2 -V 2 )b-(T 3 -V 3 )c-(T 4 -V 4 )d-(T 5 -V 5 )e-(T 6 -V 6 )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; T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from a covalent bond, (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w
  • T 1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl
  • T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from a covalent bond, (C 1 - C 12 )alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG) n , (AA) p , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V 1 , V 2 , V 3 , V
  • T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 are selected from the following: wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CO-; T 2 is (AA)p and V 2 is absent (e.g., a covalent bond); T 3 is PABC and V 3 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and d, e, and f are each 0; or wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-; T 2 is (PEG) n and V 2 is -CO-; T 3 is (AA)p and V 3 is absent (e.g., a covalent bond); T 4 is PABC and V 4 is absent (e.g., a covalent bond);
  • the left-hand side of the above linker structure for the first linker L A is 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 L A is attached to the first drug or active agent.
  • L B is a second linker comprising -(T 7 - V 7 )g-(T 8 -V 8 )h-(T 9 -V 9 )i-(T 10 -V 10 )j-(T 11 -V 11 )k-(T 12 -V 12 )l-(T 13 -V 13 )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.
  • T 7 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl
  • T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each independently selected from (C1-C12)alkyl, substituted (C 1 -C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR 13 OH)x-, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a disulfide, a hydrazine, and an ester; and V 7 , V 8 , V 9
  • Any convenient tether groups may be utilized for T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 .
  • any of the tether groups described above in relation to T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 may be used for the tether groups T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 .
  • Any convenient linking functional groups may be utilized for V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 .
  • each R 13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.
  • alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R 13 .
  • each R 15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • 94 NAI-1540479824 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 R 15 .
  • various possible substituents are as described above for R 15 .
  • one or more of the tether groups T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 is each optionally substituted with a glycoside or glycoside derivative.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • 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.
  • the phenyl ring may be substituted with one or more additional groups selected from a glycoside and a glycoside derivative.
  • the glycoside or glycoside derivative is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • L B In some embodiments of L B : g, h, i, j, and k are each 1; l and m are each 0; T 7 is a covalent bond; T 8 , T 9 , T 10 , T 11 , and T 12 are 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 , -(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, an acetal group, a hydrazine, and an ester; and V 7 , V 8 , V 9
  • T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 are each optionally substituted with a glycoside.
  • MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside.
  • the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc.
  • T 7 , T 8 , T 9 , T 10 , T 11 , T 12 , and T 13 and V 7 , V 8 , V 9 , V 10 , V 11 , V 12 , and V 13 are selected from the following: wherein: T 7 is absent (e.g., a covalent bond) and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CO-; T 9 is (AA) p and V 9 is absent (e.g., a covalent bond); T 10 is PABC and V 10 is absent (e.g., a covalent bond); and k, l, and m are each 0; or wherein: T 7 is absent (e.g., a covalent bond) and V 7 is -NHCO-; T 8 is (C1-C12)alkyl and V 8 is -CONH-; T 9 is (PEG)n and V 9 is -CO-;
  • the left-hand side of the above linker structure for the second linker L B is 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 L B is attached to the second drug or active agent.
  • the conjugate is an antibody-drug conjugate where the ROR1 antibody and the drugs are linked together by linkers as described above.
  • the linker m(e.g., L A and/or L B ) 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a 100 NAI-1540479824 cleavable linker includes a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety.
  • 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.
  • 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 substantially reduces the amount or prevents the release of the drug from the antibody.
  • 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.
  • 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.
  • substantially 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.
  • the second cleavable moiety can protect the first cleavable moiety from cleavage.
  • 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.
  • 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.
  • cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of 101 NAI-1540479824 the 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).
  • the cleavable moieties included in the cleavable linker may each be an enzymatically cleavable moiety.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • the enzyme can be an esterase.
  • the enzymatically cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase enzyme.
  • the enzyme can be a glycosidase.
  • the enzymatically cleavable moiety is a cleavable moiety (e.g., a glycoside or glycoside derivative) that can be cleaved by a glycosidase enzyme.
  • the enzymatically cleavable moiety is an ester bond.
  • the first cleavable moiety described above 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.
  • 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.
  • the second cleavable moiety includes a glycoside or glycoside derivative.
  • the enzymatically cleavable moiety is sugar moiety, such as a glycoside (or glyosyl) or glycoside derivative.
  • 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.
  • 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.
  • the first cleavable moiety includes an ester
  • the second cleavable moiety includes a glycoside or glycoside derivative.
  • 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.
  • 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. [00378]
  • 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).
  • the glycosidic bond is an O-glycosidic bond (an O-glycoside).
  • the glycoside or glycoside derivative can be cleaved from the cleavable linker it is 103 NAI-1540479824 attached to by an enzyme (e.g., through enzymatically mediated hydrolysis of the glycosidic bond).
  • a glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme that is able to carry out the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker.
  • an 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an ROR1-ADC is represented by Formula (I): wherein: Ab represents the antibody that binds to ROR1; Z 1 , Z 2 , and Z 4 are each independently CR 4 ; Z 3 is C-L B -W 2 ; R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (C1-C12)alkyl; L A is a first linker wherein: T 1 is (C 1 -C 12 )alkyl and V 1 is -CONH-; T 2 is substituted (C1-C12)alkyl and V 2 is -CO-; T 3 is (AA)p where p is an integer from 1-20 and V 3 is a covalent bond; T 4 is PABC and V 4 is a covalent bond; 104 NAI-1540479824 a, b, c, and d are each 1; e and f are each 0; and L B is a second linker wherein: T 7 is the antibody that binds to ROR1
  • W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • an ROR1-ADC is represented by Formula (I): wherein: Ab represents the antibody that binds to ROR1; Z 1 , Z 2 , and Z 4 are each independently CR 4 ; Z 3 is C-L B -W 2 ; R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (C1-C12)alkyl; L A is a linker wherein: T 1 is (C 1 -C 6 )alkyl and V 1 is -CONH-; T 2 is (C 1 -C 6 )alkylene substituted with -NHCO(PEG) k , wherein k is an integer from 2 to 10, and V 2 is -CO-; T 3 is (AA)2 and V 3 is a covalent bond; T 4 is PABC substituted with a glycoside and
  • an ROR1-ADC is represented by Formula (I): wherein: Ab represents the antibody that binds to ROR1; Z 1 , Z 2 , and Z 4 are each independently CR 4 ; Z 3 is C-L B -W 2 ; R 1 , R 2 , R 3 and R 4 are each selected from hydrogen and (C 1 -C 12 )alkyl; L A is a linker wherein: T 1 is (C1-C6)alkyl and V 1 is -CONH-; T 2 is (C 1 -C 6 )alkylene substituted with -NHCO(PEG) t , wherein (PEG) t is integer from 2 to 10, optionally 8, and V 2 is -CO-; T 3 is (AA)2 and V 3 is a covalent bond; 106 NAI-1540479824 T 4 is PABC substituted with a glycoside and V 4 is a covalent bond; a, b, c, and d are each
  • the PABC of one or both of T 4 and T 11 is substituted with a glucuronide.
  • one or both of T 1 and T 8 is ethyl.
  • one or both of T 2 and T 9 is C5 alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 5 to 10.
  • one or both of W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • the PABC of one or both of T 4 and T 11 is substituted with a glucuronide.
  • one or both of T 1 and T 8 is ethyl.
  • T 2 and T 9 is C 5 alkylene substituted with -NHCO(PEG) t , wherein (PEG) t is integer from 5 to 10.
  • W 1 and W 2 are camptothecin analogues, for example, belotecan.
  • s is an integer from 1 to 4. In further embodiments, s is 4.
  • an ROR1-ADC is represented by Formula (II): 107 NAI-1540479824
  • Ab represents the antibody that binds to ROR1; and s is an integer from 1 to 10. [00387] In some embodiments, s is an integer from 1 to 4. [00388] 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. [00389] 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.
  • 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.
  • amino acids may be referred to by their standard name, their standard three letter abbreviation and/or their standard one letter 108 NAI-1540479824 abbreviation, 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.
  • Alanine or Ala or A Cysteine or Cys or C
  • 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.
  • described herein are ROR1 antibodies that bind to ROR1, including an ROR1 polypeptide, an ROR1 polypeptide fragment, an ROR1 peptide or an ROR1 epitope.
  • 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.
  • 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.
  • 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.
  • an ROR1 antibody binds an ROR1 extracellular epitope exposed on a cell such as a cancer cell.
  • described herein is an ROR1 antibody that binds to ROR1, such as human ROR1 or a portion thereof.
  • ROR1 is a human ROR1.
  • an ROR1 antibody is a human ROR1 antibody (e.g., an antibody that binds to human ROR1).
  • ROR1 antibodies bind to both human and cyno ROR1. In other embodiments, ROR1 antibodies bind to human ROR1 but not to cyno ROR1.
  • 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 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10 -8 M or less, e.g., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M).
  • KD dissociation constant
  • 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-1540479824 antibody 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.
  • RIA for example, performed with the Fab version of an 109 NAI-1540479824 antibody 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 ®
  • 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.
  • the ROR1 antibody provided herein does not bind to ROR2 (e.g., human ROR2, cyno ROR2, mouse ROR2, and/or rat ROR2).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 110 NAI-1540479824 an ROR1 antibody as used herein is disclosed in US Patent Application Publication No. US20210155692A1, which is incorporated by reference in its entirety.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CDRs are according to exemplary numbering.
  • the CDRs are according to IMGT numbering.
  • the CDRs are according to Kabat numbering.
  • the CDRs are according to AbM numbering.
  • 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.
  • 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-1540479824 13, 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.
  • 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.
  • the ROR1 binding agent provided herein 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.
  • 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.
  • 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.
  • 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-1540479824 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.
  • 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.
  • 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.
  • the antibody further comprises one or more framework regions of SEQ ID NOs: 25 and/or 26.
  • 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.
  • the antibody provided herein is a humanized antibody. Framework regions described herein are determined based upon the boundaries of the CDR numbering system.
  • 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.
  • 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
  • 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.
  • ROR1 antibodies e.g., antibodies such as monospecific or bispecific antibodies
  • human ROR1 antibodies described herein comprise a VH region or VH domain.
  • ROR1 antibodies e.g., antibodies such as monospecific or bispecific antibodies
  • ROR1 antibodies include human ROR1 antibodies, described herein comprise a VL region or VL domain.
  • ROR1 antibodies e.g., antibodies such as monospecific or bispecific antibodies
  • ROR1 antibodies have a combination of (i) a VH domain or VH region; and (ii) a VL domain or VL region.
  • 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.
  • 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.
  • the ROR1 antibody provided herein comprises CDRs of any antibody or fragment thereof provided herein, for example in Table 1.
  • 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 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).
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25:33893402 (1997).
  • 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.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • NCBI National Center for Biotechnology Information
  • 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-1540479824 without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • 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.
  • 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.
  • a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in a reference amino acid sequence.
  • substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs and/or constant regions).
  • 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%).
  • 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%).
  • ROR1 e.g., human ROR1
  • 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%).
  • 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%).
  • ROR1 e.g., human ROR1
  • 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%).
  • ROR1 e.g., human ROR1
  • the amino terminus of a VH and/or VL 116 NAI-1540479824 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%).
  • ROR1 e.g., human ROR1
  • 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%).
  • ROR1 e.g., human ROR1
  • ROR1 e.g., human ROR1
  • the ROR1 antibodies, including human ROR1 antibodies, presented herein that bind to ROR1 further comprise conservative sequence modifications.
  • 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.
  • 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)).
  • 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%.
  • 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.
  • 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.
  • 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).
  • 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).
  • the amino acid sequence modifications do not include any modification within an SDR.
  • 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).
  • 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%).
  • ROR1 e.g
  • 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.
  • conformational and crystal structure of ROR1 antibodies bound to ROR1 may be employed to identify the epitopes.
  • the present disclosure provides an antibody that specifically binds to the same epitope as any of the ROR1 antibodies provided herein.
  • the ROR1 antibody provided herein 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.
  • 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 [00419]
  • the ROR1 antibody provided herein further comprises an Fc or a variant thereof.
  • the Fc comprises an amino acid sequence as set forth in SEQ ID NO: 38.
  • the Fc variant is a silent Fc (sFc).
  • 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.
  • 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”).
  • 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.
  • 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”).
  • EEM methionine residue position Leu358
  • the ROR1 antibody provided herein specifically binds to ROR1 competitively with any one of the anti-ROR1 antibodies or fragments thereof described herein.
  • 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.
  • 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.
  • the ROR1 antibody comprises six CDRs of the antibody designated A27.
  • the ROR1 antibody comprises six CDRs as listed in one column of Table 1.
  • 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-1540479824 light chain variable regions as set forth in SEQ ID NO:26.
  • 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.
  • the ROR1 antibody is an IgG, for example IgG1, IgG2, IgG3, or IgG4.
  • the ROR1 antibody is an IgG1.
  • the ROR1 antibody comprises a kappa ( ⁇ ) light chain (e.g., a kappa ( ⁇ ) antibody).
  • the ROR1 antibody comprises a lambda ( ⁇ ) light chain (e.g., a lambda ( ⁇ ) antibody).
  • the ROR1 antibody is an IgG1 kappa antibody.
  • 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).
  • SEC size exclusion chromatography
  • HIC hydrophobic interaction chromatography
  • SMAC standup monolayer adsorption chromatography
  • the antibodies are superior developability based on measurement of monomer percentage, solubility, and/or antibody aggregation or precipitation.
  • ROR1 antibodies e.g., antibodies such as monospecific or bispecific antibodies
  • 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 GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO:31) [00426]
  • the unconverted sulfatase motif comprises the amino acid sequence of LCTPSR (SEQ ID NO:100).
  • 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-1540479824 the 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) [00428]
  • 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 PVLDSDGSFFLYSKLTVDKSRWQGNVFSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO:33) [00429]
  • 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) [00431]
  • the C terminus of the VL is conjugated directly or indirectly to the N terminus of the CL.
  • 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).
  • 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.
  • 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.
  • 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.
  • ROR1 antibodies e.g., monospecific or bispecific antibodies
  • 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.
  • 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.
  • ROR1 antibodies e.g., monospecific or bispecific antibodies
  • 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.
  • ROR1 antibodies e.g., monospecific or bispecific antibodies
  • ROR1 antibodies including human ROR1 antibodies, described herein comprise: (a) a heavy chain having the amino acid sequence of SEQ ID NO:41, and (b) a light chain having the amino acid sequence of SEQ ID NO:28.
  • ROR1 antibodies e.g., monospecific or bispecific antibodies
  • an ROR1 binding protein comprising any one of the anti-ROR1 antibodies described herein.
  • the ROR1 binding protein is an antibody comprising two heavy chains and two light chains.
  • the ROR1 binding protein is an antibody comprising two heavy chains comprising a same VH region and two light chains comprising a same VL region.
  • the ROR1 binding protein is a monoclonal antibody, including a mouse, chimeric, humanized, or human antibody.
  • the anti- ROR1 antibody is an antibody fragment, e.g., an scFv.
  • the ROR1 binding protein is a fusion protein comprising the anti-ROR1 antibody provided herein.
  • the ROR1 binding protein is a multispecific antibody comprising the anti-ROR1 antibody or fragment thereof provided herein.
  • Other exemplary ROR1 binding molecules are described in more detail in the following sections.
  • the anti-ROR1 antibody or antigen-binding protein may incorporate any of the features, singly or in combination, as described in the sections below.
  • the amino acid sequence of an ROR1 antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to a 2-formylglycine (fGly) residue by action of a formylglycine generating enzyme (FGE) either in vivo (e.g., at the time of translation of an aldehyde tag- containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system
  • FGE formylglycine generating enzyme
  • Such sulfatase motifs may also be referred to herein as an FGE-modification site. 7.4.1 Sulfatase motifs [00439] A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 amino acid residues in length, usually no more than 6 amino acid residues in length.
  • Sulfatase motifs provided in an Ig polypeptide are at least 5 or 6 amino acid residues, and can be, for example, from 5 to 16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acid residues in length.
  • ROR1 antibodies of interest include those where one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide for a sequence of a sulfatase motif in the ROR1 antibody.
  • amino acid residues such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or
  • the ROR1 antibody includes a modification (insertion, addition, deletion, and/or substitution/replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the ROR1 antibody.
  • a modification insertion, addition, deletion, and/or substitution/replacement of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the ROR1 antibody.
  • the total number of modifications of residues can be reduced, e.g., by site-specification modification (insertion, addition, deletion, substitution/replacement) of amino acid residues flanking the native amino acid residues to provide a sequence of the desired sulfatase motif.
  • the extent of modification of the native amino acid sequence of the target antibody is minimized, so as to minimize the number of 126 NAI-1540479824 amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target antibody may minimize the impact such modifications may have upon antibody function and/or structure.
  • aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to a “consensus sulfatase motif”)
  • aldehyde tags can thus comprise a minimal sulfatase motif of 5 or 6 residues or can be longer and comprise a minimal sulfatase motif which can be flanked at the N- and/or C-terminal sides of the motif by additional amino acid residues.
  • Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.
  • An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C- terminus of a native, wild-type Ig heavy chain.
  • An aldehyde tag can be present within a CH1 domain of an Ig heavy chain.
  • An aldehyde tag can be present within a CH2 domain of an Ig heavy chain.
  • an aldehyde tag can be present within a CH3 domain of an Ig heavy chain.
  • An aldehyde tag can be present in an Ig light chain constant region, e.g., in a kappa light chain constant region or a lambda light chain constant region.
  • the ROR1 antibody as disclosed herein comprises a sulfatase motif.
  • the sulfatase motif used may be described by the formula: X 1 Z 10 X 2 Z 20 X 3 Z 30 (V) wherein: Z 10 is cysteine or serine (which can also be represented by (C/S)); Z 20 is either a proline or alanine residue (which can also be represented by (P/A)); Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 is present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar
  • the amino acid sequence of an antibody heavy and/or light chain can be modified to provide a sequence of at least 5 amino acids of the formula X 1 Z 10 X 2 Z 20 X 3 Z 30 , wherein: Z 10 is cysteine or serine; Z 20 is a proline or alanine residue; Z 30 is an aliphatic amino acid or a basic amino acid; X 1 is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the ROR1 antibody, X 1 is present; X 2 and X 3 are each independently any amino acid.
  • the sulfatase motif is generally selected so as to be capable of conversion by a selected FGE, e.g., an FGE present in a host cell in which the aldehyde-tagged antibody is expressed or an FGE which is to be contacted with the aldehyde-tagged antibody in a cell- free in vitro method.
  • FGE e.g., an FGE present in a host cell in which the aldehyde-tagged antibody is expressed or an FGE which is to be contacted with the aldehyde-tagged antibody in a cell- free in vitro method.
  • the sulfatase motif can be of the formula: X 1 CX 2 PX 3 Z 30 (VI) wherein: X 1 may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target ROR1 antibody, X 1 is present; X 2 and X 3 independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than
  • sulfatase motifs include LCTPSR (SEQ ID NO:100), MCTPSR (SEQ ID NO:101), VCTPSR (SEQ ID NO:102), LCSPSR (SEQ ID NO:103), LCAPSR (SEQ ID NO:104), LCVPSR (SEQ ID NO:105), LCGPSR (SEQ ID NO:106), ICTPAR (SEQ ID NO:107), LCTPSK (SEQ ID NO:108), MCTPSK (SEQ ID NO:109), VCTPSK (SEQ ID NO:110), LCSPSK (SEQ ID NO:111), LCAPSK (SEQ ID NO:112), LCVPSK (SEQ ID NO:113), LCGPSK (SEQ ID NO:114), LCTPSA (SEQ ID NO:115), ICTPAA (SEQ ID NO:116), MCTPSA (SEQ ID NO:117), VCTPSA (SEQ ID NO:118), LCSPSA (SEQ ID NO:
  • the ROR1 antibody as disclosed herein comprises a fGly- containing sulfatase motif.
  • FGE fGly-containing sulfatase motif
  • the fGly-containing sulfatase motif can be of the formula: X 1 (fGly)X 2 Z 20 X 3 Z 30 (VII) wherein: fGly is the formylglycine residue; Z 20 is either a proline or alanine residue (which can also be represented by (P/A)); Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X 1 may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g.,
  • fGly-containing sulfatase motifs include L(fGly)TPSR (SEQ ID NO:123), M(fGly)TPSR (SEQ ID NO:124), V(fGly)TPSR (SEQ ID NO:125), L(fGly)SPSR (SEQ ID NO:126), L(fGly)APSR (SEQ ID NO:127), L(fGly)VPSR (SEQ ID NO:128), L(fGly)GPSR (SEQ ID NO:129), I(fGly)TPAR (SEQ ID NO:130), L(fGly)TPSK (SEQ ID NO:131), M(fGly)TPSK (SEQ ID NO:132), V(fGly)TPSK (SEQ ID NO:133), L(fGly)SPSK (SEQ ID NO:134), L(fGly)APSK (SEQ ID NO:135), L(fGly)VPSK (SEQ ID NO:123), M(fGly)TPSR (SEQ ID
  • the ROR1 antibody Ab of any formula as described herein comprises a fGly'-containing sulfatase motif.
  • the ROR1 antibody Ab of any formula as described herein comprises a light chain (such as SEQ ID NO:28) and a heavy chain (such as a variant thereof of SEQ ID NO:27 further comprising one or more sequences of Formula (VIII), for example SEQ ID NO:41 or 44).
  • the ROR1 antibody containing the fGly residue may be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above) of a linker attached to the drug or active agent to produce an fGly’-containing sulfatase motif.
  • a reactive moiety e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above
  • the term fGly refers to the amino acid residue of the sulfatase motif that is coupled to the drug or active agent through a linker (e.g., a branched linker) as described herein.
  • the fGly’-containing sulfatase motif can be of the formula: X 1 (fGly’)X 2 Z 20 X 3 Z 30 (VIII) wherein: fGly’ is the amino acid residue coupled to the drug or active agent through a linker (e.g., a branched linker) as described herein; Z 20 is either a proline or alanine residue (which can also be represented by (P/A)); Z 30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V
  • fGly’-containing sulfatase motifs include 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
  • amino acid sequence of an antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an fGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system).
  • a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an fGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system).
  • the antibody used to generate a conjugate of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region.
  • Ig antibodies are referred to herein as “target Ig polypeptides” or “target antibodies.”
  • target Ig polypeptides or “target antibodies.”
  • the site in an antibody into which a sulfatase motif is introduced can be any convenient site.
  • an antibody heavy chain constant region can include Ig constant regions of any heavy chain isotype, non-naturally occurring Ig heavy chain constant regions (including consensus Ig heavy chain constant regions).
  • An Ig constant region amino acid sequence can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent a solvent-accessible loop region of the Ig constant region.
  • An Ig constant region amino acid sequence can be modified by insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above.
  • an aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain).
  • an aldehyde-tagged Ig heavy chain constant region e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain.
  • the aldehyde- tagged Ig heavy chain constant region can include heavy chain constant region sequences of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant of same, e.g., human heavy chain constant region sequences or mouse heavy chain constant region sequences, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly- modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art.
  • an aldehyde-tagged antibody comprises an aldehyde-tagged Ig light chain constant region.
  • the aldehyde-tagged Ig light chain constant region can include constant region sequences of a kappa light chain, a lambda light chain, e.g., human kappa or lambda light chain constant regions, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly- modified antibody.
  • Exemplary constant regions include human gamma 1 and gamma 3 regions.
  • a constant region may have a wild-type amino acid sequence, or it may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to a wild-type amino acid sequence.
  • the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain.
  • an isolated aldehyde- tagged antibody can comprise a heavy chain constant region amino acid sequence modified to 132 NAI-1540479824 include a sulfatase motif as described above, where the sulfatase motif is in or adjacent to a surface-accessible loop region of the antibody heavy chain constant region.
  • a sulfatase motif can be provided within or adjacent to one or more of these amino acid sequences of such modification sites of an Ig heavy chain.
  • an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif adjacent and N-terminal and/or adjacent and C-terminal to these modification sites.
  • an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modifications sites.
  • an Ig heavy chain polypeptide amino acid sequence may be modified to include two motifs, which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids.
  • two motifs which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids.
  • selected amino acid residues of the modification sites of an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide a sulfatase motif at the modification site.
  • An antibody used in an antibody-drug conjugate of the present disclosure can have any of a variety of antigen-binding specificities, including but not limited to, e.g., an antigen present on a cancer cell; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on a diseased cell; and the like.
  • an antibody conjugate can bind an antigen, where the antigen is present on the surface of the cell.
  • An antibody conjugate of the present disclosure can bind antigen with a suitable binding affinity, e.g., from 5 x 10 -6 M to 10 -7 M, from 10 -7 M to 5 x 10 -7 M, from 5 x 10 -7 M to 10 -8 M, from 10 -8 M to 5 x 10 -8 M, from 5 x 10 -8 M to 10 -9 M, or a binding affinity greater than 10 -9 M.
  • a suitable binding affinity e.g., from 5 x 10 -6 M to 10 -7 M, from 10 -7 M to 5 x 10 -7 M, from 5 x 10 -7 M to 10 -8 M, from 10 -8 M to 5 x 10 -8 M, from 5 x 10 -8 M to 10 -9 M, or a binding affinity greater than 10 -9 M.
  • a subject antibody conjugate can bind an antigen present on a cancer cell (e.g., a tumor-specific antigen; an antigen that is over-expressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
  • a subject antibody 133 NAI-1540479824 conjugate can be specific for an antigen on a cancer cell, where the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).
  • a subject antibody conjugate can bind an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the virus; where the antigen is expressed on a cell type that is infected by a virus; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor.
  • a subject antibody conjugate can bind an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, such as a viral fusion inhibitor.
  • the antibody that binds to ROR1 comprises a heavy chain which has been modified to include unconverted sulfatase motifs (e.g., any one of SEQ ID NOs:100-122) in the CH1 region and in the CT region, and therefore comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALCTPSR GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAK
  • the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. Additionally or alternatively, either or both of the sulfatase motifs are converted and conjugated to a linker-payload in an ROR1-ADC as disclosed herein.
  • the antibody that binds to ROR1 comprises a heavy chain which has been modified to include formylglycine residues in the CH1 and CT regions and therefore comprises the amino acid sequence of: 134 NAI-1540479824 EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL(fGly)T PSRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
  • the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody.
  • an ROR1-ADC comprises a heavy chain comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL(fGly’) TPSRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQV
  • each underline portion comprises the fGly- containing sulfatase motif of SEQ ID NO:146 but, in any embodiment, may be independently replaced with any one of SEQ ID NOs:147-168.
  • the ROR1-ADC comprises an ROR1 antibody, wherein the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in 135 NAI-1540479824 SEQ ID NO: 41, and the ROR1 antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain.
  • the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody.
  • the antibody that binds to ROR1 comprises a heavy chain which has been modified to include an unconverted sulfatase motif (e.g., any one of SEQ ID NOs:100-122), and therefore comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSLCTPSRNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKPK
  • the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. [00473] Additionally or alternatively, the sulfatase motif is converted and conjugated to a linker-payload in an ROR1-ADC as disclosed herein.
  • the antibody that binds to ROR1 comprises a heavy chain which has been modified to include a formylglycine residue and therefore comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSL(fGly)TPSRNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK 136 NAI-1540479824 FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
  • the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. Additionally or alternatively, the sulfatase motif is conjugated to a linker-payload in an ROR1-ADC as disclosed herein.
  • an ROR1-ADC comprises a heavy chain comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSL(fGly’)TPSRNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
  • the underline portion comprises the fGly-containing sulfatase motif of SEQ ID NO:146 but, in any embodiment, may be replaced with any one of SEQ ID NOs:147-168.
  • the ROR1-ADC comprises an ROR1 antibody, wherein the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 44, and the ROR1 antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain.
  • the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody.
  • Other suitable sulfatase motif and their location in an antibody can also be used, such as International Publication Nos. WO2012097333, WO2017189432, and WO2018169953, each of which is incorporated herein by reference in its entirety.
  • an ROR1-ADC is prepared from an ROR1 antibody, wherein the antibody comprises (i) a heavy chain that is inserted with one or more fGly-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID No: 40 or 44, and (ii) a light chain, such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28.
  • any ROR1-ADC as disclosed herein comprises an ROR1 antibody conjugated to a linker-payload as disclosed herein (such as (Ia) or (IIa)), wherein the antibody comprises (i) a heavy chain that is inserted with one or more fGly’-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 44, and (ii) a light chain, such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28.
  • Ab of any ADC formula as disclosed herein comprises (i) a heavy chain that is inserted with one or more fGly’-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 44, and (ii) a light chain, such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28.
  • a heavy chain that is inserted with one or more fGly’-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 44
  • a light chain such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28.
  • a hydrazinyl-pyrrolo coupling moiety which can interchangeably be referred to herein as an aza-hydrazino-iso-Pictet-Spengler (azaHIPS) coupling moiety, upon conjugation to a formyl-glycine, forms a pyridazine-pyrrolo coupling moiety as shown above.
  • An ROR1 antibody can include a 2-formylglycine residue (fGly) that is reacted with azaHIPS coupling 138 NAI-1540479824 moiety, thus conjugating the two together.
  • a drug can be coupled directly or indirectly (e.g., through a linker moiety) to the azaHIPS moiety at any location of the azaHIPs moiety (e.g., Z 1 , Z 2 , Z 3 , Z 4 , or Q 1 ).
  • R 2 and R 3 can each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl.
  • substituent such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted
  • Z 1 , Z 2 , Z 3 , and Z 4 can be as described herein, such as for Formula (I).
  • Q 1 may be -L A -W 1 , as described herein, such as for Formula (I).
  • Formula (Ia) below represents a hydrazinyl-pyrrolo coupling moiety that can be used to link an ROR1 antibody and a drug in an ROR1-ADC. (Ia). Accordingly, provided is an ADC produced by conjugating an ROR1 antibody as disclosed herein to one or more of a linker-payload represented by Formula (Ia).
  • R 30 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.
  • Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 4 , N and C-L B -W 2 , wherein at least one Z 1 , Z 2 , Z 3 and Z 4 is C-L B -W 2 ;
  • R 30 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;
  • R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyl, acyl, acyl, acyl,
  • Formula (X) below represents a hydrazinyl-pyrrolo coupling moiety that can be used to link an ROR1 antibody and a drug in any ROR1-ADC described herein: wherein: Z 1 , Z 2 , Z 3 and Z 4 are each independently selected from CR 4 , N and C-L B -W 2 , wherein at least one Z 1 , Z 2 , Z 3 and Z 4 is C-L B -W 2 ; 140 NAI-1540479824 R 30 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, substituted alken
  • each represents the point of attachment to a pyridazine-pyrrolo coupling moiety as described herein (e.g., in any of Formulas as disclosed herein); * in linker (L-3-a) represents the point of attachment to W 1 ; * in linker (L-3-b) represents the point of attachment to W 2 ; each R 5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R 6 is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl,
  • X 1 is selected from the group consisting of C, N, O and S
  • Y 1 and Y 2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y 1 and Y 2 are cyclically linked; each R 5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alky
  • each substituent is as defined herein, such as with respect to Formula (I).
  • R 1 is hydrogen
  • Z 1 is X 1 -Y 1 , wherein X 1 represents an atom in the ring of Formula (I) and is selected from the group consisting of C, N, O and S
  • Z 2 is C-Y 2
  • Y 1 and Y 2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl
  • each represents the point of attachment to a pyridazine-pyrrolo coupling moiety as described herein (e.g., any of Formulas as disclosed herein); * in linker (L-4-a) represents the point of attachment to W 1 ; * in linker (L-4-b) represents the point of attachment to W 2 ; each R 5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R 6’ and R 6” are each independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted substituted heterocyclyl; each R 6’ and R 6” are
  • each substituent is as defined herein, such as with respect to Formula (I).
  • the ROR1-ADC is represented by Formula (XIV-4): , (XIV-4) wherein each substituent is as defined with respect to Formula (XIV-3).
  • R 7 is an enzymatically cleavable moiety comprising a sugar moiety, such as a 147 NAI-1540479824 glycoside or glycosyl.
  • each substituent is as defined herein, such as with respect to Formula (I).
  • each represents the point of attachment to a pyridazine-pyrrolo coupling moiety as described herein (e.g., any of Formulas as disclosed herein); * in linker (L-5-a) represents the point of attachment to W 1 ; * in linker (L-5-b) represents the point of attachment to W 2 ; each R 5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; R 6’ and R 6” are independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalky
  • each substituent is as defined herein, such as with respect to Formula (I).
  • the ROR1-ADC is represented by Formula (XIV-5): , 149 NAI-1540479824 wherein each substituent is as defined with respect to Formula (XIV-3).
  • each substituent is as defined herein, such as with respect to Formula (I).
  • each substituent is as defined herein, such as with respect to Formula (I).
  • the ROR1-ADC is represented by Formula (XIV-6): , (XIV-6) wherein each substituent is as defined with respect to Formula (XIV-3).
  • each substituent is as defined herein, such as with respect to Formula (I).
  • each substituent is as defined herein, such as with respect to Formula (I).
  • the ROR1-ADC is represented by Formula (XIV-7): , (XIV-7) wherein each substituent is as defined with respect to Formula (XIV-3).
  • each substituent is as defined herein, such as with respect to Formula (I).
  • L 2a is -CO-.
  • L 2b is -CO-. In some embodiments, both L 2a and L 2b are -CO-.
  • W 1 and W 2 are belotecan.
  • both W 1 and W 2 are belotecan.
  • W 1 comprises a camptothecin or an analog thereof, such as belotecan.
  • W 1 comprises belotecan.
  • W 2 comprises a camptothecin or an analog thereof, such as belotecan. In yet further embodiments, W 2 comprises belotecan. Additionally or alternatively, W 1 and W 2 are the same. In other embodiments, W 1 and W 2 are different.
  • an ROR1-ADC of Formula (II) may be prepared by conjugating one or more linker-payloads of Formula (IIa), shown below, to an ROR1 antibody:
  • s is an integer from 1 to 10, for example 1 to 8. In some embodiments, s is 2. In some embodiments, s is 4.
  • Ab is an ROR antibody as disclosed herein.
  • ROR antibody as disclosed herein.
  • 154 NAI-1540479824 An exemplary scheme for making an ROR1-ADC is shown in Scheme A: 155 NAI-1540479824
  • the linker-drug is conjugated to an ROR1 antibody.
  • the ROR1 antibody has a recognition motif, for example, a L(C/S)TPSR (SEQ ID NO:99) recognition motif in each of the CH1 constant region and c-terminus (CT) of the heavy chain (e.g., SEQ ID NO:29, or 40, or 41 as described above).
  • the cysteine residue of a L(C/S)TPSR (SEQ ID NO:99) recognition motif can be converted to a formylglycine (fGly) and subsequently conjugated to an azaHIPs moiety through a Pictet-Spengler reaction (fGly’).
  • the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 28.
  • Scheme B Another exemplary scheme for making an ROR1-ADC is shown in Scheme B: 156 NAI-1540479824
  • the linker-drug is conjugated to an ROR1 antibody.
  • the ROR1 antibody has a recognition motif, for example, a LCTPSR (SEQ ID NO:100) recognition motif in the CH1 constant region of the heavy chain (e.g., SEQ ID NO:30, 42, 43, or 44 as described above).
  • the cysteine residue of a LCTPSR (SEQ ID NO:100) recognition motif can be converted to a formylglycine (fGly) and subsequently conjugated to an azaHIPs moiety through a Pictet-Spengler reaction.
  • the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 44 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 28.
  • the ROR1-ADC is ADC-8, wherein the ROR1-ADC is of Formula (II), s is 4, the antibody Ab is A27 as described herein and the conjugation sites of the pyridazine-pyrrolo moiety to the antibody are in the CH1 and CT regions of each heavy chain of the antibody, as shown in Scheme A. Accordingly, the DAR of ADC-8 is 8.
  • the antibody Ab of ADC-8 comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:41 and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:28.
  • the ROR1-ADC is ADC-4, wherein the ROR1-ADC is of Formula (II), s is 2, and the antibody Ab is A27 as described herein and the conjugation sites 157 NAI-1540479824 of the pyridazine-pyrrolo moiety to the antibody are in the CH1 regions of each heavy chain of the antibody, as shown in Scheme B. Accordingly, the DAR of ADC-8 is 4.
  • an ROR1-ADC includes one or more linker-drug conjugated to each heavy chain constant region of an ROR1 antibody via a pyridazine-pyrrolo coupling moiety and for example, is characterized by a stoichiometric ratio of antibody to drug (DAR) 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.
  • DAR antibody to drug
  • the ROR1-ADC is an ADC as described below in Table 3.
  • Table 3 Exemplary ROR1-ADCs 7.5 DRUGS FOR CONJUGATION
  • a conjugate or a compound of the present disclosure can include as substituents W 1 and W 2 a drug or active agent. Any of a number of drugs are suitable for use or can be modified to be rendered suitable for use, as a reactive partner to conjugate to an antibody. Examples of drugs include small molecule drugs and peptide drugs.
  • “Small molecule drug” as used herein refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa.
  • a small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom.
  • the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as a camptothecin, or an analog or derivative thereof, or a pharmaceutically active camptothecin moiety and/or a portion thereof.
  • a topoisomerase inhibitor (e.g., camptothecin, or analog or derivative thereof) conjugated to the ROR1 antibody can be any of a variety of topoisomerase inhibitors, for example camptothecin or 158 NAI-1540479824 camptothecin moieties such as, but not limited to, camptothecin and analogs and derivatives thereof as described herein.
  • drugs that find use in the conjugates and compounds described herein include, but are not limited to, a topoisomerase inhibitor, for example camptothecin or a camptothecin derivative, such as SN-38, Belotecan, Exatecan, 9- aminocamptothecin (9-AC), topotecan, des-Me-topotecan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/012325, the disclosure of which is incorporated herein by reference. [00518] In other embodiments, the drug or active agent can be a maytansine.
  • camptothecin or a camptothecin derivative such as SN-38, Belotecan, Exatecan, 9- aminocamptothecin (9-AC), topotecan, des-Me-topotecan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/
  • Maytansine, “maytansine moiety,” “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof.
  • a maytansine conjugated to the ROR1 antibody can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein (e.g., deacylmaytansine).
  • the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or a portion thereof.
  • An auristatin conjugated to the ROR1 antibody can be any of a variety of auristatin moieties such as, but not limited to, an auristatin and analogs and derivatives thereof as described herein.
  • examples of drugs that find use in the conjugates and compounds described herein include but are not limited to an auristatin or an auristatin derivative, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like.
  • the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and/or a portion thereof.
  • a duocarmycin conjugated to the ROR1 antibody can be any of a variety of duocarmycin moieties such as, but not limited to, a duocarmycin and analogs and derivatives thereof as described herein.
  • Examples of drugs that find use in the conjugates and compounds described herein include but are not limited to a duocarmycin or a duocarmycin derivative, such as duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like.
  • the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin.
  • the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulatory agent, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide.
  • a cytotoxin can include any compound that leads to cell death (e.g., necrosis or apoptosis) or a decrease in cell viability.
  • Kinase inhibitors can include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandet
  • selective estrogen receptor modulators include, but are not limited to, Endoxifen, Tamoxifen, Afimoxifene, Toremifene, and the like.
  • Immunostimulatory agents can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, and the like.
  • TLR agonists include, but are not limited to, imiquimod, resiquimod, and the like.
  • Oligonucleotide dugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, volanesorsen, inotersen, tofersen, tominersen, and the like.
  • Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like.
  • Cytokines include, but are not limited to, Albinterferon Alfa-2B, Aldesleukin, ALT- 801, Anakinra, Ancestim, Avotermin, Balugrastim, Bempegaldesleukin, Binetrakin, Cintredekin Besudotox, CTCE-0214, Darbepoetin alfa, Denileukin diftitox, Dulanermin, Edodekin alfa, Emfilermin, Epoetin delta, Erythropoietin, Human interleukin-2, Interferon alfa, Interferon alfa-2c, Interferon alfa-n1, Interferon alfa-n3, Interferon alfacon-1, Interferon beta-1a, Interferon beta-1b, Interferon gamma-1b, Interferon Kappa, Interleukin-1 alpha, Interleukin-10, Interleukin-7, Lenograstim, Lerid
  • Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like. 160 NAI-1540479824
  • “Peptide drug” as used herein refers to amino acid-containing polymeric compounds and is meant to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics.
  • the peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source).
  • Peptide drugs can range in molecular weight and can be from 200 Da to 10 kDa or greater in molecular weight.
  • Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti-angiogenic peptides; peptides that activate B cells; peptides that activate T cells; anti-viral peptides; peptides that inhibit viral fusion; peptides that increase production of one or more lymphocyte populations; anti- microbial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti-inflammatory peptides; peptides that regulate glucose metabolism; an anti-thrombotic peptide; an anti-nociceptive peptide; a vasodilator peptide; a platelet aggregation inhibitor; an analgesic; and the like.
  • drugs that find use in the conjugates and compounds described herein include, but are not limited to Tubulysin M, Calicheamicin, a STAT3 inhibitor, alpha-Amanitin, an aurora kinase inhibitor, belotecan, and an anthracycline.
  • Other examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent.
  • the ROR1 antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent.
  • Cancer chemotherapeutic agents include non-peptidic (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.
  • 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.
  • dolastatin 10 or auristatin PE can be included in an antibody-drug conjugate of the present disclosure.
  • Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and 161 NAI-1540479824 benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).
  • PBD pyrrolobenzodiazepine
  • Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (CytoxanTM), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.
  • alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclopho
  • Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6- thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10- propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine.
  • CYTOSAR-U cytarabine
  • cytosine arabinoside including, but not limited to, fluorouracil (5-FU), floxuridine (FudR), 6- thioguanine, 6-
  • Suitable natural products and their derivatives include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g., etoposide, teniposide, etc.; antibiotics, e.g., anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phen
  • anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • 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 162 NAI-1540479824 sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.
  • Hormone modulators and steroids that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g., prednisone, dexamethasone, etc.; estrogens and pregestins, e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g., aminoglutethimide; 17 ⁇ -ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl- testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine,
  • Estrogens stimulate proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation.
  • Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis- DDP), carboplatin, etc.; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc.
  • metal complexes e.g., cisplatin (cis- DDP), carboplatin, etc.
  • ureas e.g., hydroxyurea
  • hydrazines e.g., N-methylhydrazine
  • epidophyllotoxin e.g., a topoisomerase inhibitor
  • 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 TM (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat.
  • Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere ⁇ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel- xylose). 163 NAI-1540479824 [00543] Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No.
  • 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- ⁇ ; (7) IFN- ⁇ ; (8) colony- stimulating factors; and (9) inhibitors of angiogenesis.
  • RTK tyrosine kinase
  • tumor-associated antigen antagonists such as antibodies that bind specifically to a tumor antigen
  • apoptosis receptor agonists such as antibodies that bind specifically to a tumor antigen
  • interleukin-2 interleukin-2
  • IFN- ⁇ IFN- ⁇
  • IFN- ⁇ IFN- ⁇
  • (8) colony- stimulating factors and (9) inhibitors of angiogenesis.
  • examples of drugs include small molecule drugs, such
  • the ROR1 antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agent.
  • a cancer chemotherapeutic agent such as a microtubule affecting agent.
  • the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid.
  • Embodiments of the present disclosure include conjugates where an antibody is conjugated to two or more drug moieties, such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties.
  • the drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein.
  • the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2.
  • DAR drug-to-antibody ratio
  • the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the 164 NAI-1540479824 conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). By average is meant the arithmetic mean.
  • the two drugs or active agents attached to the branched linker are the same drug or active agent. For example, a first branch of a branched linker may be attached to a drug, or an active agent and a second branch of the branched linker may be attached to the same drug or the same active agent as the first branch.
  • the two drugs or active agents attached to the branched linker are different drugs or active agents.
  • a first branch of a branched linker may be attached to a first drug, or a first active agent and a second branch of the branched linker may be attached to a second drug or a second active agent different from the first drug or the first active agent attached to the first branch.
  • the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect.
  • 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.
  • 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,
  • 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).
  • 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.
  • 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.
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an auristatin (e.g., MMAE) as described herein.
  • auristatin e.g., MMAE
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an iRGD peptide as described herein.
  • the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and an iRGD peptide as described herein.
  • the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein.
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein.
  • a kinase inhibitor e.g., Sorafenib, Lapatinib, Gefitinib, and the like
  • the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.
  • the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein.
  • Drugs to be conjugated to an ROR1 antibody may be modified to incorporate a reactive partner for reaction with the ROR1 antibody.
  • the drug is a peptide drug
  • the reactive moiety e.g., aminooxy or hydrazide can be positioned at an N-terminal region, the N-terminus, a C-terminal region, the C-terminus, or at a position internal to the peptide.
  • an example of a method involves synthesizing a peptide drug having an aminooxy group.
  • the peptide is synthesized from a Boc-protected precursor.
  • An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N- Boc group.
  • the amino group of the peptide reacts with 3-(2,5- dioxopyrrolidin-1-yloxy)propanoic acid.
  • Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker.
  • peptide coupling reagents that can be used include, but not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP 166 NAI-1540479824 (1-benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP (benzo
  • HOBt and DIC can be used as peptide coupling reagents.
  • Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group.
  • Deprotection of the N-oxysuccinimide group occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA.
  • reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine.
  • the product and intermediates can be purified by conventional means, such as HPLC purification.
  • HPLC purification HPLC purification.
  • pH and steric hindrance e.g., the accessibility of the amino acid residue to reaction with a reactive partner of interest
  • modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with an ROR1 antibody present in or on a living cell, the conditions are selected so as to be physiologically compatible.
  • the pH can be dropped temporarily for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (e.g., from about 30 min to 1 hour).
  • Physiological conditions for conducting modification of antibodies on a cell surface can be similar to those used in a 167 NAI-1540479824 ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040).
  • Small molecule compounds containing, or modified to contain, an ⁇ -nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the antibody-drug conjugates of the present disclosure.
  • ROR1-ADCs can be included in a pharmaceutical composition for administration, e.g., to a subject for treating a disease, disorder, or condition.
  • a pharmaceutical composition comprises ROR1-ADCs which, on average, can exhibit a drug-to-antibody ratio (“DAR”) of about 0.1 to about 20, or from 0.5 to 20, or from 1 to 20.
  • DAR drug-to-antibody ratio
  • Methods to determine DAR are well known to the skilled person and include methods using Reverse Phase Chromatography, or HPLC- MS.
  • a pharmaceutical composition comprising an ROR1-ADC exhibits a DAR of about 0.1 to about 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2, or from 2 to 20, or from 2 to 19, or from 2 to 18, or from 2 to 17, or from 2 to 16, or from 2 to 15, or from 2 to 14, or from 2 to 13, or from 2 to 12, or from 2 to 11, or from 2 to 10, or from 2 to 9, or from 2 to 8, or from 2 to 7, or from 2 to 6, or from 2 to 5, or from 2 to 4, or from 2 to 3, or from 3 to 20, or from 3 to 19, or from 3 to 18, or from 3 to 17, or from 3 to 16, or from 1 to 16,
  • the composition exhibits a DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the composition exhibits a DAR of 1 to 10. In certain embodiments, the composition exhibits a DAR of 1 to 5 (e.g., 3 or 3.5 or 4). In certain embodiments, the composition exhibits a DAR of 5 to 10 (e.g., 6, or 6.5, or 7, or 7.5 or 8).
  • a pharmaceutical composition comprises an ROR1-ADC comprising an ROR1 antibody as described herein, including A27 (see, e.g., Tables 1-2), and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises ROR1-ADC ADC-4 or ADC-8 (see, e.g., Table 3), and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising an ROR1-ADC of any formula as disclosed herein (such as Formula (I), (II), (XIV-3), (XIV- 4), (XIV-5), (XIV-6), or (XIV-7)) and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a population of ROR1-ADCs, wherein the ROR1-ADCs are of a same formula as disclosed herein (such as any one of Formula (I), (II), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), yet, their s are different.
  • the pharmaceutical composition may exhibit various DAR, such as about 1 to about 10, about 1 to about 8, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about
  • An ROR1-ADC can be formulated in any of a variety of different ways.
  • An ROR1- ADC of the present disclosure can be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, e.g., oral, topical, or parenteral administration.
  • an ROR1-ADC is provided as a liquid injectable (such as in those embodiments where they it can be administered intravenously or directly into a tissue)
  • an ROR1-ADC can be provided as a ready-to-use dosage form, or as a storage-stable powder for reconstitution or liquid composed of pharmaceutically acceptable excipients and excipients.
  • ROR1-ADCs can be provided in a pharmaceutical composition comprising an effective amount of an ROR1-ADC and a pharmaceutically acceptable excipient (e.g., saline).
  • the pharmaceutical composition can optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like).
  • the formulations are suitable for administration to a mammal, such as those that are suitable for administration to a human.
  • pharmaceutical compositions that contain an effective amount of an ROR1-ADC described herein and a pharmaceutically acceptable excipient.
  • the ROR1-ADC comprises an ROR1 antibody as described herein, including A27, as described in any one of Tables 1-2.
  • a pharmaceutical composition comprises an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)) and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises an effective amount of ADC-4 or ADC-8 (see, e.g., Table 3) and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)) and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises an effective amount of ADC-4 or ADC-8 (see, e.g., Table 3) and a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances that are suitable for administration into a human or veterinary subject (e.g., a physiologically acceptable and/or pharmacologically acceptable).
  • the pharmaceutically acceptable excipient can be co-mingled with one or more of the active components, e.g., a hybrid molecule, and with each other, 170 NAI-1540479824 when more than one pharmaceutically acceptable excipient is present in the pharmaceutical composition, in a manner so as not to substantially impair the desired pharmaceutical efficacy.
  • compositions of the invention can additionally contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable buffering agents including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
  • compositions of the invention can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the antibody or antigen-binding fragment of the invention into association with an excipient that constitutes one or more accessory ingredients.
  • the pharmaceutical composition is prepared by uniformly and intimately bringing the active agent into association with a liquid excipient, a finely divided solid excipient, or both, and then, if necessary, shaping the product.
  • a composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the composition, which preferably is isotonic with the blood of the recipient.
  • This aqueous preparation can be formulated of known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
  • acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed, such as synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid can be used in the preparation of injectables.
  • compositions of the invention can be carried out in accordance with methods well known in the art.
  • the delivery systems useful in the context of the invention include time-released, delayed 171 NAI-1540479824 release, and sustained release systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
  • the composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician, and can be particularly suitable for some compositions of the invention.
  • release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
  • polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are lipids such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides; hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides
  • hydrogel release systems such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides
  • sylastic systems such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides
  • peptide-based systems such as fatty acids or neutral fats
  • wax coatings such as those described in U.S.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • an ROR1-ADC or pharmaceutical composition is suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic method.
  • Components can be provided as concentrates (including lyophilized compositions), which can be further diluted prior to use, or they can be provided at the concentration of use.
  • ROR1-ADC for use of an ROR1-ADC in vivo, single dosages can be provided in sterilized containers having the desired amount and concentration of components. 7.7 METHODS OF TREATMENT [00569] Also provided herein are methods of treating, preventing, or alleviating an ROR1- mediated disease, disorder, or condition, including one or more symptoms of the ROR1- mediated disease, disorder, or condition with an ROR1-ADC comprising an ROR1 antibody, and a drug conjugated directly or indirectly thereto. Also provided herein are methods of killing tumor cells with an ROR1-ADC comprising an ROR1 antibody, and a drug conjugated directly or indirectly thereto.
  • a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of Formula (I).
  • a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC comprising a camptothecin analogue (e.g., belotecan), or any derivative thereof.
  • a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)).
  • a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of ADC-4 or ADC-8 (see, e.g., Table 3). In some embodiments, the contacting is in vivo or in vitro. [00571] In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC comprising a camptothecin analogue (e.g., belotecan), or any derivative thereof.
  • a camptothecin analogue e.g., belotecan
  • a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)).
  • the contacting is in vivo or in vitro.
  • the method of contacting the tumor cell with an ROR1-ADC comprises contacting the tumor cell with a composition comprising the ROR1-ADC and one or more pharmaceutically acceptable excipients.
  • the ROR1-ADC in the composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8.
  • a method of treating a cancer in a subject in need thereof comprising administering an effective amount of an ROR1-ADC to the subject, wherein the ROR1-ADC comprises an ROR1 antibody and a drug conjugated thereto via a linker, as described herein.
  • the ROR1 antibody is A27, as described herein, such as in Tables 1-2.
  • the ROR1 is ADC-4 or ADC-8 (see, e.g., Table 3).
  • a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)).
  • a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)).
  • the method of treating cancer in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising the ROR1- ADC and a pharmaceutically acceptable excipient.
  • the ROR1-ADC in the pharmaceutical composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8.
  • the method of treating cancer in a subject in need thereof is effective to enhance the removal of the cancer cells, for example, tumor cells, in the subject.
  • “Enhancing” the removal of cancer or tumor cells does not require a 100% enhancement of removal. Any enhancement in the rate of removal is contemplated.
  • “modulating” tumor growth refers to reducing the size of the tumor, slowing tumor growth, or inhibiting an increase in the size of an existing tumor. Complete abolition of a tumor is not required; any decrease in tumor size or slowing of tumor growth constitutes a beneficial biological effect in a subject.
  • tumor cell removal can be enhanced by, for example, at least about 5%, at least about 10% or at least about 20% compared to levels of removal observed in the absence of the method (e.g., in a biologically matched control subject or specimen that is not exposed to the agent of the method).
  • the effect is detected by, for example, a reduction in tumor size, a decrease or maintenance of the levels of tumor markers, or reduction or maintenance of a tumor cell population.
  • removal of tumor cells is enhanced by, for example, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared to the removal of tumor cells in the absence of an ROR1 antibody of the method.
  • the present disclosure also provides a method of modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject in need thereof, the method comprising administering to the subject an ROR1-ADC comprising an ROR1 antibody and a drug linked thereto via a linker, as described herein, in an amount effective to modulate the tumor growth in the subject.
  • a method of modulating tumor growth in a subject in need thereof comprising administering an effective amount of an ROR1-ADC comprising an ROR1 antibody and a drug conjugated thereto via a linker, as described herein.
  • the antibody that binds to ROR1 is A27, as described in any one of Tables 1-2.
  • the ROR1-ADC is ADC-4 or ADC-8 (see, e.g., Table 3).
  • a method of modulating tumor growth in a subject in need thereof comprises 174 NAI-1540479824 administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)).
  • a method of modulating tumor growth in a subject in need thereof comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV- 7), or (II)).
  • the method of modulating tumor growth in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising the ROR1-ADC and one or more pharmaceutically acceptable excipients.
  • the ROR1-ADC in the pharmaceutical composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8.
  • the cancer and tumor cells described herein that may be treated and/or killed by the methods described herein express an ROR1 antigen, for example, as on a surface of the cancer or tumor cell.
  • a tumor or cancer cell may overexpress an ROR1 antigen.
  • the term “overexpress” means to transcribe and translate more genetic product than normal (such as in normal cells), the process of which is often a characteristic of cancer cells.
  • the present disclosure also provides a method of alleviating or reducing side effects associated with cancer comprising administering an effective amount of an ROR1-ADC to a subject in need thereof.
  • a method of alleviating or reducing side effects associated with cancer comprises administering an effective amount of an ROR1-ADC comprising an ROR1 antibody and a drug conjugated thereto via a linker, as described herein.
  • the antibody that binds to ROR1 is A27, as described in any one of Tables 1-2.
  • a method of alleviating or reducing side effects associated with cancer comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)).
  • a method of alleviating or reducing side effects associated with cancer comprises administering to the subject an effective amount of ADC-4 or ADC-8 (see, e.g., Table 3).
  • a method of alleviating or reducing side effects associated with cancer comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)). 175 NAI-1540479824 [00585]
  • the method of alleviating or reducing side effects associated with cancer in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising an ROR1-ADC as disclosed herein and one or more pharmaceutically acceptable excipients.
  • the ROR1-ADC in the pharmaceutical composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8.
  • an ROR1-ADC comprising an ROR1 antibody can be used to treating, preventing, or alleviating an ROR1-mediated disease, disorder, or condition, including one or more symptoms of the ROR1-mediated disease, disorder, or condition with an ROR1-ADC comprising an ROR1 antibody.
  • a method of detecting ROR1 antigen in a subject having or suspected of having a disease or condition comprising: (a) administering to the subject any antibody above; and (b) detecting the presence or the level of ROR1 antigen in the subject.
  • a method for treating a subject with cancer comprises administering a therapeutically effective amount of the ADC as disclosed herein or the pharmaceutical composition as disclosed herein to the subject.
  • the cancer is an ROR1 antigen expressing cancer.
  • the disease or condition is cancer.
  • the cancer may be a cancer from the bladder, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), bone, bone marrow, brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), breast, colon, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastrointestinal, gum, head, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia, renal cell carcinoma), liver, lung, na
  • the cancer may be a neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal 176 NAI-1540479824 cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma;
  • the cancer is triple negative breast cancer, non-small cell lung cancer, or mantle cell lymphoma.
  • An ROR1-ADC comprising an ROR1 antibody of the present disclosure may be administered to a subject per se or in the form of a pharmaceutical composition for the treatment of, e.g., cancer, autoimmunity, transplantation rejection, post-traumatic immune responses, graft-versus-host disease, ischemia, stroke, and infectious diseases (e.g., by targeting viral antigens, such as gp120 of HIV).
  • an ROR1-ADC as described herein may be used in a method of treating a subject with cancer in combination with one or more additional therapies.
  • the additional therapies that may be used in combination with an ROR1-ADC described herein include but are not limited to: (i) surgery; (ii) radiotherapy; (iii) endocrine therapy; (iv) immunotherapy (including adjuvant therapy and cell therapy such as CAR T-cell therapy); and (v) chemotherapy, including cytotoxic agents and chemotherapeutic agents.
  • Any therapy that has an activity against a cancer may be used in combination with an ROR1-ADC provided herein. Examples of such agents for cancer treatment can be found, for instance, at www.cancer.gov/about-cancer/treatment/drugs and in publicly available sources such as Cancer Principles and Practice of Oncology by V. T. Devita and S.
  • the additional therapy is a radiotherapy including, for example, gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes.
  • Radiotherapy may comprise 179 NAI-1540479824 radiation or associated administration of radiopharmaceuticals.
  • the source of radiation may be either external or internal to the subject being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)).
  • EBRT external beam radiation therapy
  • BT brachytherapy
  • radioactive elements include, e.g., radium, cesium-137, iridium-192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.
  • the additional therapy is an immunotherapy.
  • Immunotherapy also called biological response modifier therapy, biologic therapy, biotherapy, immune therapy, or biological therapy
  • Immunotherapy is treatment that uses parts of the immune system to fight disease. Immunotherapy can help the immune system recognize cancer cells or enhance a response against cancer cells.
  • Immunotherapies include active and passive immunotherapies.
  • Active immunotherapies including immunotherapeutic agents, stimulate the body's own immune system (e.g., vaccines) while passive immunotherapies, including immunotherapeutic agents, generally use immune system components created outside of the body (e.g., antibodies), antibodies conjugated with drugs, toxins, or radionuclides, and targeted therapeutics.
  • immunotherapeutic agents include immune checkpoint inhibitors.
  • the immune checkpoint inhibitor used in methods of treatment can totally or partially reduce, inhibit, interfere with, or modulate one or more checkpoint proteins which regulate T-cell activation or function.
  • Immune checkpoint inhibitors include antibodies or are derived from antibodies.
  • the checkpoint inhibitor is an OX40 (CD134) agonist.
  • the checkpoint inhibitor is an anti-OX40 antibody.
  • the anti-OX40 antibody is anti-OX-40.
  • the anti-OX40 antibody is MEDI6469.
  • the checkpoint inhibitor is a CD40 agonist.
  • the checkpoint inhibitor is an anti-CD40 antibody. In some embodiments, the anti-CD40 antibody is CF-870,893. [00599] In certain embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti CTLA 4 antibodies include, but are not limited to, those described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238.
  • the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP- 180 NAI-1540479824 675,206).
  • the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101).
  • Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the trade name YervoyTM.
  • the checkpoint inhibitor is a PD-1/PD-L1 inhibitor.
  • PD-l/PD-L1 inhibitors include, but are not limited to, those described in US Patent Nos.7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Patent Application Publication Nos. WO2003042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699.
  • the checkpoint inhibitor is a PD-1 inhibitor.
  • the PD-1 inhibitor is an anti-PD-1 antibody.
  • the anti- PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106) or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab).
  • the anti-PD-1 antibody is nivolumab.
  • Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is marketed under the trade name OpdivoTM.
  • the anti-PD-1 antibody is pembrolizumab.
  • Pembrolizumab is a humanized monoclonal IgG4 antibody and is marketed under the trade name KeytrudaTM.
  • the anti-PD-1 antibody is CT-011, a humanized antibody. CT-011 administered alone has failed to show response in treating acute myeloid leukemia (AML) at relapse.
  • the anti-PD-1 antibody is AMP-224, a fusion protein.
  • the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody in which the ability to bind Fc gamma receptor I is specifically engineered out, and which has a unique binding signature to PD-1 with high affinity and superior target specificity.
  • the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody.
  • the anti- PD-L1 antibody is MEDI4736 (durvalumab). In some embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In some embodiments, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A, and Tecentriq®). [00603] In certain embodiments, the checkpoint inhibitor is a PD-L2 inhibitor. In some embodiments, the PD-L2 inhibitor is an anti-PD-L2 antibody. In some embodiments, the anti- PD-L2 antibody is rHIgM12B7A.
  • the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor.
  • the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007, 179, 4202-4211).
  • the LAG-3 inhibitor is BMS-986016. 181 NAI-1540479824
  • the checkpoint inhibitor is a B7 inhibitor.
  • the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor.
  • the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834).
  • the checkpoint inhibitor is a TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).
  • the checkpoint inhibitor is a GITR agonist. In some embodiments, the checkpoint inhibitor is an anti-GITR antibody.
  • the anti-GITR antibody is TRX518.
  • the checkpoint inhibitor is a CD137 agonist. In some embodiments, the checkpoint inhibitor is an anti-CD137 antibody. In some embodiments, the anti-CD137 antibody is urelumab. In some embodiments, the anti-CD137 antibody is PF- 05082566. [00609] In certain embodiments, the checkpoint inhibitor is recombinant human interleukin- 15 (rhIL-15). [00610] In certain embodiments, the checkpoint inhibitor is an IDO inhibitor. In some embodiments, the IDO inhibitor is INCB024360. In some embodiments, the IDO inhibitor is indoximod.
  • exemplary immunotherapies include adjuvant therapies, including immunotherapeutic agents such as cytokines, chemokines, interferons, interleukins, or lymphokines.
  • immunotherapeutic agents such as cytokines, chemokines, interferons, interleukins, or lymphokines.
  • cytokines such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL- 12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors (including TNF-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide (alum); Bacille Calmette-Guerin (BCG); Keyhole limpet hemocyanin
  • exemplary immunotherapies include cell therapies, for example, a population of immune cells, such as leukocytes (nucleated white blood cells), comprising (e.g., expressing) a receptor that binds to an antigen of interest.
  • a leukocyte of the present disclosure may be, for example, a neutrophil, eosinophil, basophil, lymphocyte, or a 182 NAI-1540479824 monocyte.
  • a leukocyte is a lymphocyte.
  • lymphocytes include T cells, B cells, Natural Killer (NK) cells or NKT cells.
  • a T- cell is a CD4+ Th (T helper) cell, a CD8+ cytotoxic T cell, a ⁇ T cell or a regulatory (suppressor) T cell.
  • an immune cell is a dendritic cell.
  • the cell therapies are CAR-T cell therapies.
  • a bispecific CAR is comprised of two distinct antigen recognition domains present in tandem on a single transgenic receptor (referred to as a TanCAR; see, e.g., Grada Z et al. Molecular Therapy Nucleic Acids 2013; 2:e105, incorporated herein by reference in its entirety).
  • methods comprise delivering to a tumor a combination comprising an ROR1- ADC and an immunotherapeutic agent, wherein the immunotherapeutic agent is an engineered nucleic acid that encodes an antigen, or delivering to a tumor an engineered nucleic acid that induces expression of a self-antigen, and delivering to the tumor an immune cell expressing a bispecific CAR that binds to two antigens, one of which is encoded by the engineered nucleic acid.
  • Other exemplary immunotherapies include immunotherapeutic agents such as cancer vaccines, which can be used to elicit an immune response in a subject against a cancer antigen.
  • An exemplary method involves administering to a subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigenic polypeptide or an immunogenic fragment thereof, in combination with administering an ROR1-ADC either in the same composition or a separate composition, administered at the same time, or sequentially dosed, wherein the anti-antigenic polypeptide antibody titer in the subject is increased following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer.
  • the additional therapies include chemotherapy such as one or more cytotoxic agents or one or more chemotherapeutic agents.
  • a cytotoxic agent can inhibit or prevent a cellular function and/or cause cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • radioactive isotopes e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153
  • the additional therapy includes one or more chemotherapeutic agents.
  • Chemotherapeutic agents include chemical compounds useful in the treatment of cancer.
  • Chemotherapeutic agents include (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iii) anti-androgens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, including those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation; (viii) vaccines such as gene therapy vaccines.
  • Chemotherapeutic agents can also include antibodies.
  • Exemplary kinase inhibitors include erlotinib (Tarceva®), gefitinib (Iressa®), dasatinib (Sprycel®), nilotinib (Tasigna®), crizotinib (Xalkori®), ruxolitinib (Jakafi®), vemurafenib (Zelboraf®), vandetanib (Caprelsa®), pazopanib (Votrient®), afatinib, alisertib, amuvatinib, axitinib, baricitinib, bosutinib, brivanib, canertinib, cabozantinib (Cabometyx®), cediranib, ceritinib, crenolanib, dabrafenib, dacomitinib, danusertib, do
  • an ROR1-ADC is used in combination with a HSP90 inhibitor (e.g., XL888), liver X receptor (LXR) modulators, retinoid-related orphan receptor gamma (RORy) modulators, a CK1 inhibitor, a CK1- ⁇ inhibitor, a Wnt pathway inhibitor (e.g., SST-215), or a mineralocorticoid receptor inhibitor, (e.g., esaxerenone or XL-550) for the treatment of cancer.
  • HSP90 inhibitor e.g., XL888
  • LXR liver X receptor
  • RORy retinoid-related orphan receptor gamma
  • CK1 inhibitor e.g., CK1- ⁇ inhibitor
  • Wnt pathway inhibitor e.g., SST-215
  • mineralocorticoid receptor inhibitor e.g., esaxerenone or XL-550
  • Kinase inhibitors can be tyrosine kinase inhibitors, such as the EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitor such as Mubritonib (TAK165, Takeda); CP- 724.714, (Axon Medchem BV, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI- 166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling;
  • kits comprising an ROR1-ADC provided herein, or a composition (e.g., a pharmaceutical composition) provided herein, packaged into suitable packaging material.
  • a kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • packaging material refers to a physical structure housing the components of the kit.
  • Kits provided herein can include labels or inserts. Labels or inserts include “printed matter,” e.g., paper or cardboard, separate or affixed to a component, a kit or packing 185 NAI-1540479824 material (e.g., a box), or attached to, for example, an ampoule, tube, or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media, or memory type cards. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, and date.
  • Kits provided herein can additionally include other components. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Kits can also be designed for cold storage.
  • a kit can further be designed to contain antibodies provided herein, or cells that contain nucleic acids encoding the antibodies provided herein.
  • the cells in the kit can be maintained under appropriate storage conditions until ready to use.
  • range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise.
  • This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
  • reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth.
  • Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.
  • reference to a range of 25- 250, 250-500, 500-1,000, 1,000-2,500, 2,500-5,000, 5,000-25,000, 25,000-50,000 includes any numerical value or range within or encompassing such values, e.g., 25, 26, 27, 28, 29...250, 251, 252, 253, 254...500, 501, 502, 503, 504..., etc.
  • a series of ranges are disclosed throughout this document. The use of a series of ranges includes combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
  • ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5- 20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth.
  • molecular weight is weight average molecular weight
  • temperature is in degrees Celsius
  • pressure is at or near atmospheric.
  • average is meant the arithmetic mean.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • Curves plotted on top of the sensorgrams in the slides were generated by simulating from the optimal averaged parameters, and the faded bands surrounding these curves display the impact of toggling these parameters by their respective standard deviations.
  • Binding interactions with maximum RU ⁇ 100 were ranked as nonbinders, since this was not significantly above some baseline/bulk shifts.
  • R 2 values were determined from averaging values from the top 3 antigen concentrations used in the determination of the KD. The significance and thresholds for R 2 varied with signal amplitude and intrinsic noise, but for this dataset, R 2 values less than 0.75 denoted increasingly inadequate description of the binding process by one-step kinetic model and therefore the obtained parameters were highly uncertain well outside of the listed standard deviations.
  • Patent No.7,985,783 B2 each of which is incorporated in its entirety herein by reference. Briefly, an antibody containing a heterologous sulfatase motif was contacted with a formylglycine- generating enzyme to convert a cysteine or serine in the motif to a formylglycine having an aldehyde moiety, thereby generating an “aldehyde-tagged antibody.” Any formylglycine- generating enzyme can be used, such as Mycobacterium tuberculosis or Mycobacterium tuberculosis. [00646] Example 3.
  • Linker-Payload Preparation [00647] Synthetic reagents were purchased from Sigma-Aldrich, Acros, AK Scientific, or other commercial sources and used as received. Anhydrous solvents were obtained from commercial sources in sealed bottles. Compound 12 (previously reported in Chuprakov et al. Bioconjugate Chem.2021, 32, 746-754) was obtained commercially from Shanghai Medicilon and used without purification. Belotecan 13 as well as compounds 16 and 17 were obtained from commercial sources and used as received. Column chromatography was performed using a Biotage Isolera or Biotage Selekt chromatography system.
  • LRMS Low-resolution mass spectra
  • Agilent Technology 6120 Quadrupole LC/MS equipped with Agilent 1260 Infinity HPLC system, G1314 variable wavelength detector, and Agilent Poroshell 120 SB C18, 4.6 mm x 50 mm column at room temperature using 10-100% gradient of water and acetonitrile containing 0.1% formic acid.
  • HPLCs were monitored at 254 or 205 nm.
  • Belotecan hydrochloride 13 (2.35 g, 5.0 mmol) was suspended in a mixture of 30 mL of anhydrous DMF and 1.75 mL of DIPEA (10 mmol). The resulting mixture was stirred and treated with HOAt (0.68 g, 5 mmol), followed by PNP-carbonate 12 (5.1 g, 5 mmol) in small portions at room temperature. Reaction mixture was stirred at RT for 8 h until starting materials were judged fully consumed based on HPLC analysis.
  • Reaction mixture was stirred at 0 °C for 1 h, then another 10 mL of 2 M LiOH solution was added and stirring continued for 15 minutes before warming the reaction mixture to room temperature and adding another 10 mL of 2 M lithium hydroxide and 5 mL of methanol.
  • the resulting mixture was stirred for 1 h at room temperature, then quenched by adding 2 M aqueous HCl solution to pH 2-3 and let stir for 30 minutes.
  • the mixture was transferred to a separatory funnel and washed with MTBE (2x50 mL). Aqueous layer was separated and directly purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA).
  • reaction mixture was treated with a separately prepared solution of bis-PFP ester 11 ester (1.0 g, 1.1 mmol in 2 mL of DMA) in a few small portions with 10 minutes intervals between additions. After the addition was complete, reaction mixture was stirred for 15 minutes at room temperature and treated with 2.1 mL of piperidine (22 mmol). After 20 minutes, reaction mixture was directly purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA).
  • ion exchange chromatography is further used to purify the ROR1-ADC samples.
  • DAR drug-to-antibody ratio
  • PLRP polymeric reverse phase chromatography
  • the HIC column (Tosoh Bioscience, Cat. No.14947) was run with 1.5 M ammonium sulfate and 25 mM sodium phosphate (pH 7.0) as mobile phase A and 25% isopropanol and 18.75 mM sodium phosphate (pH 7.0) as mobile phase B.
  • the PLRP column (Agilent, Cat. No.
  • HIC Hydrophobic Interaction Chromatography
  • Reverse Phase HPLC was performed as described below: Samples were analyzed on an Agilent 1100 Instrument equipped with a 50 x 2.1 mm PL1912- 1802 (8 ⁇ m, 1000 ⁇ ) column from Agilent held at 80 °C. Samples were prepared by adding 10 ⁇ L of a mixture of 0.5 mM DTT, 50 ⁇ L of 8 M guanidine HCl, 130 mM tris, and 1 mM EDTA (pH 7.6) to a 20 ⁇ g sample and adding PBS to a final volume of 100 ⁇ L.
  • Example 5 In Vitro Cytotoxicity [00688] In vitro cytotoxicity of the ADC prepared as described in Examples 1-4 was further assessed. [00689] hROR1 expressing HEK cells (1500 cells/well in 100 ⁇ L of media) were plated in Costar (Corning) 3610 plate (individually packed, sterile, white wall clear bottom, TC treated, VWR# 29444-010) and incubated overnight.
  • Dilution series of the tested articles (such as, ADC-8) were made. Belotecan and isotype control (FITC antibody conjugated to the ADC-8 linker payload) were tested in parallel and served as controls.
  • the final top concentration was 100 nM for free drugs and 10 nM for ADC-8.
  • ADC-8 was serially diluted 1:4.
  • Starting with a top concentration of 600 nM free drug or 60 nM ADC (6x the final top concentration) each of the samples was serially diluted 5 times (at 1:6 for free drug and 1:4 for ADC).20 ⁇ L of each sample were added to 100 ⁇ L cells.
  • ADC In Vivo Efficacy in MDA-MB-231 TNBC Xenograft Model [00693] CB.17 female SCID mice were implanted (flank) with 1.0 x 10 7 of MDA-MB-231 tumor cells in 50% MATRIGEL® (Corning Life Sciences) matrix (1:1 cell to Matrigel) on Day -13. When the average tumor volume reached about 200 mm 3 on Day 1, mice were randomized into respective treatment groups (10 mice per group). The mice then received intravenous injections of the tested ADC (at 7.5 mg/kg) on Day 1 and Day 8 (Q7D x 2).
  • Control FITC ADC (FITC conjugated to the ADC-8 linker-payload), and a benchmark ADC (which comprises a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1 (CAS # 1643432-38-5), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE)) were tested in parallel, serving as controls. 200 NAI-1540479824 Body weight and tumor volume were measured twice per week. The study was ended on Day 45. [00694] Tumor volumes were measured and %TGI was calculated.
  • TGITV% [1-(Ti-T0)/(Vi-V0)] ⁇ 100%.
  • Ti means tumor volume of the treated groups at day i following treatment
  • T0 means tumor volume of the treated groups at day 0
  • V i means tumor volume of the vehicle groups at day i following treatment
  • V 0 means tumor volume of the vehicle groups at day 0
  • %TGI result was analyzed with One Way ANOVA with Dunnett’s post hoc test as appropriate, while **** indicated p ⁇ 0.0001 and NS indicated p > 0.05.
  • ADC In Vivo Efficacy in JEKO-1 MCL Xenograft Model
  • CB.17 female SCID mice were inoculated subcutaneously in the right up flank region with Jeko-1 tumor cells (5 ⁇ 10 6 ) in 0.1 ml of PBS with Matrigel (1:1) for tumor development. When the tumor volumes reached about 150 ⁇ 250 mm 3 on Day 1, mice were randomized into respective treatment groups (8 mice per group). The mice then received intravenous injections of vehicle or the tested ADC (7.5 mg/kg) on Day 1 and Day 8.
  • Control FITC ADC (FITC conjugated to the ADC-8 linker-payload), and a benchmark ADC (which comprises a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1 (CAS # 1643432-38-5), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE)) were tested in parallel, serving as controls.
  • MMAE antimicrotubule cytotoxin monomethyl auristatin E
  • TGITV% [1-(Ti-T0)/(Vi-V0)] ⁇ 100%.
  • T i means tumor volume of the treated groups at day i following treatment
  • T 0 means tumor volume of the treated groups at day 0
  • Vi means tumor volume of the vehicle groups at day i following treatment
  • V0 means tumor volume of the vehicle groups at day 0
  • the %TGI result was analyzed with One Way ANOVA with Dunnett’s post hoc test as appropriate, while **** indicated p ⁇ 0.0001.
  • ADC In Vivo Efficacy in Patient-Derived Xenograft (PDX) Models [00703] CB.17 female SCID mice are implanted (flank) with patient tumor cells in 50% MATRIGEL® (Corning Life Sciences) matrix (1:1 cell to Matrigel). When the average tumor volume reaches about 200 mm 3 on Day 1, mice are randomized into respective treatment groups and receive intravenous injections of vehicle or the tested ADC (10 mg/kg) every three weeks (Q3W). Tumor volumes are measured and %TGI is calculated.
  • TGITV% [1-(Ti-T0)/(Vi-V0)] ⁇ 100%.
  • Ti means tumor volume of the treated groups at day i following treatment
  • T 0 means tumor volume of the treated groups at Day 0
  • V i means tumor volume of the vehicle groups at day i following treatment
  • V 0 means tumor volume of the vehicle groups at day 0
  • the %TGI result is analyzed with One Way ANOVA with Dunnett’s post hoc test as appropriate.
  • female athymic nude-Foxn1nu mice were used.
  • NSCLC non-small cell lung cancer
  • ADC Pharmacokinetics Study in Rats [00709] Sprague-Dawley rats were dosed intravenously with a single dose of 5 mg/kg of the tested ADC on Day 0 after 16 hours of fasting. The rats’ body weights were measured on Day -3. Day 0, Day 7, Day 14, and Day21.100 ⁇ L plasma was collected at 30 min (on Day 0), 6 h (on Day 0), 24 h (on Day 1), 48 h (on Day 2), Day 4, Day 7, Day 10, and Day14, saved in bullet tubes with K 2 EDTA, and stored at -20 °C until end of the study.
  • tAb and tADC Total antibody and total ADC concentrations (referred to herein as tAb and tADC, respectively) were quantified using MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (having an assay MRD of 1:100 and a quantitation range of 10 ng/mL (LLOQ) – 10240 ng/mL (ULOQ)). A 1000X dilution linearity was used, while the assay controls included 34 ng/mL (LQC), 800 ng/mL (MQC), 8000 ng/mL (HQC).
  • conjugates were captured with an anti-human IgG (Fc specific), F(ab′) 2 fragment, highly cross adsorbed-Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 ⁇ g/mL and detected with a Sulfo TAG-labeled goat anti- human antibody (R32AJ) at 0.15 ⁇ g/mL.
  • Fc specific anti-human IgG
  • F(ab′) 2 fragment highly cross adsorbed-Biotin antibody produced in goat
  • R32AJ Sulfo TAG-labeled goat anti- human antibody
  • conjugates were captured with an anti-human IgG (Fc specific), F(ab′) 2 fragment, highly cross adsorbed- Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 ⁇ g/mL and detected with a Sulfo TAG-labeled anti-Belotecan (Clone 1H11, Catalent) at 1 ⁇ g/mL.
  • Fc specific anti-human IgG
  • F(ab′) 2 fragment highly cross adsorbed- Biotin antibody produced in goat
  • SAB37012678 Sulfo TAG-labeled anti-Belotecan
  • Pooled Sprague Dawley rat plasma was used as a negative control (NC).
  • the assay buffer used was 1xPBS+1%BSA+0.1%Tween, while the read buffer was 1X MSD Read Buffer T (MSD R92TC-1). Data were analyzed and plotted using GraphPad Prism.
  • ADC-8 displayed good in vivo stability when administered at a single dose of 5 mg/kg. 203 NAI-1540479824 Table E5. Rat PK results.
  • Example 10 ADC: Toxicity and Toxicokinetics (TK) Study in Rats [00714] Female Sprague-Dawley rats were dosed intravenously with the tested ADC via i.v. bolus once per week for four weeks (Q1W x 4) as detailed in Table E6 below.100 ⁇ L plasma was collected at various time points post-dose, saved in bullet tubes with EDTA and stored at -20 °C until end of the study.
  • tAb and tADC were quantified using MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (having an assay MRD of 1:100 and a quantitation range of 10 ng/mL (LLOQ) – 10240 ng/mL (ULOQ)).
  • conjugates were captured with an anti-human IgG (Fc specific), F(ab′) 2 fragment, highly cross adsorbed-Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 ⁇ g/mL and detected with a Sulfo TAG-labeled goat anti- human antibody (R32AJ) at 0.15 ⁇ g/mL.
  • Fc specific anti-human IgG
  • F(ab′) 2 fragment highly cross adsorbed-Biotin antibody produced in goat
  • R32AJ Sulfo TAG-labeled goat anti- human antibody
  • conjugates were captured with an anti-human IgG (Fc specific), F(ab′) 2 fragment, highly cross adsorbed- Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 ⁇ g/mL and detected 204 NAI-1540479824 with a Sulfo TAG-labeled anti-Belotecan (Clone 1H11, Catalent) at 1 ⁇ g/mL.
  • Fc specific anti-human IgG
  • F(ab′) 2 fragment highly cross adsorbed- Biotin antibody produced in goat
  • SAB3701268 Sulfo TAG-labeled anti-Belotecan
  • Sulfo TAG-labeled anti-Belotecan Clone 1H11, Catalent
  • ADC-8 was well-tolerated and having a projected maximum tolerated dose (MTD) of ⁇ 60 mg/kg, while ADC-4 was also well-tolerated and having an MTD of > 90 mg/kg.
  • MTD projected maximum tolerated dose
  • FIG.5A ADC-8 exhibited a linear PK profile, while the total mAb and total ADC curves overlapped, suggesting good in vivo stability.
  • This TK analysis further showed that following the Q1W x 4 administration, there was accumulation with AR (accumulation ratios) of 1.94 to 2.0.
  • ADC-4 also showed that total ADC and total antibody were overlapped, suggesting stable ADC.

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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 [00001] 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 [00002] 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 [00003] 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 [00004] 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-1540479824 antibody 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. [00005] 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. [00006] 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 [00007] 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. [00008] 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. [00009] 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. [00010] 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. [00011] 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-1540479824 molecule 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. [00012] 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. [00013] 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. [00014] 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 linker
Figure imgf000005_0001
wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3, and R4 are each selected from hydrogen and alkyl; LA is a first linker comprising: -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-(T5-V5)e-(T6-V6)f-, 3 NAI-1540479824 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 T6 are 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 V6 are 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 R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LB is 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 T13 are 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-1540479824 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 V13 are 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 R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; s is an integer from 1 to 10; W1 is a first drug; and W2 is a second drug. [00015] In some embodiments, Z1 is CR4. [00016] In some embodiments, Z3 is C-LB-W2. [00017] In some embodiments, one or both of W1 and W2 are camptothecin analogues, for example, belotecan. [00018] In some embodiments, LA comprises: -(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 T6 are 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-1540479824 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 V6 are 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 R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00019] In some embodiments of LA: T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6 are 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 V6 are 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:
Figure imgf000008_0001
integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000008_0002
integer from 1 to 6 and r is 0 or 1; 6 NAI-1540479824 4-amino-piperidine each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups 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. [00020] In some embodiments, T1, T2, T3, T4, T5, and T6 are each optionally substituted with a glycoside. [00021] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. [00022] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [00023] In some embodiments, LA is a linker wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABC and V4 is 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. [00024] 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. [00025] In some embodiments, LB comprises: -(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 T13 are 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 V13 are 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 R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00026] In some embodiments, T7, T8, T9, T10, T11, T12, and T13 are each optionally substituted with a glycoside. [00027] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. [00028] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [00029] In some embodiments of LB: T7 is a covalent bond; T8, T9, T10, T11, and T12 are 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 V12 are 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-1540479824 wherein:
Figure imgf000011_0001
integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000011_0002
each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups 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. [00030] In some embodiments, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12 are each optionally substituted with a glycoside. [00031] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. [00032] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [00033] In some embodiments, LB is a linker wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is substituted (C1-C12)alkyl and V9 is -CO-; T10 is (AA)p and V10 is absent (e.g., a covalent bond); T11 is PABC and V11 is 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. [00034] 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. [00035] In some embodiments, an ROR1-ADC is represented by Formula (I): 9 NAI-1540479824 wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3 and R4 are each selected from hydrogen and (C1-C12)alkyl; LA is a first linker wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is (AA)p where p is an integer from 1-20 and V3 is a covalent bond; T4 is PABC and V4 is a covalent bond; a, b, c, and d are each 1; e and f are each 0; and LB is a second linker wherein: T7 is a covalent bond and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is substituted (C1-C12)alkyl and V9 is -CO-; T10 is (AA)p where p is an integer from 1-20 and V10 is a covalent bond; T11 is PABC and V11 is 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; W1 is a first drug; and W2 is a second drug. [00036] In some embodiments, one or both of W1 and W2 are camptothecin analogues, for example, belotecan. [00037] In some embodiments, an ROR1-ADC is represented by Formula (I): 10 NAI-1540479824 wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3 and R4 are each selected from hydrogen and (C1-C12)alkyl; LA is a linker wherein: T1 is (C1-C6)alkyl and V1 is -CONH-; T2 is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is
Figure imgf000013_0001
integer from 2 to 10, optionally 8, and V2 is -CO-; T3 is (AA)2 and V3 is a covalent bond; T4 is PABC substituted with a glycoside and V4 is a covalent bond; a, b, c, and d are each 1; and e and f are each 0; and LB is a linker wherein: T7 is a covalent bond and V7 is -NHCO-; T8 is (C1-C6)alkyl and V8 is -CONH-; T9 is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is
Figure imgf000013_0002
integer from 2 to 10, optionally 8, and V9 is -CO-; T10 is (AA)2 and V10 is a covalent bond; T11 is PABC substituted with a glycoside and V11 is 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-1540479824 W1 is a first drug; and W2 is a second drug. [00038] In some embodiments, the PABC of one or both of T4 and T11 is substituted with a glucuronide. In some embodiments, one or both of T1 and T8 is ethyl. In some embodiments, one or both of T2 and T9 is C5 alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is
Figure imgf000014_0001
integer from 5-10, optionally 8. In some embodiments, one or both of W1 and W2 are camptothecin analogues, for example, belotecan. [00039] In some embodiments, an ROR1-ADC is represented by Formula (II):
Figure imgf000014_0002
wherein: Ab represents the antibody that binds to ROR1; and s is an integer from 1 to 10. [00040] In some embodiments, s is an integer from 1 to 8. In some embodiments, s is 2. In some embodiments, s is 4. [00041] Formula (II) may be prepared by conjugating one or more linker-payloads of Formula (IIa), shown below, with an ROR1 antibody: 12 NAI-1540479824
[00042] 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. [00043] 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. [00044] 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. [00045] 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-1540479824 framework 4 (FR4) sequence, for example, as set forth in any one of SEQ ID NOs: 25 and 26. [00046] In some embodiments, an ROR1-ADC is represented by Formula (I) or (II) wherein Ab comprises human framework sequences. [00047] 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. [00048] 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. [00049] 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. [00050] 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. [00051] 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-1540479824 ADC of Formula (I) or (II) and a pharmaceutically acceptable excipient, wherein the ROR1 antibody is as described in any embodiment herein. [00052] 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 [00053] FIG.1 provides exemplary in vitro cytotoxicity results as detailed in Example 5. [00054] 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. [00055] 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. [00056] FIG.4 provides exemplary pharmacokinetics (PK) results in rats as detailed in Example 9. [00057] 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. [00058] 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 [00059] 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 [00060] 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-1540479824 Sambrook 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. [00061] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art-recognized meanings. [00062] “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-). [00063] The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1 carbon 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. [00064] “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. [00065] “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. [00066] The term “alkane” refers to alkyl group and alkylene group, as defined herein. [00067] 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. [00068] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein. [00069] “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. [00070] 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. [00071] The term “alkoxyamino” refers to the group –NH-alkoxy, wherein alkoxy is defined herein. [00072] 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. [00073] 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. [00074] 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 [00075] 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. [00076] “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. [00077] 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. [00078] “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). [00079] 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. [00080] “Alkynyloxy” refers to the group –O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like. [00081] “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)- [00082] “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 R20 is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [00083] “Aminocarbonyl” or the term “aminoacyl” refers to the group -C(O)NR21R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [00084] “Aminocarbonylamino” refers to the group –NR21C(O)NR22R23 where R21, R22, and R23 are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group. [00085] 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 [00086] 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. [00087] “Aminosulfonyl” refers to the group –SO2NR21R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21 and R22 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein. [00088] “Sulfonylamino” refers to the group –NR21SO2R22, wherein R21 and R22 independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21 and R22 are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [00089] “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-1540479824 amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2- aryl, -SO2-heteroaryl and trihalomethyl. [00090] “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. [00091] “Amino” refers to the group –NH2. [00092] 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. [00093] The term “azido” refers to the group –N3. [00094] “Carboxyl,” “carboxy” or “carboxylate” refers to –CO2H or salts thereof. [00095] “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. [00096] “(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-1540479824 cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. [00097] “Cyano” or “nitrile” refers to the group –CN. [00098] “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. [00099] 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. [00100] “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. [00101] 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. [00102] “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. [00103] “Cycloalkoxy” refers to –O-cycloalkyl. [00104] “Cycloalkenyloxy” refers to –O-cycloalkenyl. 22 NAI-1540479824 [00105] “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo. [00106] “Hydroxy” or “hydroxyl” refers to the group –OH. [00107] “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. [00108] 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. [00109] “Heteroaryloxy” refers to –O-heteroaryl. [00110] “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-1540479824 oxide, -S(O)-, or -SO2- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein. [00111] 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. [00112] 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. [00113] “Heterocyclyloxy” refers to the group –O-heterocyclyl. [00114] The term “heterocyclylthio” refers to the group heterocyclic-S-. [00115] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein. [00116] The term “hydroxyamino” refers to the group -NHOH. [00117] “Nitro” refers to the group –NO2. [00118] “Oxo” refers to the atom (=O). [00119] “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-1540479824 substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4- methylphenyl-SO2-. [00120] “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. [00121] “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. [00122] 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. [00123] “Thiol” refers to the group -SH. [00124] “Thioxo” or the term “thioketo” refers to the atom (=S). [00125] “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. [00126] The term “substituted thioalkoxy” refers to the group -S-substituted alkyl. [00127] 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 [00128] 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. [00129] 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. [00130] 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. [00131] 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, =N2 or =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)R70 and -NR70C(NR70)NR80R80, where R60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70 is independently hydrogen or R60; each R80 is independently R70 or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3 alkyl substitution; and each M+ is a counter ion with a net single positive charge. Each M+ may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as +N(R60)4; or an alkaline earth ion, such as [Ca2+]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-1540479824 examples, -NR80R80 is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N- methyl-piperazin-1-yl and N-morpholinyl. [00132] 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)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+. [00133] 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)R70 and -NR70C(NR70)NR80R80, where R60, R70, R80 and M+ are as previously defined. [00134] 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. [00135] 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 [00136] 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)-. [00137] 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. [00138] 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. [00139] 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. [00140] “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 [00141] “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. [00142] “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. [00143] 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. [00144] “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. [00145] 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). [00146] 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. [00147] “Patient” refers to human and non-human subjects, especially mammalian subjects. [00148] 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-1540479824 development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient. [00149] In some embodiments, the term “treating,” or “treatment” excludes a prophylactic treatment. [00150] 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. [00151] “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. [00152] “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. [00153] 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. [00154] The term “subject” refers to human and non-human subjects, especially mammalian subjects. [00155] 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. [00156] 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-1540479824 stereochemistry 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. [00157] 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. [00158] 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. [00159] 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. [00160] 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-1540479824 embodiments, 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). [00161] Thus, in one embodiment, the term “ROR1” or “ROR1 antigen” as used herein refers to ROR1 (e.g., human ROR1). [00162] 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-1540479824 and 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. [00163] 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. [00164] 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. [00165] 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. [00166] 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-1540479824 bispecific antibody may bind to two different epitopes on the same antigen (e.g., epitopes on ROR). [00167] 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. [00168] 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. [00169] 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. [00170] 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. [00171] 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-1540479824 requires 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. [00172] 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. [00173] 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. [00174] 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 KA that 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-1540479824 that 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 KD of 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.” [00175] 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 “KD value” 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-1540479824 measured 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. [00176] 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-1540479824 occur (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. [00177] 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. [00178] 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. [00179] 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 [00180] 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. [00181] 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. [00182] 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”)). [00183] 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”). [00184] 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-1540479824 CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALKAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:39). [00185] 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”) [00186] 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. [00187] 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). [00188] 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 [00189] 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. [00190] 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. [00191] 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. [00192] 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 [00193] 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). [00194] 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-1540479824 binding 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. [00195] 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. [00196] 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-1540479824 present 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.
Figure imgf000048_0001
[00197] 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. [00198] 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. [00199] 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 [00200] 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. [00201] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. [00202] 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. [00203] 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). [00204] 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. [00205] 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)). [00206] 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-1540479824 cyclophosphamide (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. [00207] 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. [00208] 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. [00209] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine. [00210] 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. [00211] 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-1540479824 medroxyprogesterone 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. [00212] 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. [00213] 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). [00214] 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-1540479824 taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No.5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No.5,821,263; and taxol derivative described in U.S. Patent No.5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No.5,824,701. [00215] 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. [00216] 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. [00217] 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. [00218] 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-1540479824 antimitotic agent, such as an auristatin or an active auristatin analog or derivative thereof. In some embodiments, the drug is a DNA alkylating agent. [00219] 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. [00220] “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-1540479824 formulations, 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. [00221] 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. [00222] 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. [00223] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise. [00224] 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. [00225] 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 [00226] 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. [00227] 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). [00228] 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. [00229] 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. [00230] 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. [00231] 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-1540479824 such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. [00232] 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. [00233] 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. [00234] 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 [00235] 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. [00236] 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-1540479824 antibody (e.g., at an internal site of the antibody). Combinations of the above conjugation sites are also possible. [00237] 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). [00238] 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. [00239] 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-1540479824 antibody. 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. [00240] 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. [00241] 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.
Figure imgf000058_0001
56 NAI-1540479824 [00242] 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. [00243] 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 R21 and R22 are each independently selected from any of the substituents described for R’ and R” above. [00244] 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. [00245] 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-1540479824 drug, 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. [00246] 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. [00247] 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. [00248] 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). [00249] 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. [00250] 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-1540479824 imaging agent or tracer to detect the location of the ADC in vivo, while a second payload may be a drug or active agent that provides a therapeutic activity. [00251] 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. [00252] 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 [00253] 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’. [00254] 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). [00255] 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 [00256] Aspects of the present disclosure include a conjugate of Formula (I):
Figure imgf000063_0001
wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, Z3 and Z4 are each independently selected from CR4, N and C-LB-W2, wherein at least one Z1, Z2, Z3 and Z4 is C-LB-W2; R1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R2 and R3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, 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 R2 and R3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl; each R4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LA is a first linker; LB is a second linker; s is an integer from 1 to 10; W1 is a first drug; and 61 NAI-1540479824 W2 is a second drug. [00257] The substituents related to conjugates of Formula (I) are described in more detail below. [00258] In certain embodiments, Z1, Z2, Z3 and Z4 are each independently selected from CR4, N and C-LB-W2, wherein at least one Z1, Z2, Z3 and Z4 is C-LB-W2. In certain embodiments, Z1 is CR4. In certain embodiments, Z1 is N. In certain embodiments, Z1 is C- LB-W2. In certain embodiments, Z2 is CR4. In certain embodiments, Z2 is N. In certain embodiments, Z2 is C-LB-W2. In certain embodiments, Z3 is CR4. In certain embodiments, Z3 is N. In certain embodiments, Z3 is C-LB-W2. In certain embodiments, Z4 is CR4. In certain embodiments, Z4 is N. In certain embodiments, Z4 is C-LB-W2. In some embodiments, each of Z1, Z3, and Z4 is CR4. In some embodiments, Z3 is C-LB-W2. [00259] Combinations of various Z1, Z2, Z3 and Z4 are possible. For example, in some instances, Z1 is C-LB-W2, Z2 is CR4, Z3 is CR4, and Z4 is CR4. In some instances, Z1 is CR4, Z2 is C-LB-W2, Z3 is CR4, and Z4 is CR4. In some instances, Z1 is CR4, Z2 is CR4, Z3 is C-LB- W2, and Z4 is CR4. In some instances, Z1 is CR4, Z2 is CR4, Z3 is CR4, and Z4 is C-LB-W2. [00260] In certain embodiments, R1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl. In certain embodiments, R1 is hydrogen. In certain embodiments, R1 is 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, R1 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R1 is alkynyl or substituted alkynyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R1 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R1 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R1 is 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 C3-5 substituted cycloalkyl. In certain embodiments, R1 is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl or C3-8 substituted heterocyclyl, such as a 62 NAI-1540479824 C3-6 heterocyclyl or C3-6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl. [00261] In certain embodiments, R2 and R3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, 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 R2 and R3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl. [00262] In certain embodiments, R2 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R2 is hydrogen. In certain embodiments, R2 is 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, R2 is methyl. In certain embodiments, R2 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R2 is alkynyl or substituted alkynyl. In certain embodiments, R2 is alkoxy or substituted alkoxy. In certain embodiments, R2 is amino or substituted amino. In certain embodiments, R2 is carboxyl or carboxyl ester. In certain embodiments, R2 is acyl or acyloxy. In certain embodiments, R2 is acyl amino or amino acyl. In certain embodiments, R2 is alkylamide or substituted alkylamide. In certain embodiments, R2 is sulfonyl. In certain embodiments, R2 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R2 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R2 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R2 is 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 C3-5 substituted cycloalkyl. In certain embodiments, R2 is heterocyclyl or substituted heterocyclyl, such as a C3-6 heterocyclyl or C3- 6 substituted heterocyclyl, or a C3-5 heterocyclyl or C3-5 substituted heterocyclyl. 63 NAI-1540479824 [00263] In certain embodiments, R3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R3 is hydrogen. In certain embodiments, R3 is 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, R3 is methyl. In certain embodiments, R3 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R3 is alkynyl or substituted alkynyl. In certain embodiments, R3 is alkoxy or substituted alkoxy. In certain embodiments, R3 is amino or substituted amino. In certain embodiments, R3 is carboxyl or carboxyl ester. In certain embodiments, R3 is acyl or acyloxy. In certain embodiments, R3 is acyl amino or amino acyl. In certain embodiments, R3 is alkylamide or substituted alkylamide. In certain embodiments, R3 is sulfonyl. In certain embodiments, R3 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R3 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R3 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R3 is 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 C3-5 substituted cycloalkyl. In certain embodiments, R3 is 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. [00264] In certain embodiment, both R2 and R3 are methyl. [00265] In certain embodiments, R2 and R3 are optionally cyclically linked to form a 5 or 6- membered heterocyclyl. In certain embodiments, R2 and R3 are cyclically linked to form a 5 or 6-membered heterocyclyl. In certain embodiments, R2 and R3 are cyclically linked to form a 5-membered heterocyclyl. In certain embodiments, R2 and R3 are cyclically linked to form a 6-membered heterocyclyl. [00266] In certain embodiments, each R4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, 64 NAI-1540479824 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. [00267] The various possibilities for each R4 are described in more detail as follows. In certain embodiments, R4 is hydrogen. In certain embodiments, each R4 is hydrogen. In certain embodiments, R4 is halogen, such as F, Cl, Br, or I. In certain embodiments, R4 is F. In certain embodiments, R4 is Cl. In certain embodiments, R4 is Br. In certain embodiments, R4 is I. In certain embodiments, R4 is alkyl or substituted alkyl, such as 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, R4 is methyl. In certain embodiments, R4 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R4 is alkynyl or substituted alkynyl. In certain embodiments, R4 is alkoxy or substituted alkoxy. In certain embodiments, R4 is amino or substituted amino. In certain embodiments, R4 is carboxyl or carboxyl ester. In certain embodiments, R4 is acyl or acyloxy. In certain embodiments, R4 is acyl amino or amino acyl. In certain embodiments, R4 is alkylamide or substituted alkylamide. In certain embodiments, R4 is sulfonyl. In certain embodiments, R4 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R4 is aryl or substituted aryl, such as 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 (e.g., phenyl or substituted phenyl). In certain embodiments, R4 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5-8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R4 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3- 8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R4 is 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. [00268] In certain embodiments, LA is a first linker. Examples of linkers that can be used in the conjugates of the present disclosure are described in more detail below. [00269] In certain embodiments, LB is 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 [00270] In certain embodiments, W1 is 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. [00271] In certain embodiments, W2 is 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. [00272] 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. [00273] 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. [00274] For example, as shown in Formula (I) above, LA is attached to the antibody Ab through a conjugation moiety, and thus the antibody Ab is indirectly bonded to the linker LA through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As described above, the antibody Ab is an ROR1 antibody, and thus LA is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the ROR1 antibody, e.g., the linker LA is indirectly bonded to the ROR1 antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. [00275] Any convenient linker may be utilized for the first linker LA in the subject conjugates and compounds. In certain embodiments, the first linker LA may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the first linker LA may include an alkyl or substituted alkyl group. In certain embodiments, the first linker LA may include an alkenyl or substituted alkenyl group. 66 NAI-1540479824 In certain embodiments, the first linker LA may include an alkynyl or substituted alkynyl group. In certain embodiments, the first linker LA may include an alkoxy or substituted alkoxy group. In certain embodiments, the first linker LA may include an amino or substituted amino group. In certain embodiments, the first linker LA may include a carboxyl or carboxyl ester group. In certain embodiments, the first linker LA may include an acyl amino group. In certain embodiments, the first linker LA may include an alkylamide or substituted alkylamide group. In certain embodiments, the first linker LA may include an aryl or substituted aryl group. In certain embodiments, the first linker LA may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the first linker LA may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the first linker LA may include a heterocyclyl or substituted heterocyclyl group. [00276] In certain embodiments, the first linker LA may include a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where 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. [00277] In some embodiments, LA is a first linker described by the formula:
Figure imgf000069_0001
wherein L1, L2, L3, L4, L5 and L6 are 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. [00278] 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 [00279] In certain embodiments, the linker subunit L1 is 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. [00280] 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, L5 and 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). [00281] 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, L1 comprises a polyethylene glycol. In some embodiments, L1 comprises a modified polyethylene glycol. In some embodiments, L1 comprises an amino acid residue. In some embodiments, L1 comprises an alkyl group or a substituted alkyl. In some embodiments, L1 comprises an aryl group or a substituted aryl group. In some embodiments, L1 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00282] 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, L2 comprises a polyethylene glycol. In some embodiments, L2 comprises a modified polyethylene glycol. In some embodiments, L2 comprises an amino acid residue. In some embodiments, L2 comprises an alkyl group or a substituted alkyl. In some embodiments, L2 comprises an aryl group or a substituted aryl group. In some embodiments, L2 comprises a diamine (e.g., a linking group comprising an alkylene diamine). 68 NAI-1540479824 [00283] 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, L3 comprises a polyethylene glycol. In some embodiments, L3 comprises a modified polyethylene glycol. In some embodiments, L3 comprises an amino acid residue. In some embodiments, L3 comprises an alkyl group or a substituted alkyl. In some embodiments, L3 comprises an aryl group or a substituted aryl group. In some embodiments, L3 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00284] 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, L4 comprises a polyethylene glycol. In some embodiments, L4 comprises a modified polyethylene glycol. In some embodiments, L4 comprises an amino acid residue. In some embodiments, L4 comprises an alkyl group or a substituted alkyl. In some embodiments, L4 comprises an aryl group or a substituted aryl group. In some embodiments, L4 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00285] 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, L5 comprises a polyethylene glycol. In some embodiments, L5 comprises a modified polyethylene glycol. In some embodiments, L5 comprises an amino acid residue. In some embodiments, L5 comprises an alkyl group or a substituted alkyl. In some embodiments, L5 comprises an aryl group or a substituted aryl group. In some embodiments, L5 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00286] 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, L6 comprises a polyethylene glycol. In some embodiments, L6 comprises a modified polyethylene glycol. In some embodiments, L6 comprises an amino acid residue. In some embodiments, L6 comprises an alkyl group or a substituted alkyl. In some embodiments, L6 comprises an aryl group or a substituted aryl group. In some embodiments, L6 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00287] In some embodiments, LA is a first linker comprising: -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-(L6)f-, 69 NAI-1540479824 wherein:
Figure imgf000072_0001
-(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. [00288] 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. [00289] As described above, in certain embodiments, L1 is 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, T1 is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). In certain embodiments, V1 is 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-1540479824 embodiments, 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. [00290] 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. [00291] For example, as shown in Formula (I) above, LB is attached to the antibody Ab through a conjugation moiety, and thus the antibody Ab is indirectly bonded to the second linker LB through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. As described above, the antibody Ab is an ROR1 antibody, and thus LB is attached through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety to the ROR1 antibody, e.g., the linker LB is indirectly bonded to the ROR1 antibody through the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety. [00292] Any convenient linker may be utilized for the second linker LB in the subject conjugates and compounds. In certain embodiments, the second linker LB may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl amino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, the second linker LB may include an alkyl or substituted alkyl group. In certain embodiments, the second linker LB may include an alkenyl or substituted alkenyl group. In certain embodiments, the second linker LB may include an alkynyl or substituted alkynyl group. In certain embodiments, the second linker LB may include an alkoxy or substituted alkoxy group. In certain embodiments, the second linker LB may include an amino or substituted amino group. In certain embodiments, the second linker LB may include a 71 NAI-1540479824 carboxyl or carboxyl ester group. In certain embodiments, the second linker LB may include an acyl amino group. In certain embodiments, the second linker LB may include an alkylamide or substituted alkylamide group. In certain embodiments, the second linker LB may include an aryl or substituted aryl group. In certain embodiments, the second linker LB may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the second linker LB may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the second linker LB may include a heterocyclyl or substituted heterocyclyl group. [00293] In certain embodiments, the second linker LB may include a polymer. For example, the polymer may include a polyalkylene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol (e.g., where 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. [00294] In some embodiments, LB is a second linker described by the formula:
Figure imgf000074_0001
wherein L7, L8, L9, L10, L11, L12 and L13 are 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. [00295] 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. [00296] In certain embodiments, the linker subunit L7 is attached to the hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). 72 NAI-1540479824 In 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. [00297] 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, L12 and 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). [00298] 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, L7 comprises a polyethylene glycol. In some embodiments, L7 comprises a modified polyethylene glycol. In some embodiments, L7 comprises an amino acid residue. In some embodiments, L7 comprises an alkyl group or a substituted alkyl. In some embodiments, L7 comprises an aryl group or a substituted aryl group. In some embodiments, L7 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00299] 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, L8 comprises a polyethylene glycol. In some embodiments, L8 comprises a modified polyethylene glycol. In some embodiments, L8 comprises an amino acid residue. In some embodiments, L8 comprises an alkyl group or a substituted alkyl. In some embodiments, L8 comprises an aryl group or a substituted aryl group. In some embodiments, L8 comprises a diamine (e.g., a linking group comprising an alkylene diamine). 73 NAI-1540479824 [00300] 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, L9 comprises a polyethylene glycol. In some embodiments, L9 comprises a modified polyethylene glycol. In some embodiments, L9 comprises an amino acid residue. In some embodiments, L9 comprises an alkyl group or a substituted alkyl. In some embodiments, L9 comprises an aryl group or a substituted aryl group. In some embodiments, L9 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00301] 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, L10 comprises a polyethylene glycol. In some embodiments, L10 comprises a modified polyethylene glycol. In some embodiments, L10 comprises an amino acid residue. In some embodiments, L10 comprises an alkyl group or a substituted alkyl. In some embodiments, L10 comprises an aryl group or a substituted aryl group. In some embodiments, L10 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00302] 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, L11 comprises a polyethylene glycol. In some embodiments, L11 comprises a modified polyethylene glycol. In some embodiments, L11 comprises an amino acid residue. In some embodiments, L11 comprises an alkyl group or a substituted alkyl. In some embodiments, L11 comprises an aryl group or a substituted aryl group. In some embodiments, L11 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00303] 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, L12 comprises a polyethylene glycol. In some embodiments, L12 comprises a modified polyethylene glycol. In some embodiments, L12 comprises an amino acid residue. In some embodiments, L12 comprises an alkyl group or a substituted alkyl. In some embodiments, L12 comprises an aryl group or a substituted aryl group. In some embodiments, L12 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00304] 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-1540479824 a substituted aryl group, or a diamine. In some embodiments, L13 comprises a polyethylene glycol. In some embodiments, L13 comprises a modified polyethylene glycol. In some embodiments, L13 comprises an amino acid residue. In some embodiments, L13 comprises an alkyl group or a substituted alkyl. In some embodiments, L13 comprises an aryl group or a substituted aryl group. In some embodiments, L13 comprises a diamine (e.g., a linking group comprising an alkylene diamine). [00305] In some embodiments, LB is a second linker comprising:
Figure imgf000077_0001
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-;
Figure imgf000077_0002
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. [00306] 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 [00307] As described above, in certain embodiments, L7 is 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, T7 is attached to the hydrazinyl-indolyl or a hydrazinyl- pyrrolo-pyridinyl conjugation moiety (e.g., as shown in Formula (I) above). In certain embodiments, V7 is 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. [00308] 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 T13 each 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 [00309] 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-C12 alkyl, or C1-C10 alkyl, or C1-C6 alkyl, or C1-C3 alkyl. In some instances, (C1-C12)alkyl is a C2-alkyl. For example, (C1-C12)alkyl may be an alkylene or substituted alkylene, such as C1-C12 alkylene, or C1-C10 alkylene, or C1-C6 alkylene, or C1-C3 alkylene. In some instances, (C1-C12)alkyl is a 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). [00310] In certain embodiments, substituted (C1-C12)alkyl is a straight chain or branched substituted alkyl group that includes from 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some instances, substituted (C1-C12)alkyl may be a substituted alkyl, such as substituted C1-C12 alkyl, or substituted C1-C10 alkyl, or substituted C1-C6 alkyl, or substituted C1-C3 alkyl. In some instances, substituted (C1-C12)alkyl is a substituted C2-alkyl. For example, substituted (C1-C12)alkyl may be a substituted alkylene, such as substituted C1-C12 alkylene, or substituted C1-C10 alkylene, or substituted C1-C6 alkylene, or substituted C1-C3 alkylene. In some instances, substituted (C1-C12)alkyl is a substituted 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-C12 alkylene (e.g., C3-alkylene) substituted with a -CONHCH2CH2SO3H group, or may include C1-C12 alkylene (e.g., C5-alkylene) substituted with a -NHCOCH2SO3H group. [00311] In some embodiments, substituted (C1-C12)alkyl may include C1-C12 alkylene (e.g., C3-alkylene or C5-alkylene) substituted with a (PEG)t group as described herein (e.g., -NHCO(PEG)t, wherein
Figure imgf000079_0001
indicates the point of attachment 77 NAI-1540479824 to carbonyl group of -NHCO-, and t is an integer), such as -NHCO(CH2CH2O)3CH3 or -NHCO(CH2CH2O)5CH3 or -NHCO(CH2CH2O)8CH3. [00312] 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). [00313] 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). [00314] 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:
Figure imgf000080_0001
, 78 NAI-1540479824 where y is an integer from 1 to 6, r is 0 or 1, and each R12 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl. In certain embodiments, any two adjacent R12 groups of the EDA may be cyclically linked, e.g., to form a piperazinyl ring. In certain embodiments, y is 1 and the two adjacent R12 groups are an alkyl group, cyclically linked to form a piperazinyl ring. In certain embodiments, y is 1 and the adjacent R12 groups are selected from hydrogen, an alkyl (e.g., methyl) and a substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH). [00315] 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:
Figure imgf000081_0001
where R12 is selected from hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety (e.g., a polyethylene glycol or a modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R12 is a polyethylene glycol moiety. In certain embodiments, R12 is a carboxy modified polyethylene glycol. [00316] In certain embodiments, R12 includes 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, R17 is 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, R17 is COOH. In certain embodiments, R17 is OH. In certain embodiments, R17 is OCH3. [00317] In certain embodiments, (PEG)k is (PEG)t having the following structure:
Figure imgf000082_0001
, wherein t is an integer from 2 to 10. In certain embodiments, t is 8. [00318] 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)n is described by the structure:
Figure imgf000082_0002
, 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. [00319] 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-1540479824 from 1 to 20, from 1 to 12 or from 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain embodiments, p is 1. In certain embodiments, p is 2. [00320] In further embodiments, (AA)p comprises a dipeptide of valine-alanine. [00321] 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. [00322] 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, R13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R13 is hydrogen. In certain embodiments, R13 is 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, R13 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 81 NAI-1540479824 substituted alkenyl. In certain embodiments, R13 is alkynyl or substituted alkynyl. In certain embodiments, R13 is alkoxy or substituted alkoxy. In certain embodiments, R13 is amino or substituted amino. In certain embodiments, R13 is carboxyl or carboxyl ester. In certain embodiments, R13 is acyl or acyloxy. In certain embodiments, R13 is acyl amino or amino acyl. In certain embodiments, R13 is alkylamide or substituted alkylamide. In certain embodiments, R13 is sulfonyl. In certain embodiments, R13 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R13 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R13 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5- 8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R13 is 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 C3-5 substituted cycloalkyl. In certain embodiments, R13 is 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. [00323] In certain embodiments, R13 is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R13. [00324] 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. [00325] 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). [00326] In some embodiments, a tether group includes a MABO group described by the following structure: 82 NAI-1540479824 . [00327] In some embodiments, a tether group includes a MABC group described by the following structure:
Figure imgf000085_0001
. [00328] In some embodiments, a tether group includes a PABO group described by the following structure:
Figure imgf000085_0002
. [00329] In some embodiments, a tether group includes a PABC group described by the following structure:
Figure imgf000085_0003
. [00330] In some embodiments, a tether group includes a PAB group described by the following structure: . [00331] In some embodiments, a tether group includes a PABA group described by the following structure:
Figure imgf000085_0004
[00332] In some embodiments, a tether group includes a PAP group described by the following structure: 83 NAI-1540479824 . [00333] In some embodiments, a tether group includes a PHP group described by the following structure:
Figure imgf000086_0001
. [00334] In certain embodiments, each R14 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00335] In certain embodiments, R14 is hydrogen. In certain embodiments, each R14 is hydrogen. In certain embodiments, R14 is 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, R14 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R14 is alkynyl or substituted alkynyl. In certain embodiments, R14 is alkoxy or substituted alkoxy. In certain embodiments, R14 is amino or substituted amino. In certain embodiments, R14 is carboxyl or carboxyl ester. In certain embodiments, R14 is acyl or acyloxy. In certain embodiments, R14 is acyl amino or amino acyl. In certain embodiments, R14 is alkylamide or substituted alkylamide. In certain embodiments, R14 is sulfonyl. In certain embodiments, R14 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R14 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R14 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5- 8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R14 is 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 C3-5 substituted cycloalkyl. In certain embodiments, R14 is heterocyclyl or substituted heterocyclyl, such as C3-8 heterocyclyl 84 NAI-1540479824 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. [00336] 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. [00337] In certain embodiments, one or more of the tether groups T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12, and/or T13 is each optionally substituted with a glycoside or glycoside derivative. For example, in some instances, T1, T2, T3, T4, T5, and T6 are each optionally substituted with a glycoside. In some instances, T7, T8, T9, T10, T11, T12 , and T13 are 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. [00338] 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. [00339] For example, in some embodiments, the glycoside or glycoside derivative is selected from the following structures: 85 NAI-1540479824 [00340] Regarding the linking functional groups, V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, and V13 any convenient linking functional groups may be utilized in the subject linkers. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, thiophosphoraidate, and the like. In some embodiments, V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, and V13 are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, - OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, 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 R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00342] In certain embodiments, R15 is hydrogen. In certain embodiments, each R15 is hydrogen. In certain embodiments, R15 is 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, R15 is alkenyl or substituted alkenyl, such as C2-6 alkenyl or C2-6 substituted alkenyl, or C2-4 alkenyl or C2-4 substituted alkenyl, or C2-3 alkenyl or C2-3 substituted alkenyl. In certain embodiments, R15 is alkynyl or substituted alkynyl. In certain embodiments, R15 is alkoxy or substituted alkoxy. In certain embodiments, R15 is amino or substituted amino. In certain embodiments, R15 is carboxyl or carboxyl ester. In certain 86 NAI-1540479824 embodiments, R15 is acyl or acyloxy. In certain embodiments, R15 is acyl amino or amino acyl. In certain embodiments, R15 is alkylamide or substituted alkylamide. In certain embodiments, R15 is sulfonyl. In certain embodiments, R15 is thioalkoxy or substituted thioalkoxy. In certain embodiments, R15 is aryl or substituted aryl, such as C5-8 aryl or C5-8 substituted aryl, such as a C5 aryl or C5 substituted aryl, or a C6 aryl or C6 substituted aryl. In certain embodiments, R15 is heteroaryl or substituted heteroaryl, such as C5-8 heteroaryl or C5- 8 substituted heteroaryl, such as a C5 heteroaryl or C5 substituted heteroaryl, or a C6 heteroaryl or C6 substituted heteroaryl. In certain embodiments, R15 is cycloalkyl or substituted cycloalkyl, such as C3-8 cycloalkyl or C3-8 substituted cycloalkyl, such as a C3-6 cycloalkyl or C3-6 substituted cycloalkyl, or a C3-5 cycloalkyl or C3-5 substituted cycloalkyl. In certain embodiments, R15 is 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. [00343] In certain embodiments, each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, 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. [00344] As described above, in some embodiments, LA is 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. [00345] In some embodiments, in the first linker LA: T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6 are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)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 V6 are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; 87 NAI-1540479824 wherein:
Figure imgf000090_0001
integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000090_0002
, where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine
Figure imgf000090_0003
AA is an amino acid residue, where p is an integer from 1 to 20; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00346] In some embodiments, LA comprises: -(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 T6 are 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-1540479824 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 V6 are 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 R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00347] In some embodiments of LA: T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6 are 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 V6 are 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:
Figure imgf000091_0001
integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000091_0002
, where y is an integer from 1 to 6 and r is 0 or 1; 89 NAI-1540479824 4-amino-piperidine each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring; a, b, c, and d are each 1; and e and f are 0. [00348] In some embodiments, T1, T2, T3, T4, T5, and T6 are each optionally substituted with a glycoside. [00349] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. [00350] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [00351] In certain embodiments, T1, T2, T3, T4, T5, and T6 and V1, V2, V3, V4, V5, and V6 are selected from the following: wherein: T1 is (C1-C12)alkyl and V1 is -CO-; T2 is (AA)p and V2 is absent (e.g., a covalent bond); T3 is PABC and V3 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and d, e, and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is (PEG)n and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABC and V4 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-; T2 is an amino acid analog and V2 is -NH-; T3 is (PEG)n and V3 is -CO-; T4 is (AA)p and V4 is absent (e.g., a covalent bond); 90 NAI-1540479824 T5 is PABC and V5 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is (PEG)n and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABC and V4 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABC and V4 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is (PEG)n and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABA and V4 is -CO-; T5 is (C1-C12)alkyl and V5 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-; T2 is 4AP and V2 is -CO-; T3 is (C1-C12)alkyl and V3 is -CO-; T4 is (AA)p and V4 is absent (e.g., a covalent bond); T5 is PABC and V5 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: 91 NAI-1540479824 T1 is (C1-C12)alkyl and V1 is -CO-; T2 is 4AP and V2 is -CO-; T3 is (C1-C12)alkyl and V3 is -O-; T4 is (C1-C12)alkyl and V4 is -CO-; T5 is (AA)p and V5 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and T6 is PABC and V6 is absent (e.g., a covalent bond); or wherein: T1 is (C1-C12)alkyl and V1 is -CO-; T2 is an amino acid analog and V2 is absent (e.g., a covalent bond); T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABC and V4 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is (PEG)n and V2 is -CONH-; T3 is substituted (C1-C12)alkyl and V3 is -CO-; T4 is (AA)p and V4 is absent (e.g., a covalent bond); T5 is PABC and V5 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CO-; T2 is an (AA)p and V2 is -NH-; T3 is (PEG)n and V3 is -CO-; T4 is (AA)p and V4 is absent (e.g., a covalent bond); T5 is PABC and V5 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and f is 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is (PEG)n and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); 92 NAI-1540479824 T4 is PAP and V4 is -C(O)O-; p is an integer from 1 to 10; and e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is (AA)p and V3 is absent (e.g., a covalent bond); T4 is PABC and V4 is absent (e.g., a covalent bond); p is an integer from 1 to 10; and e and f are each 0; or wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is PABC and V3 is absent (e.g., a covalent bond); and d, e, and f are each 0. [00352] In certain embodiments, the left-hand side of the above linker structure for the first linker LA is 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 LA is attached to the first drug or active agent. [00353] As described above, in some embodiments, LB is 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. [00354] In some embodiments, in the second linker LB: T7 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T8, T9, T10, T11, T12, and T13 are each independently selected from (C1-C12)alkyl, substituted (C1-C12)alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)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 V13 are each independently selected from a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2- and -P(O)OH-, wherein q is an integer from 1 to 6; wherein: 93 NAI-1540479824 integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000096_0001
, where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine
Figure imgf000096_0002
AA is an amino acid residue, where p is an integer from 1 to 20; and each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring; each R13 is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. [00355] 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 T6 may be used for the tether groups T7, T8, T9, T10, T11, T12, and T13. [00356] 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 V6 may be used for the linking functional groups V7, V8, V9, V10, V11, V12 , and V13. [00357] In certain embodiments, each R13 is 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. [00358] In certain embodiments, each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, 94 NAI-1540479824 alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are as described above for R15. In these embodiments, various possible substituents are as described above for R15. [00359] In certain embodiments of the second linker LB, one or more of the tether groups T7, T8, T9, T10, T11, T12 , and T13 is 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. [00360] 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. [00361] In some embodiments, T7, T8, T9, T10, T11, T12 , and T13 are each optionally substituted with a glycoside. [00362] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. [00363] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [00364] In some embodiments of LB: g, h, i, j, and k are each 1; l and m are each 0; T7 is a covalent bond; T8, T9, T10, T11, and T12 are 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 V12 are 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:
Figure imgf000098_0001
integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000098_0002
, where y is an integer from 1 to 6 and r is 0 or 1; 4-amino-piperidine
Figure imgf000098_0003
each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring. [00365] In some embodiments, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12 are each optionally substituted with a glycoside. [00366] In some embodiments, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally substituted with a glycoside. [00367] In some embodiments, the glycoside is selected from a glucuronide, a galactoside, a glucoside, a mannoside, a fucoside, O-GlcNAc, and O-GalNAc. [00368] In certain embodiments, T7, T8, T9, T10, T11, T12, and T13 and V7, V8, V9, V10, V11, V12, and V13 are selected from the following: wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is (AA)p and V9 is absent (e.g., a covalent bond); T10 is PABC and V10 is absent (e.g., a covalent bond); and k, l, and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is (PEG)n and V9 is -CO-; 96 NAI-1540479824 T10 is (AA)p and V10 is absent (e.g., a covalent bond); and T11 is PABC and V11 is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is an amino acid analog and V9 is -NH-; T10 is (PEG)n and V10 is -CO-; T11 is (AA)p and V11 is absent (e.g., a covalent bond); T12 is PABC and V12 is absent (e.g., a covalent bond); and m is 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is (PEG)n and V9 is -CO-; T10 is (AA)p and V10 is absent (e.g., a covalent bond); T11 is PABC and V11 is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is substituted (C1-C12)alkyl and V9 is -CO-; T10 is (AA)p and V10 is absent (e.g., a covalent bond); T11 is PABC and V11 is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is (PEG)n and V9 is -CO-; T10 is (AA)p and V10 is absent (e.g., a covalent bond); T11 is PABA and V11 is -CO-; T12 is (C1-C12)alkyl and V12 is absent (e.g., a covalent bond); and m is 0; or wherein: 97 NAI-1540479824 T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is 4AP and V9 is -CO-; T10 is (C1-C12)alkyl and V10 is -CO-; T11 is (AA)p and V11 is absent (e.g., a covalent bond); T12 is PABC and V12 is absent (e.g., a covalent bond); and m is 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is 4AP and V9 is -CO-; T10 is (C1-C12)alkyl and V10 is -O-; T11 is (C1-C12)alkyl and V11 is -CO-; T12 is (AA)p and V12 is absent (e.g., a covalent bond); and T13 PABC and V13 is absent (e.g., a covalent bond); or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is an amino acid analog and V9 is absent (e.g., a covalent bond); T10 is (AA)p and V10 is absent (e.g., a covalent bond); T11 is PABC and V11 is absent (e.g., a covalent bond); and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is (PEG)n and V9 is -CONH-; T10 is substituted (C1-C12)alkyl and V10 is -CO-; T11 is (AA)p and V11 is absent (e.g., a covalent bond); T12 is PABC and V12 is absent (e.g., a covalent bond); and m is 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is (AA)p and V9 is -NH-; 98 NAI-1540479824 T10 is (PEG)n and V10 is -CO-; T11 is (AA)p and V11 is absent (e.g., a covalent bond); T12 is PABC and V12 is absent (e.g., a covalent bond); and m is 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is (PEG)n and V9 is -CO-; T10 is (AA)p and V10 is absent (e.g., a covalent bond); T11 is PAP and V11 is -C(O)O-; and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CO-; T9 is (AA)p and V9 is absent (e.g., a covalent bond); T10 is PABC and V10 is absent (e.g., a covalent bond); T11 is PAP and V11 is -C(O)O-; and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is substituted (C1-C12)alkyl and V9 is -CO-; T10 is PABC and V10 is absent (e.g., a covalent bond); and k, l, and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is absent (e.g., a covalent bond); T9 is heteroaryl and V9 is absent (e.g., a covalent bond); T10 is (C1-C12)alkyl and V10 is -CONH-; T11 is (PEG)n and V11 is -CO-; and l and m are each 0; or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is absent (e.g., a covalent bond); 99 NAI-1540479824 T9 is heteroaryl and V9 is absent (e.g., a covalent bond); T10 is (C1-C12)alkyl and V10 is -CONH-; T11 is substituted (C1-C12)alkyl and V11 is -CO-; T12 is (AA)p and V12 is absent (e.g., a covalent bond); and T13 PAB and V13 is absent (e.g., a covalent bond); or wherein: T7 is absent (e.g., a covalent bond) and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is absent (e.g., a covalent bond); T9 is heteroaryl and V9 is absent (e.g., a covalent bond); T10 is (C1-C12)alkyl and V10 is -CONH-; T11 is substituted (C1-C12)alkyl and V11 is -CO-; T12 is (AA)p and V12 is absent (e.g., a covalent bond); and T13 PABC and V13 is absent (e.g., a covalent bond). [00369] In certain embodiments, the left-hand side of the above linker structure for the second linker LB is 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 LB is attached to the second drug or active agent. [00370] 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., LA and/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. [00371] 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-1540479824 cleavable linker includes a first cleavable moiety and a second cleavable moiety that hinders cleavage of the first cleavable moiety. By “hinders cleavage” is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood or substantially inhibits the cleavage of the first cleavable moiety, thus substantially reducing the amount or preventing the cleavage of the cleavable linker. For instance, the presence of uncleaved second cleavable moiety can hinder cleavage of the first cleavable moiety. The hinderance of cleavage of the first cleavable moiety by the presence of the second cleavable moiety, in turn, substantially reduces the amount or prevents the release of the drug from the antibody. For example, the premature release of the drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired target site of action for the drug. [00372] 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. [00373] 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-1540479824 the 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). [00374] 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. [00375] 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 [00376] 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. [00377] 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. [00378] 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-1540479824 attached to by an enzyme (e.g., through enzymatically mediated hydrolysis of the glycosidic bond). A glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any convenient enzyme that is able to carry out the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker. An example of an enzyme that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that attaches the glycoside or glycoside derivative to the cleavable linker is a 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. [00379] In some embodiments, an ROR1-ADC is represented by Formula (I):
Figure imgf000106_0001
wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3 and R4 are each selected from hydrogen and (C1-C12)alkyl; LA is a first linker wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is (AA)p where p is an integer from 1-20 and V3 is a covalent bond; T4 is PABC and V4 is a covalent bond; 104 NAI-1540479824 a, b, c, and d are each 1; e and f are each 0; and LB is a second linker wherein: T7 is a covalent bond and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is substituted (C1-C12)alkyl and V9 is -CO-; T10 is (AA)p where p is an integer from 1-20 and V10 is a covalent bond; T11 is PABC and V11 is 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; W1 is a first drug; and W2 is a second drug. [00380] In some embodiments, one or both of W1 and W2 are camptothecin analogues, for example, belotecan. [00381] In some embodiments, an ROR1-ADC is represented by Formula (I):
Figure imgf000107_0001
wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3 and R4 are each selected from hydrogen and (C1-C12)alkyl; LA is a linker wherein: T1 is (C1-C6)alkyl and V1 is -CONH-; T2 is (C1-C6)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10, and V2 is -CO-; T3 is (AA)2 and V3 is a covalent bond; T4 is PABC substituted with a glycoside and V4 is a covalent bond; 105 NAI-1540479824 a, b, c, and d are each 1; and e and f are each 0; and LB is a linker wherein: T7 is a covalent bond and V7 is -NHCO-; T8 is (C1-C6)alkyl and V8 is -CONH-; T9 is (C1-C6)alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 2 to 10, and V9 is -CO-; T10 is (AA)2 and V10 is a covalent bond; T11 is PABC substituted with a glycoside and V11 is 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; W1 is a first drug; and W2 is a second drug. [00382] In some embodiments, an ROR1-ADC is represented by Formula (I):
Figure imgf000108_0001
wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3 and R4 are each selected from hydrogen and (C1-C12)alkyl; LA is a linker wherein: T1 is (C1-C6)alkyl and V1 is -CONH-; T2 is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is
Figure imgf000108_0002
integer from 2 to 10, optionally 8, and V2 is -CO-; T3 is (AA)2 and V3 is a covalent bond; 106 NAI-1540479824 T4 is PABC substituted with a glycoside and V4 is a covalent bond; a, b, c, and d are each 1; and e and f are each 0; and LB is a linker wherein: T7 is a covalent bond and V7 is -NHCO-; T8 is (C1-C6)alkyl and V8 is -CONH-; T9 is (C1-C6)alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is
Figure imgf000109_0001
integer from 2 to 10, optionally 8, and V9 is -CO-; T10 is (AA)2 and V10 is a covalent bond; T11 is PABC substituted with a glycoside and V11 is 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; W1 is a first drug; and W2 is a second drug. [00383] In some embodiments, the PABC of one or both of T4 and T11 is substituted with a glucuronide. In some embodiments, one or both of T1 and T8 is ethyl. In some embodiments, one or both of T2 and T9 is C5 alkylene substituted with -NHCO(PEG)k, wherein k is an integer from 5 to 10. In some embodiments, one or both of W1 and W2 are camptothecin analogues, for example, belotecan. [00384] In some embodiments, the PABC of one or both of T4 and T11 is substituted with a glucuronide. In some embodiments, one or both of T1 and T8 is ethyl. In some embodiments, one or both of T2 and T9 is C5 alkylene substituted with -NHCO(PEG)t, wherein (PEG)t is
Figure imgf000109_0002
integer from 5 to 10. In some embodiments, one or both of W1 and W2 are camptothecin analogues, for example, belotecan. [00385] In some embodiments, s is an integer from 1 to 4. In further embodiments, s is 4. [00386] In some embodiments, an ROR1-ADC is represented by Formula (II): 107 NAI-1540479824
wherein: Ab represents the antibody that binds to ROR1; and s is an integer from 1 to 10. [00387] In some embodiments, s is an integer from 1 to 4. [00388] 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. [00389] 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 [00390] 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-1540479824 abbreviation, 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. [00391] 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. [00392] 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. [00393] 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., 10-8 M or less, e.g., from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M). 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-1540479824 antibody 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. [00394] 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. [00395] 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-1540479824 an ROR1 antibody as used herein is disclosed in US Patent Application Publication No. US20210155692A1, which is incorporated by reference in its entirety. [00396] 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. [00397] 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. [00398] 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. [00399] 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-1540479824 13, 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. [00400] 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. [00401] 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. [00402] 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. [00403] 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. [00404] 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-1540479824 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. [00405] 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. [00406] 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. [00407] 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 [00408] 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. [00409] 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. [00410] 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 [00411] 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-1540479824 without allowing gaps. In calculating percent identity, typically only exact matches are counted. [00412] 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). [00413] 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-1540479824 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%). 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. [00414] 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-1540479824 embodiments, 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). [00415] 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). [00416] 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%). [00417] 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. [00418] 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 [00419] 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. [00420] 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. [00421] 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. [00422] 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-1540479824 light 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. [00423] 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. [00424] 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. [00425] 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) [00426] 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). [00427] 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-1540479824 the 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) [00428] 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) [00429] 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. [00430] 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) [00431] In further embodiments, the C terminus of the VL is conjugated directly or indirectly to the N terminus of the CL. 121 NAI-1540479824 [00432] 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. [00433] 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. [00434] 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:41, 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 122 NAI-1540479824 comprise: (a) a heavy chain having the amino acid sequence of SEQ ID NO:44, and (b) a light chain having the amino acid sequence of SEQ ID NO:28. [00435] In some embodiments, provided herein is an ROR1 binding protein comprising any one of the anti-ROR1 antibodies described herein. In some embodiments, the ROR1 binding protein is an antibody comprising two heavy chains and two light chains. In some embodiments, the ROR1 binding protein is an antibody comprising two heavy chains comprising a same VH region and two light chains comprising a same VL region. [00436] In some embodiments, the ROR1 binding protein is a monoclonal antibody, including a mouse, chimeric, humanized, or human antibody. In some embodiments, the anti- ROR1 antibody is an antibody fragment, e.g., an scFv. In some embodiments, the ROR1 binding protein is a fusion protein comprising the anti-ROR1 antibody provided herein. In other embodiments, the ROR1 binding protein is a multispecific antibody comprising the anti-ROR1 antibody or fragment thereof provided herein. [00437] Other exemplary ROR1 binding molecules are described in more detail in the following sections. In some embodiments, the anti-ROR1 antibody or antigen-binding protein according to any of the above embodiments may incorporate any of the features, singly or in combination, as described in the sections below. 123 NAI-1540479824 ) 6 3 : D) T 0 ) S 1 7 V ) 5 ) 9 ) 3 Y : F 3 : A 1 S : S 1 : 2 R : V F A T R
Figure imgf000126_0001
Figure imgf000126_0002
D I GV D G V A S S G A YS I Y D I S D I S D I YD I P K Q E P D AQ I Q Q S Q A S ( Y I E A S Y S Q YQ A G ( VE S ( A E E Q R S ( S ( QE S Q P ( R K V Q ) ) Q )2 ) 7 D 2 ) 6 ) 0 T ) 2 W H 5 2 Y : : F 1 1 2 2 O T : : : R : I : W N S O V G N G O A N S O O P O YO A S N S N S N Y N V D I G YD I Y I YD I V D I A S G S D I YD K D I AS Q P Q Y V Q S QQ Q Y I Q F T Q S E S Y ( I E S ( R E E E Q A S ( S ( S ( QE S F E V ( G S S S ( Q AS S ) Y ) ) ) ) ) S A 1 : D 6 : Y1 1 V 5 9 2 AV R O TGO D : A 1 : 1 : T 2 N S K N F C T C O S O S O R : P O S V T V N S N Y N S N L L I T ) D G I GV S D I GD V L S A S S R T V 6 2 : Q Y I D I S D I YD I L P DQ Y I Q Q S G R S Q A Q Y GQ DO E Y A I E YE A E S E QQ E GG G N S ( A S ( VS ( R S ( S ( QS ( P W QY V S D I 1 2 3 1 2 3 V D AQ E R HR HR D LR LR LR L F S G L S ( VD VD VD VD VD : V C C C C C C e c GG : S K n GY I e c S P I E 4 R e u S EY n e S 2 u QV 8 9 R. 7 Dq D. q e e V L V R q e T K T 4 0 C S Cq L e S S Q H A S M 4 QG 5 1- V VV C L I Q E Y V D G I A N Table 2: Antibody Clone A27 and its Variants Heavy Chain and Light Chain Sequences
Figure imgf000127_0001
125 NAI-1540479824
Figure imgf000128_0001
7.4 CONJUGATION [00438] In certain embodiments, the amino acid sequence of an ROR1 antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to a 2-formylglycine (fGly) residue by action of a formylglycine generating enzyme (FGE) either in vivo (e.g., at the time of translation of an aldehyde tag- containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system). Such sulfatase motifs may also be referred to herein as an FGE-modification site. 7.4.1 Sulfatase motifs [00439] A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 amino acid residues in length, usually no more than 6 amino acid residues in length. Sulfatase motifs provided in an Ig polypeptide are at least 5 or 6 amino acid residues, and can be, for example, from 5 to 16, 6-16, 5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10, 5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define a sulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acid residues in length. [00440] In certain embodiments, ROR1 antibodies of interest include those where one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues have been inserted, deleted, substituted (replaced) relative to the native amino acid sequence to provide for a sequence of a sulfatase motif in the ROR1 antibody. In certain embodiments, the ROR1 antibody includes a modification (insertion, addition, deletion, and/or substitution/replacement) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the ROR1 antibody. Where an amino acid sequence native to the ROR1 antibody contains one or more residues of the desired sulfatase motif, the total number of modifications of residues can be reduced, e.g., by site-specification modification (insertion, addition, deletion, substitution/replacement) of amino acid residues flanking the native amino acid residues to provide a sequence of the desired sulfatase motif. In certain embodiments, the extent of modification of the native amino acid sequence of the target antibody is minimized, so as to minimize the number of 126 NAI-1540479824 amino acid residues that are inserted, deleted, substituted (replaced), or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target antibody may minimize the impact such modifications may have upon antibody function and/or structure. [00441] It should be noted that while aldehyde tags of particular interest are those comprising at least a minimal sulfatase motif (also referred to a “consensus sulfatase motif”), it will be readily appreciated that longer aldehyde tags are both contemplated and encompassed by the present disclosure and can find use in the compositions and methods of the present disclosure. Aldehyde tags can thus comprise a minimal sulfatase motif of 5 or 6 residues or can be longer and comprise a minimal sulfatase motif which can be flanked at the N- and/or C-terminal sides of the motif by additional amino acid residues. Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues. [00442] An aldehyde tag can be present at or near the C-terminus of an Ig heavy chain; e.g., an aldehyde tag can be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C- terminus of a native, wild-type Ig heavy chain. An aldehyde tag can be present within a CH1 domain of an Ig heavy chain. An aldehyde tag can be present within a CH2 domain of an Ig heavy chain. An aldehyde tag can be present within a CH3 domain of an Ig heavy chain. An aldehyde tag can be present in an Ig light chain constant region, e.g., in a kappa light chain constant region or a lambda light chain constant region. [00443] In some embodiments, the ROR1 antibody as disclosed herein comprises a sulfatase motif. [00444] In certain embodiments, the sulfatase motif used may be described by the formula: X1Z10X2Z20X3Z30 (V) wherein: Z10 is cysteine or serine (which can also be represented by (C/S)); Z20 is either a proline or alanine residue (which can also be represented by (P/A)); Z30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X1 is present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M, S or 127 NAI-1540479824 V, with the proviso that when the sulfatase motif is at the N-terminus of the target ROR1 antibody, X1 is present; and X2 and X3 independently can be any amino acid, though usually an aliphatic amino acid, a polar, uncharged amino acid, or a sulfur containing amino acid (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. [00445] The amino acid sequence of an antibody heavy and/or light chain can be modified to provide a sequence of at least 5 amino acids of the formula X1Z10X2Z20X3Z30, wherein: Z10 is cysteine or serine; Z20 is a proline or alanine residue; Z30 is an aliphatic amino acid or a basic amino acid; X1 is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the ROR1 antibody, X1 is present; X2 and X3 are each independently any amino acid. [00446] The sulfatase motif is generally selected so as to be capable of conversion by a selected FGE, e.g., an FGE present in a host cell in which the aldehyde-tagged antibody is expressed or an FGE which is to be contacted with the aldehyde-tagged antibody in a cell- free in vitro method. [00447] For example, where the FGE is a eukaryotic FGE (e.g., a mammalian FGE, including a human FGE), the sulfatase motif can be of the formula: X1CX2PX3Z30 (VI) wherein: X1 may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, S or V, with the proviso that when the sulfatase motif is at the N-terminus of the target ROR1 antibody, X1 is present; X2 and X3 independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G, or C, e.g., S, T, A, V or G; and 128 NAI-1540479824 Z30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I. [00448] Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO:100), MCTPSR (SEQ ID NO:101), VCTPSR (SEQ ID NO:102), LCSPSR (SEQ ID NO:103), LCAPSR (SEQ ID NO:104), LCVPSR (SEQ ID NO:105), LCGPSR (SEQ ID NO:106), ICTPAR (SEQ ID NO:107), LCTPSK (SEQ ID NO:108), MCTPSK (SEQ ID NO:109), VCTPSK (SEQ ID NO:110), LCSPSK (SEQ ID NO:111), LCAPSK (SEQ ID NO:112), LCVPSK (SEQ ID NO:113), LCGPSK (SEQ ID NO:114), LCTPSA (SEQ ID NO:115), ICTPAA (SEQ ID NO:116), MCTPSA (SEQ ID NO:117), VCTPSA (SEQ ID NO:118), LCSPSA (SEQ ID NO:119), LCAPSA (SEQ ID NO:120), LCVPSA (SEQ ID NO:121), and LCGPSA (SEQ ID NO:122). 7.4.2 fGly-containing Sequences [00449] In some embodiments, the ROR1 antibody as disclosed herein comprises a fGly- containing sulfatase motif. [00450] Upon action of FGE on the antibody heavy and/or light chain, the serine or the cysteine in the sulfatase motif is modified to fGly. Thus, the fGly-containing sulfatase motif can be of the formula: X1(fGly)X2Z20X3Z30 (VII) wherein: fGly is the formylglycine residue; Z20 is either a proline or alanine residue (which can also be represented by (P/A)); Z30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; X1 may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target ROR1 antibody, X1 is present; and X2 and X3 independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. 129 NAI-1540479824 [00451] Specific examples of fGly-containing sulfatase motifs include L(fGly)TPSR (SEQ ID NO:123), M(fGly)TPSR (SEQ ID NO:124), V(fGly)TPSR (SEQ ID NO:125), L(fGly)SPSR (SEQ ID NO:126), L(fGly)APSR (SEQ ID NO:127), L(fGly)VPSR (SEQ ID NO:128), L(fGly)GPSR (SEQ ID NO:129), I(fGly)TPAR (SEQ ID NO:130), L(fGly)TPSK (SEQ ID NO:131), M(fGly)TPSK (SEQ ID NO:132), V(fGly)TPSK (SEQ ID NO:133), L(fGly)SPSK (SEQ ID NO:134), L(fGly)APSK (SEQ ID NO:135), L(fGly)VPSK (SEQ ID NO:136), L(fGly)GPSK (SEQ ID NO:137), L(fGly)TPSA (SEQ ID NO:138), I(fGly)TPAA (SEQ ID NO:139), M(fGly)TPSA (SEQ ID NO:140), V(fGly)TPSA (SEQ ID NO:141), L(fGly)SPSA (SEQ ID NO:142), L(fGly)APSA (SEQ ID NO:143), L(fGly)VPSA (SEQ ID NO:144), and L(fGly)GPSA (SEQ ID NO:145). [00452] In some embodiments, the ROR1 antibody Ab of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)) comprises a fGly'-containing sulfatase motif. In further embodiments, the ROR1 antibody Ab of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV- 7), or (II)) comprises a light chain (such as SEQ ID NO:28) and a heavy chain (such as a variant thereof of SEQ ID NO:27 further comprising one or more sequences of Formula (VIII), for example SEQ ID NO:41 or 44). [00453] As described above, to produce the conjugate, the ROR1 antibody containing the fGly residue may be conjugated to a drug or active agent by reaction of the fGly with a reactive moiety (e.g., a hydrazinyl-indolyl or a hydrazinyl-pyrrolo-pyridinyl conjugation moiety, as described above) of a linker attached to the drug or active agent to produce an fGly’-containing sulfatase motif. As used herein, the term fGly’ refers to the amino acid residue of the sulfatase motif that is coupled to the drug or active agent through a linker (e.g., a branched linker) as described herein. Thus, the fGly’-containing sulfatase motif can be of the formula: X1(fGly’)X2Z20X3Z30 (VIII) wherein: fGly’ is the amino acid residue coupled to the drug or active agent through a linker (e.g., a branched linker) as described herein; Z20 is either a proline or alanine residue (which can also be represented by (P/A)); Z30 is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), usually lysine), or an aliphatic amino acid (alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I; 130 NAI-1540479824 X1 may be present or absent and, when present, can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., L, M, V, S or T, e.g., L, M or V, with the proviso that when the sulfatase motif is at the N-terminus of the target ROR1 antibody, X1 is present; and X2 and X3 independently can be any amino acid, e.g., an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid, (e.g., other than an aromatic amino acid or a charged amino acid), e.g., S, T, A, V, G or C, e.g., S, T, A, V or G. [00454] Specific examples of fGly’-containing sulfatase motifs include 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). [00455] As noted above, the amino acid sequence of an antibody is modified to include a sulfatase motif that contains a serine or cysteine residue that is capable of being converted (oxidized) to an fGly residue by action of an FGE either in vivo (e.g., at the time of translation of an aldehyde tag-containing protein in a cell) or in vitro (e.g., by contacting an aldehyde tag-containing protein with an FGE in a cell-free system). The antibody used to generate a conjugate of the present disclosure include at least an Ig constant region, e.g., an Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain), or an Ig light chain constant region. Such Ig antibodies are referred to herein as “target Ig polypeptides” or “target antibodies.” [00456] The site in an antibody into which a sulfatase motif is introduced can be any convenient site. As noted above, in some instances, the extent of modification of the native amino acid sequence of the target polypeptide is minimized, so as to minimize the number of amino acid residues that are inserted, deleted, substituted (replaced), and/or added (e.g., to the N- or C-terminus). Minimizing the extent of amino acid sequence modification of the target 131 NAI-1540479824 antibody may minimize the impact such modifications may have upon antibody function and/or structure. [00457] An antibody heavy chain constant region can include Ig constant regions of any heavy chain isotype, non-naturally occurring Ig heavy chain constant regions (including consensus Ig heavy chain constant regions). An Ig constant region amino acid sequence can be modified to include an aldehyde tag, where the aldehyde tag is present in or adjacent a solvent-accessible loop region of the Ig constant region. An Ig constant region amino acid sequence can be modified by insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, or more than 16 amino acids, to provide an amino acid sequence of a sulfatase motif as described above. [00458] In some cases, an aldehyde-tagged antibody comprises an aldehyde-tagged Ig heavy chain constant region (e.g., at least a CH1 domain; at least a CH1 and a CH2 domain; a CH1, a CH2, and a CH3 domain; or a CH1, a CH2, a CH3, and a CH4 domain). The aldehyde- tagged Ig heavy chain constant region can include heavy chain constant region sequences of an IgA, IgM, IgD, IgE, IgG1, IgG2, IgG3, or IgG4 isotype heavy chain or any allotypic variant of same, e.g., human heavy chain constant region sequences or mouse heavy chain constant region sequences, a hybrid heavy chain constant region, a synthetic heavy chain constant region, or a consensus heavy chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly- modified Ig polypeptide. Allotypic variants of Ig heavy chains are known in the art. See, e.g., Jefferis and Lefranc (2009) MAbs 1:4. [00459] In some cases, an aldehyde-tagged antibody comprises an aldehyde-tagged Ig light chain constant region. The aldehyde-tagged Ig light chain constant region can include constant region sequences of a kappa light chain, a lambda light chain, e.g., human kappa or lambda light chain constant regions, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, etc., modified to include at least one sulfatase motif that can be modified by an FGE to generate an fGly- modified antibody. Exemplary constant regions include human gamma 1 and gamma 3 regions. With the exception of the sulfatase motif, a constant region may have a wild-type amino acid sequence, or it may have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90% or at least 95% identical) to a wild-type amino acid sequence. [00460] In some embodiments the sulfatase motif is at a position other than, or in addition to, the C-terminus of the Ig polypeptide heavy chain. As noted above, an isolated aldehyde- tagged antibody can comprise a heavy chain constant region amino acid sequence modified to 132 NAI-1540479824 include a sulfatase motif as described above, where the sulfatase motif is in or adjacent to a surface-accessible loop region of the antibody heavy chain constant region. [00461] A sulfatase motif can be provided within or adjacent to one or more of these amino acid sequences of such modification sites of an Ig heavy chain. For example, an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif adjacent and N-terminal and/or adjacent and C-terminal to these modification sites. Alternatively, or in addition, an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modifications sites. In some embodiments, an Ig heavy chain polypeptide amino acid sequence may be modified to include two motifs, which may be adjacent to one another, or which may be separated by one, two, three, four or more (e.g., from about 1 to about 25, from about 25 to about 50, or from about 50 to about 100, or more, amino acids. Alternatively, or in addition, where a native amino acid sequence provides for one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues of the modification sites of an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide a sulfatase motif at the modification site. [00462] An antibody used in an antibody-drug conjugate of the present disclosure can have any of a variety of antigen-binding specificities, including but not limited to, e.g., an antigen present on a cancer cell; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on a diseased cell; and the like. For example, an antibody conjugate can bind an antigen, where the antigen is present on the surface of the cell. An antibody conjugate of the present disclosure can bind antigen with a suitable binding affinity, e.g., from 5 x 10-6 M to 10-7 M, from 10-7 M to 5 x 10-7 M, from 5 x 10-7 M to 10-8 M, from 10-8 M to 5 x 10-8 M, from 5 x 10-8 M to 10-9 M, or a binding affinity greater than 10-9 M. [00463] As non-limiting examples, a subject antibody conjugate can bind an antigen present on a cancer cell (e.g., a tumor-specific antigen; an antigen that is over-expressed on a cancer cell; etc.), and the conjugated moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, a subject antibody 133 NAI-1540479824 conjugate can be specific for an antigen on a cancer cell, where the conjugated moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). [00464] As further non-limiting examples, a subject antibody conjugate can bind an antigen present on a cell infected with a virus (e.g., where the antigen is encoded by the virus; where the antigen is expressed on a cell type that is infected by a virus; etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor. For example, a subject antibody conjugate can bind an antigen present on a cell infected with a virus, and the conjugated moiety can be a drug, such as a viral fusion inhibitor. [00465] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include unconverted sulfatase motifs (e.g., any one of SEQ ID NOs:100-122) in the CH1 region and in the CT region, and therefore comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALCTPSR GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLCT PSRGS (SEQ ID NO:29), where each underline portion comprises the unconverted sulfatase motif of SEQ ID NO:100 but, in any embodiment, may be independently replaced with any one of SEQ ID NOs:101-122. [00466] In further embodiments, the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. Additionally or alternatively, either or both of the sulfatase motifs are converted and conjugated to a linker-payload in an ROR1-ADC as disclosed herein. [00467] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include formylglycine residues in the CH1 and CT regions and therefore comprises the amino acid sequence of: 134 NAI-1540479824 EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL(fGly)T PSRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGS L(fGly)TPSRGS (SEQ ID NO:40), wherein fGly indicates a formylglycine residue, where each underline portion comprises the fGly-containing sulfatase motif of SEQ ID NO:123 but, in any embodiment, may be independently replaced with any one of SEQ ID NOs:124-145. [00468] In further embodiments, the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. [00469] In some embodiments, an ROR1-ADC comprises a heavy chain comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL(fGly’) TPSRGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GSL(fGly’)TPSRGS (SEQ ID NO:41), wherein each (fGly’) is an amino acid (formylglycine residue) having its side chain replaced by a linker-drug as disclosed herein, for example, in the ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV- 5), (XIV-6), (XIV-7), or (II)). In SEQ ID NO:41, each underline portion comprises the fGly- containing sulfatase motif of SEQ ID NO:146 but, in any embodiment, may be independently replaced with any one of SEQ ID NOs:147-168. [00470] In further embodiments, the ROR1-ADC comprises an ROR1 antibody, wherein the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in 135 NAI-1540479824 SEQ ID NO: 41, and the ROR1 antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. [00471] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include an unconverted sulfatase motif (e.g., any one of SEQ ID NOs:100-122), and therefore comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSLCTPSRNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:42), where the underline portion comprises the unconverted sulfatase motif of SEQ ID NO:100 but, in any embodiment, may be replaced with any one of SEQ ID NOs:101-122. [00472] In further embodiments, the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. [00473] Additionally or alternatively, the sulfatase motif is converted and conjugated to a linker-payload in an ROR1-ADC as disclosed herein. [00474] In some embodiments, the antibody that binds to ROR1 comprises a heavy chain which has been modified to include a formylglycine residue and therefore comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSL(fGly)TPSRNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK 136 NAI-1540479824 FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO:43), wherein fGly is the formylglycine residue, where the underline portion comprises the fGly-containing sulfatase motif of SEQ ID NO:123 but, in any embodiment, may be replaced with any one of SEQ ID NOs:124-145. [00475] In further embodiments, the antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. Additionally or alternatively, the sulfatase motif is conjugated to a linker-payload in an ROR1-ADC as disclosed herein. [00476] In some embodiments, an ROR1-ADC comprises a heavy chain comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFKGYYIHWVRQAPGKGLEWVAAIYPYGG STDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVYIYGVFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSL(fGly’)TPSRNTKVDKKVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO:44), wherein (fGly’) is an amino acid (formylglycine residue)having its side chain replaced by a linker-drug as disclosed herein, for example, in the ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV- 7), or (II)). In SEQ ID NO:44, the underline portion comprises the fGly-containing sulfatase motif of SEQ ID NO:146 but, in any embodiment, may be replaced with any one of SEQ ID NOs:147-168. [00477] In further embodiments, the ROR1-ADC comprises an ROR1 antibody, wherein the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 44, and the ROR1 antibody further comprises a light chain comprising an amino acid sequence of SEQ ID NO:28. Additionally or alternatively, there may or may not be an additional K at the C-terminal K of the heavy chain. As it would be understood by one of 137 NAI-1540479824 skill in the art, the C-terminal residue of the heavy chain can be removed, for example, during production of the antibody. [00478] Other suitable sulfatase motif and their location in an antibody can also be used, such as International Publication Nos. WO2012097333, WO2017189432, and WO2018169953, each of which is incorporated herein by reference in its entirety. [00479] In some embodiments, an ROR1-ADC is prepared from an ROR1 antibody, wherein the antibody comprises (i) a heavy chain that is inserted with one or more fGly-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID No: 40 or 44, and (ii) a light chain, such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28. [00480] In some embodiments, any ROR1-ADC as disclosed herein comprises an ROR1 antibody conjugated to a linker-payload as disclosed herein (such as (Ia) or (IIa)), wherein the antibody comprises (i) a heavy chain that is inserted with one or more fGly’-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 44, and (ii) a light chain, such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28. [00481] In some embodiments, Ab of any ADC formula as disclosed herein (such as Formula (I), (II), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)) comprises (i) a heavy chain that is inserted with one or more fGly’-containing sulfatase motifs, such as a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 or 44, and (ii) a light chain, such as the one comprising an amino acid sequence as set forth in SEQ ID NO: 28. [00482] A general scheme for coupling an antibody to a pyridazine-pyrrolo coupling moiety is shown below.
Figure imgf000140_0001
[00483] A hydrazinyl-pyrrolo coupling moiety, which can interchangeably be referred to herein as an aza-hydrazino-iso-Pictet-Spengler (azaHIPS) coupling moiety, upon conjugation to a formyl-glycine, forms a pyridazine-pyrrolo coupling moiety as shown above. An ROR1 antibody can include a 2-formylglycine residue (fGly) that is reacted with azaHIPS coupling 138 NAI-1540479824 moiety, thus conjugating the two together. To generate an ROR1-ADC, a drug can be coupled directly or indirectly (e.g., through a linker moiety) to the azaHIPS moiety at any location of the azaHIPs moiety (e.g., Z1, Z2, Z3, Z4, or Q1). R2 and R3 can each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. Z1, Z2, Z3, and Z4 can be as described herein, such as for Formula (I). Q1 may be -LA-W1, as described herein, such as for Formula (I). [00484] Formula (Ia) below represents a hydrazinyl-pyrrolo coupling moiety that can be used to link an ROR1 antibody and a drug in an ROR1-ADC.
Figure imgf000141_0001
(Ia). Accordingly, provided is an ADC produced by conjugating an ROR1 antibody as disclosed herein to one or more of a linker-payload represented by Formula (Ia). In some embodiments, one or more of the components (such as R2, R3, Z1, Z2, Z3, Z4, LA, or W1) of Formula (Ia) are each as defined herein, such as for Formula (I). In further embodiments, R30 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some embodiments of Formula (Ia), Z1, Z2, Z3 and Z4 are each independently selected from CR4, N and C-LB-W2, wherein at least one Z1, Z2, Z3 and Z4 is C-LB-W2; R30 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; 139 NAI-1540479824 R2 and R3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, 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 R2 and R3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl; each R4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LA is a first linker as described herein; LB is a second linker as described herein; W1 is a first drug as described herein; and W2 is a second drug as described herein. [00485] Formula (X) below represents a hydrazinyl-pyrrolo coupling moiety that can be used to link an ROR1 antibody and a drug in any ROR1-ADC described herein:
Figure imgf000142_0001
wherein: Z1, Z2, Z3 and Z4 are each independently selected from CR4, N and C-LB-W2, wherein at least one Z1, Z2, Z3 and Z4 is C-LB-W2; 140 NAI-1540479824 R30 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; R2 and R3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, 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 R2 and R3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl; each R4 is independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LA is a first linker as described herein; LB is a second linker as described herein; W1 is a first drug as described herein; and W2 is a second drug as described herein. [00486] In some embodiments, provided is an ADC of Formula (I), wherein R1 is hydrogen; Z1 is X1-Y1, wherein X1 represents an atom in the ring of Formula (I) and is selected from the group consisting of C, N, O and S; Z2 is C-Y2; Y1 and Y2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y1 and Y2 are cyclically linked; Z3 is C-LB-W2; 141 NAI-1540479824 Z4 is CH; LA is linker (L-3-a); and LB is linker (L-3-b).
Figure imgf000144_0001
(L-3-b). In some embodiments of linker (L-3-a) and linker (L-3-b), each represents the point of attachment to a pyridazine-pyrrolo coupling moiety as described herein (e.g., in any of Formulas as disclosed herein); * in linker (L-3-a) represents the point of attachment to W1; * in linker (L-3-b) represents the point of attachment to W2; each R5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R6 is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R7 is a cleavable moiety; each k is an integer from 1 to 10; L1a comprises -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-, L1b comprises -(T1-V1)a-(T2-V2)b-(T3-V3)c-(T4-V4)d-, L2a comprises -(T5-V5)e-(T6-V6)f-(T7-V7)g-(T8-V8)h-; L2b comprises -(T5-V5)e-(T6-V6)f-(T7-V7)g-(T8-V8)h-; 142 NAI-1540479824 T1, T2, T3, T4, T5, T6, T7, and T8 are each independently a covalent bond, C1-C12 alkyl, substituted C1-C12 alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA)w, (PEG)n, (AA)p, -(CR13OH)m-, P4A-R12, acetal, a hydrazine, a disulfide, or an ester; V1, V2, V3, V4, V5, V6, V7, and V8 are each independently a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, or -P(O)OH-; each of a, b, c, d, e, f, g, and h is independently 1 or 0; each m is independently an integer from 1 to 12; each n is independently an integer from 1 to 30; each p is independently an integer from 1 to 20; each q is independently an integer from 1 to 6; each w is independently an integer from 1 to 20; each R12 is independently 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, or substituted heterocyclyl; each R13 is independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; and each R15 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00487] Accordingly, in some embodiments, the ROR1-ADC is represented by Formula (XIV-3): 143 NAI-1540479824
wherein: X1 is selected from the group consisting of C, N, O and S; Y1 and Y2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y1 and Y2 are cyclically linked; each R5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R6 is independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R7 is a cleavable moiety; L1a comprises -(T1-V1)a-(T2-V2)b-, L2a comprises -(T5-V5)e-(T6-V6)f-; L1b comprises -(T7-V7)g-(T8-V9)h-, L2b comprises -(T12-V12)l-(T13-V13)m-; 144 NAI-1540479824 T1, T2, T5, and T6 are each independently a covalent bond, C1-C12 alkyl, substituted C1-C12 alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, EDA, (PEG)n, (AA)p, -(CR13OH)v-, 4AP, acetal, a hydrazine, a disulfide, or an ester; V1, V2, V5, and V6 are each independently a covalent bond, -CO-, -NR15-, -NR15(CH2)q-, -NR15(C6H4)-, -CONR15-, -NR15CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR15-, -NR15SO2-, or -P(O)OH-; T7, T8, T12, and T13 are 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, V12, and V13 are 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-; each of a, b, e, g, h, l, and m is independently 1 or 0; each k is an integer from 1 to 10; each p is independently an integer from 1 to 20; each q is independently an integer from 1 to 6; each v is independently an integer from 1 to 12; each z is an integer from 1 to 10; each R12 is independently 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, or substituted heterocyclyl; 145 NAI-1540479824 each R13 is independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl; and each R15 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00488] In some embodiments, provided is an ADC of Formula (I), wherein R1 is hydrogen; Z1 is X1-Y1, wherein X1 represents an atom in the ring of Formula (I) and is selected from the group consisting of C, N, O and S; Z2 is C-Y2; Y1 and Y2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y1 and Y2 are cyclically linked; Z4 is CH; Z3 is C-LB-W2; LA is linker (L-4-a); and LB is linker (L-4-b).
Figure imgf000148_0001
(L-4-b), 146 NAI-1540479824 wherein: each represents the point of attachment to a pyridazine-pyrrolo coupling moiety as described herein (e.g., any of Formulas as disclosed herein); * in linker (L-4-a) represents the point of attachment to W1; * in linker (L-4-b) represents the point of attachment to W2; each R5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R6’ and R6” are each independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; each R7 is a cleavable moiety; and L1a, L2a, L1b, and L2b are as defined herein, such as for linker (L-3-a) or (L-3-b). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00489] Accordingly, in some embodiments, the ROR1-ADC is represented by Formula (XIV-4):
Figure imgf000149_0001
, (XIV-4) wherein each substituent is as defined with respect to Formula (XIV-3). In some embodiments, R7 is an enzymatically cleavable moiety comprising a sugar moiety, such as a 147 NAI-1540479824 glycoside or glycosyl. In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00490] In some embodiments, provided is an ADC of Formula (I), wherein R1 is hydrogen; Z1 is X1-Y1, wherein X1 represents an atom in the ring of Formula (I) and is selected from the group consisting of C, N, O and S; Z2 is C-Y2; Y1 and Y2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y1 and Y2 are cyclically linked; Z4 is CH; Z3 is C-LB-W2; LA is linker (L-5-a); and LB is linker (L-5-b):
Figure imgf000150_0001
148 NAI-1540479824 (L-5-b). wherein: each represents the point of attachment to a pyridazine-pyrrolo coupling moiety as described herein (e.g., any of Formulas as disclosed herein); * in linker (L-5-a) represents the point of attachment to W1; * in linker (L-5-b) represents the point of attachment to W2; each R5 is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; R6’ and R6” are independently alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; and L1a, L2a, L1b, and L2b are as defined herein, such as for linker (L-3-a) or (L-3-b). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00491] Accordingly, in some embodiments, the ROR1-ADC is represented by Formula (XIV-5): ,
Figure imgf000151_0001
149 NAI-1540479824 wherein each substituent is as defined with respect to Formula (XIV-3). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00492] In some embodiments, provided is an ADC of Formula (I), wherein R1 is hydrogen; Z2 is C-Y2; Z1 is X1-Y1, wherein X1 represents an atom in the ring of Formula (I) and is selected from the group consisting of C, N, O and S; Y1 and Y2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y1 and Y2 are cyclically linked; Z4 is CH; Z3 is C-LB-W2; LA is linker (L-6-a); and LB is linker (L-6-b):
Figure imgf000152_0001
150 NAI-1540479824 (L-6-b), wherein: each represents the point of attachment to a pyridazine-pyrrolo coupling moiety (e.g., any of Formulas as disclosed herein); * in linker (L-6-a) represents the point of attachment to W1; * in linker (L-6-b) represents the point of attachment to W2; and L1a, L2a, L1b, and L2b are as defined herein, such as for linker (L-3-a) or (L-3-b). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00493] Accordingly, in some embodiments, the ROR1-ADC is represented by Formula (XIV-6):
Figure imgf000153_0001
, (XIV-6) wherein each substituent is as defined with respect to Formula (XIV-3). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00494] In some embodiments, provided is an ADC of Formula (I), wherein R1 is hydrogen; Z1 is X1-Y1, wherein X1 represents an atom in the ring of Formula (I) and is selected from the group consisting of C, N, O and S; Z2 is C-Y2; 151 NAI-1540479824 Y1 and Y2 are each independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 substituted heterocyclyl, and absent when adjacent to N; or Y1 and Y2 are cyclically linked; Z4 is CH; Z3 is C-LB-W2; LA is linker (L-7-a); and LB is linker (L-7-b):
Figure imgf000154_0001
(L-7-b), wherein: 152 NAI-1540479824 each represents the point of attachment to a pyridazine-pyrrolo coupling moiety (e.g., any of Formulas as disclosed herein); * in linker (L-7-a) represents the point of attachment to W1; * in linker (L-7-b) represents the point of attachment to W2; and L2a and L2b are as defined herein, such as for linker (L-3-a) or (L-3-b). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00495] Accordingly, in some embodiments, the ROR1-ADC is represented by Formula (XIV-7):
Figure imgf000155_0001
, (XIV-7) wherein each substituent is as defined with respect to Formula (XIV-3). In some embodiments, each substituent is as defined herein, such as with respect to Formula (I). [00496] In some embodiments of any Formula as disclosed herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), L2a is -CO-. Additionally or alternatively, L2b is -CO-. In some embodiments, both L2a and L2b are -CO-. [00497] In certain embodiments, at least one of W1 and W2 (e.g., in Formula (I), Formula (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)) is belotecan. In some embodiments, both W1 and W2 are belotecan. 153 NAI-1540479824 [00498] In some embodiments of any Formula as disclosed herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), W1 comprises a camptothecin or an analog thereof, such as belotecan. In further embodiments, W1 comprises belotecan. [00499] In some embodiments of any Formula as disclosed herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), W2 comprises a camptothecin or an analog thereof, such as belotecan. In yet further embodiments, W2 comprises belotecan. Additionally or alternatively, W1 and W2 are the same. In other embodiments, W1 and W2 are different. [00500] For example, an ROR1-ADC of Formula (II) may be prepared by conjugating one or more linker-payloads of Formula (IIa), shown below, to an ROR1 antibody:
Figure imgf000156_0001
[00501] In some embodiments of any Formula as disclosed herein (such as Formula (I), (II), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), s is an integer from 1 to 10, for example 1 to 8. In some embodiments, s is 2. In some embodiments, s is 4. [00502] In some embodiments of any Formula as disclosed herein (such as Formula (I), (II), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), Ab is an ROR antibody as disclosed herein. For example, see the descriptions and embodiments relating to an ROR antibody detailed below in Sections 7.3 and 7.4. 154 NAI-1540479824 [00503] An exemplary scheme for making an ROR1-ADC is shown in Scheme A:
Figure imgf000157_0001
155 NAI-1540479824 [00504] In Scheme A, the linker-drug is conjugated to an ROR1 antibody. The ROR1 antibody has a recognition motif, for example, a L(C/S)TPSR (SEQ ID NO:99) recognition motif in each of the CH1 constant region and c-terminus (CT) of the heavy chain (e.g., SEQ ID NO:29, or 40, or 41 as described above). The cysteine residue of a L(C/S)TPSR (SEQ ID NO:99) recognition motif can be converted to a formylglycine (fGly) and subsequently conjugated to an azaHIPs moiety through a Pictet-Spengler reaction (fGly’). [00505] In some embodiments, the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 41 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 28. [00506] Another exemplary scheme for making an ROR1-ADC is shown in Scheme B:
Figure imgf000158_0001
156 NAI-1540479824
[00507] In Scheme B, the linker-drug is conjugated to an ROR1 antibody. The ROR1 antibody has a recognition motif, for example, a LCTPSR (SEQ ID NO:100) recognition motif in the CH1 constant region of the heavy chain (e.g., SEQ ID NO:30, 42, 43, or 44 as described above). The cysteine residue of a LCTPSR (SEQ ID NO:100) recognition motif can be converted to a formylglycine (fGly) and subsequently conjugated to an azaHIPs moiety through a Pictet-Spengler reaction. [00508] In some embodiments, the ROR1 antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 44 and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 28. [00509] In some embodiments, the ROR1-ADC is ADC-8, wherein the ROR1-ADC is of Formula (II), s is 4, the antibody Ab is A27 as described herein and the conjugation sites of the pyridazine-pyrrolo moiety to the antibody are in the CH1 and CT regions of each heavy chain of the antibody, as shown in Scheme A. Accordingly, the DAR of ADC-8 is 8. [00510] In some embodiments, the antibody Ab of ADC-8 comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:41 and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:28. [00511] In some embodiments, the ROR1-ADC is ADC-4, wherein the ROR1-ADC is of Formula (II), s is 2, and the antibody Ab is A27 as described herein and the conjugation sites 157 NAI-1540479824 of the pyridazine-pyrrolo moiety to the antibody are in the CH1 regions of each heavy chain of the antibody, as shown in Scheme B. Accordingly, the DAR of ADC-8 is 4. [00512] In some embodiments, the antibody Ab of ADC-4 comprises a heavy chain comprising the amino acid sequence as set forth in SEQ ID NO:44 and a light chain comprising the amino acid sequence as set forth in SEQ ID NO:28. [00513] In some embodiments, an ROR1-ADC includes one or more linker-drug conjugated to each heavy chain constant region of an ROR1 antibody via a pyridazine-pyrrolo coupling moiety and for example, is characterized by a stoichiometric ratio of antibody to drug (DAR) 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. [00514] In some embodiment, the ROR1-ADC is an ADC as described below in Table 3. Table 3: Exemplary ROR1-ADCs
Figure imgf000160_0001
7.5 DRUGS FOR CONJUGATION [00515] As noted above, a conjugate or a compound of the present disclosure can include as substituents W1 and W2 a drug or active agent. Any of a number of drugs are suitable for use or can be modified to be rendered suitable for use, as a reactive partner to conjugate to an antibody. Examples of drugs include small molecule drugs and peptide drugs. [00516] “Small molecule drug” as used herein refers to a compound, e.g., an organic compound, which exhibits a pharmaceutical activity of interest and which is generally of a molecular weight of 800 Da or less, or 2000 Da or less, but can encompass molecules of up to 5kDa and can be as large as 10 kDa. A small inorganic molecule refers to a molecule containing no carbon atoms, while a small organic molecule refers to a compound containing at least one carbon atom. [00517] For example, the drug or active agent can be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as a camptothecin, or an analog or derivative thereof, or a pharmaceutically active camptothecin moiety and/or a portion thereof. A topoisomerase inhibitor (e.g., camptothecin, or analog or derivative thereof) conjugated to the ROR1 antibody can be any of a variety of topoisomerase inhibitors, for example camptothecin or 158 NAI-1540479824 camptothecin moieties such as, but not limited to, camptothecin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to, a topoisomerase inhibitor, for example camptothecin or a camptothecin derivative, such as SN-38, Belotecan, Exatecan, 9- aminocamptothecin (9-AC), topotecan, des-Me-topotecan, derivatives thereof, and the like. Additional examples of topoisomerase inhibitors that find use in the present disclosure are described in PCT/US2022/012325, the disclosure of which is incorporated herein by reference. [00518] In other embodiments, the drug or active agent can be a maytansine. “Maytansine,” “maytansine moiety,” “maytansine active agent moiety” and “maytansinoid” refer to a maytansine and analogs and derivatives thereof, and pharmaceutically active maytansine moieties and/or portions thereof. A maytansine conjugated to the ROR1 antibody can be any of a variety of maytansinoid moieties such as, but not limited to, maytansine and analogs and derivatives thereof as described herein (e.g., deacylmaytansine). [00519] In other instances, the drug or active agent can be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or a portion thereof. An auristatin conjugated to the ROR1 antibody can be any of a variety of auristatin moieties such as, but not limited to, an auristatin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include but are not limited to an auristatin or an auristatin derivative, such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), derivatives thereof, and the like. [00520] In other cases, the drug or active agent can be a duocarmycin, or an analog or derivative thereof, or a pharmaceutically active duocarmycin moiety and/or a portion thereof. A duocarmycin conjugated to the ROR1 antibody can be any of a variety of duocarmycin moieties such as, but not limited to, a duocarmycin and analogs and derivatives thereof as described herein. Examples of drugs that find use in the conjugates and compounds described herein include but are not limited to a duocarmycin or a duocarmycin derivative, such as duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065, derivatives thereof, and the like. In some embodiments, the duocarmycin is a duocarmycin analog, such as, but not limited to, adozelesin, bizelesin, or carzelesin. 159 NAI-1540479824 [00521] In certain embodiments, the drug is selected from a cytotoxin, a kinase inhibitor, a selective estrogen receptor modulator, an immunostimulatory agent, a toll-like receptor (TLR) agonist, an oligonucleotide, an aptamer, a cytokine, a steroid, and a peptide. [00522] For example, a cytotoxin can include any compound that leads to cell death (e.g., necrosis or apoptosis) or a decrease in cell viability. [00523] Kinase inhibitors can include, but are not limited to, Adavosertib, Afatinib, Axitinib, Bosutinib, Cetuximab, Cobimetinib, Crizotinib, Cabozantinib, Dacomitinib, Dasatinib, Entrectinib, Erdafitinib, Erlotinib, Fostamatinib, Gefitinib, Ibrutinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Pazopanib, Pegaptanib, Ruxolitinib, Sorafenib, Sunitinib, Tucatinib, Vandetanib, Vemurafenib, and the like. [00524] For example, selective estrogen receptor modulators include, but are not limited to, Endoxifen, Tamoxifen, Afimoxifene, Toremifene, and the like. [00525] Immunostimulatory agents can include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony stimulating factors, interferons, interleukins, and the like. TLR agonists include, but are not limited to, imiquimod, resiquimod, and the like. [00526] Oligonucleotide dugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golodirsen, viltolarsen, volanesorsen, inotersen, tofersen, tominersen, and the like. [00527] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, NOX-E36, and the like. [00528] Cytokines include, but are not limited to, Albinterferon Alfa-2B, Aldesleukin, ALT- 801, Anakinra, Ancestim, Avotermin, Balugrastim, Bempegaldesleukin, Binetrakin, Cintredekin Besudotox, CTCE-0214, Darbepoetin alfa, Denileukin diftitox, Dulanermin, Edodekin alfa, Emfilermin, Epoetin delta, Erythropoietin, Human interleukin-2, Interferon alfa, Interferon alfa-2c, Interferon alfa-n1, Interferon alfa-n3, Interferon alfacon-1, Interferon beta-1a, Interferon beta-1b, Interferon gamma-1b, Interferon Kappa, Interleukin-1 alpha, Interleukin-10, Interleukin-7, Lenograstim, Leridistim, Lipegfilgrastim, Lorukafusp alfa, Maxy-G34, Methoxy polyethylene glycol-epoetin beta, Molgramostim, Muplestim, Nagrestipen, Oprelvekin, Pegfilgrastim, Pegilodecakin, Peginterferon alfa-2a, Peginterferon alfa-2b, Peginterferon beta-1a, Peginterferon lambda-1a, Recombinant CD40-ligand, Regramostim, Romiplostim, Sargramostim, Thrombopoietin, Tucotuzumab celmoleukin, Viral Macrophage-Inflammatory Protein, and the like. [00529] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like. 160 NAI-1540479824 [00530] “Peptide drug” as used herein refers to amino acid-containing polymeric compounds and is meant to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, and proteins, as well as peptide mimetics. The peptide drugs may be obtained by chemical synthesis or be produced from a genetically encoded source (e.g., recombinant source). Peptide drugs can range in molecular weight and can be from 200 Da to 10 kDa or greater in molecular weight. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; anti-angiogenic peptides; peptides that activate B cells; peptides that activate T cells; anti-viral peptides; peptides that inhibit viral fusion; peptides that increase production of one or more lymphocyte populations; anti- microbial peptides; growth factors; growth hormone-releasing factors; vasoactive peptides; anti-inflammatory peptides; peptides that regulate glucose metabolism; an anti-thrombotic peptide; an anti-nociceptive peptide; a vasodilator peptide; a platelet aggregation inhibitor; an analgesic; and the like. [00531] Additional examples of drugs that find use in the conjugates and compounds described herein include, but are not limited to Tubulysin M, Calicheamicin, a STAT3 inhibitor, alpha-Amanitin, an aurora kinase inhibitor, belotecan, and an anthracycline. [00532] Other examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where a tumor cell expresses or overexpresses an ROR1 antigen, the ROR1 antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptidic (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. [00533] 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 antibody-drug conjugate of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1-TM1); and 161 NAI-1540479824 benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD). [00534] Agents that act to reduce cellular proliferation are known in the art and widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide. [00535] Antimetabolite agents include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6- thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10- propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatine, and gemcitabine. [00536] Suitable natural products and their derivatives, (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L- asparaginase, azathioprine; brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g., etoposide, teniposide, etc.; antibiotics, e.g., anthracycline, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g., dactinomycin; basic glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mithramycin); anthracenediones, e.g., mitoxantrone; azirinopyrrolo indolediones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, etc.; and the like. [00537] Other anti-proliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine. [00538] 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 162 NAI-1540479824 sulfate, vincristine sulfate, natural and synthetic epothilones including but not limited to, eopthilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like. [00539] Hormone modulators and steroids (including synthetic analogs) that are suitable for use include, but are not limited to, adrenocorticosteroids, e.g., prednisone, dexamethasone, etc.; estrogens and pregestins, e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocortical suppressants, e.g., aminoglutethimide; 17α-ethinylestradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyl- testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®. Estrogens stimulate proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids may inhibit T cell proliferation. [00540] Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis- DDP), carboplatin, etc.; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other anti-proliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy- 6-(3-(4-morpholinyl)propoxy)quinazoline); etc. [00541] 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^, TAXOTERETM (a formulation of docetaxel), 10-desacetyl analogs of paclitaxel and 3’N- desbenzoyl-3’N-t-butoxycarbonyl analogs of paclitaxel) may be readily prepared utilizing techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos.5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtained from a variety of commercial sources, including for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis). [00542] Paclitaxel should be understood to refer to not only the common chemically available form of paclitaxel, but analogs and derivatives (e.g., Taxotere^ docetaxel, as noted above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel- xylose). 163 NAI-1540479824 [00543] Also included within the term “taxane” are a variety of known derivatives, including both hydrophilic derivatives, and hydrophobic derivatives. Taxane derivatives include, but not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Patent No.5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamide derivatives described in U.S. Patent No.5,821,263; and taxol derivative described in U.S. Patent No.5,415,869. It further includes prodrugs of paclitaxel including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Patent No.5,824,701. [00544] 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. [00545] Examples of drugs include small molecule drugs, such as a cancer chemotherapeutic agent. For example, where a tumor cell expresses or overexpresses tyrosine-protein kinase transmembrane receptor 1 (ROR1) antigens, the ROR1 antibody can be produced as described herein to include a modified amino acid, which can be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule affecting agent. In certain embodiments, the drug is a microtubule affecting agent that has antiproliferative activity, such as a maytansinoid. [00546] Embodiments of the present disclosure include conjugates where an antibody is conjugated to two or more drug moieties, such as 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, 10 drug moieties, 11 drug moieties, 12 drug moieties, 13 drug moieties, 14 drug moieties, 15 drug moieties, 16 drug moieties, 17 drug moieties, 18 drug moieties, 19 drug moieties, or 20 or more drug moieties. The drug moieties may be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugates have an average drug-to-antibody ratio (DAR) (molar ratio) in the range of from 0.1 to 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In certain embodiments, the conjugates have an average DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the 164 NAI-1540479824 conjugates have an average DAR of 1 to 10. In certain embodiments, the conjugates have an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugates have an average DAR of 5 to 10 (e.g., 8). By average is meant the arithmetic mean. [00547] In certain embodiments, the two drugs or active agents attached to the branched linker are the same drug or active agent. For example, a first branch of a branched linker may be attached to a drug, or an active agent and a second branch of the branched linker may be attached to the same drug or the same active agent as the first branch. In other embodiments, the two drugs or active agents attached to the branched linker are different drugs or active agents. For example, a first branch of a branched linker may be attached to a first drug, or a first active agent and a second branch of the branched linker may be attached to a second drug or a second active agent different from the first drug or the first active agent attached to the first branch. [00548] In some embodiments, where two different drugs or active agents are attached to the branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. For example, in some instances, the use of two different drugs or active agents attached to the branched linker may provide a lower therapeutically effective concentration at which both payloads act, thereby increasing overall potency of the ADC. [00549] 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). [00550] 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. [00551] In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an auristatin (e.g., MMAE) as described herein. In some 165 NAI-1540479824 embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and an iRGD peptide as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and an iRGD peptide as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a kinase inhibitor (e.g., Sorafenib, Lapatinib, Gefitinib, and the like) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are an auristatin (e.g., MMAE) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein. In some embodiments, where two different drugs or active agents are attached to the branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., belotecan) as described herein and a selective estrogen receptor modulator (e.g., Endoxifen) as described herein. [00552] Drugs to be conjugated to an ROR1 antibody may be modified to incorporate a reactive partner for reaction with the ROR1 antibody. Where the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide can be positioned at an N-terminal region, the N-terminus, a C-terminal region, the C-terminus, or at a position internal to the peptide. For example, an example of a method involves synthesizing a peptide drug having an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. An amino group of a peptide can react with a compound comprising a carboxylic acid group and oxy-N- Boc group. As an example, the amino group of the peptide reacts with 3-(2,5- dioxopyrrolidin-1-yloxy)propanoic acid. Other variations on the compound comprising a carboxylic acid group and oxy-N-protecting group can include different number of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound comprising a carboxylic acid group and oxy-N- protecting group occurs through standard peptide coupling chemistry. Examples of peptide coupling reagents that can be used include, but not limited to, DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP 166 NAI-1540479824 (1-benzotriazole diethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoroformamidinium hexafluorophosphosphate), DPPA (diphenylphosphorazidate), BOP (benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium tetrafluoroborate), TSTU (O-(N-succinimidyl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridin-1- ylmethylene]- -N-methylmethanaminium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo- 3-oxazolidinyl)phosphinic chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy)- tris(pyrrolidino)phosphonium tetrafluorophopsphate), BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). As a non-limiting example, HOBt and DIC can be used as peptide coupling reagents. [00553] Deprotection to expose the amino-oxy functionality is performed on the peptide comprising an N-protecting group. Deprotection of the N-oxysuccinimide group, for example, occurs according to standard deprotection conditions for a cyclic amide group. Deprotecting conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions include a hydrazine reagent, amino reagent, or sodium borohydride. Deprotection of a Boc protecting group can occur with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. The product and intermediates can be purified by conventional means, such as HPLC purification. [00554] The ordinarily skilled artisan will appreciate that factors such as pH and steric hindrance (e.g., the accessibility of the amino acid residue to reaction with a reactive partner of interest) are of importance, modifying reaction conditions to provide for optimal conjugation conditions is well within the skill of the ordinary artisan, and is routine in the art. Where conjugation is conducted with an ROR1 antibody present in or on a living cell, the conditions are selected so as to be physiologically compatible. For example, the pH can be dropped temporarily for a time sufficient to allow for the reaction to occur but within a period tolerated by the cell (e.g., from about 30 min to 1 hour). Physiological conditions for conducting modification of antibodies on a cell surface can be similar to those used in a 167 NAI-1540479824 ketone-azide reaction in modification of cells bearing cell-surface azides (see, e.g., U.S. 6,570,040). [00555] Small molecule compounds containing, or modified to contain, an α-nucleophilic group that serves as a reactive partner with a compound or conjugate disclosed herein are also contemplated for use as drugs in the antibody-drug conjugates of the present disclosure. General methods are known in the art for chemical synthetic schemes and conditions useful for synthesizing a compound of interest (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). 7.6 FORMULATIONS & COMPOSITIONS [00556] ROR1-ADCs can be included in a pharmaceutical composition for administration, e.g., to a subject for treating a disease, disorder, or condition. A pharmaceutical composition comprises ROR1-ADCs which, on average, can exhibit a drug-to-antibody ratio (“DAR”) of about 0.1 to about 20, or from 0.5 to 20, or from 1 to 20. In other words, each antibody is conjugated to one or two linker-drug conjugates. Methods to determine DAR are well known to the skilled person and include methods using Reverse Phase Chromatography, or HPLC- MS. [00557] For example, in any embodiment, a pharmaceutical composition comprising an ROR1-ADC exhibits a DAR of about 0.1 to about 20, or from 0.5 to 20, or from 1 to 20, such as from 1 to 19, or from 1 to 18, or from 1 to 17, or from 1 to 16, or from 1 to 15, or from 1 to 14, or from 1 to 13, or from 1 to 12, or from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2, or from 2 to 20, or from 2 to 19, or from 2 to 18, or from 2 to 17, or from 2 to 16, or from 2 to 15, or from 2 to 14, or from 2 to 13, or from 2 to 12, or from 2 to 11, or from 2 to 10, or from 2 to 9, or from 2 to 8, or from 2 to 7, or from 2 to 6, or from 2 to 5, or from 2 to 4, or from 2 to 3, or from 3 to 20, or from 3 to 19, or from 3 to 18, or from 3 to 17, or from 3 to 16, or from 3 to 15, or from 3 to 14, or from 3 to 13, or from 3 to 12, or from 3 to 11, or from 3 to 10, or from 3 to 9, or from 3 to 8, or from 3 to 7, or from 3 to 6, or from 3 to 5, or from 3 to 4, or from 4 to 20, or from 4 to 19, or from 4 to 18, or from 4 to 17, or from 4 to 16, or from 4 to 15, or from 4 to 14, or from 4 to 13, or from 4 to 12, or from 4 to 11, or from 4 to 10, or from 4 to 9, or from 4 to 8, or from 4 to 7, or from 4 to 6, or from 4 to 5, or from 5 to 20, or from 5 to 19, or from 5 to 18, or from 5 to 17, or from 5 to 16, or from 5 to 15, or from 5 to 14, or 168 NAI-1540479824 from 5 to 13, or from 5 to 12, or from 5 to 11, or from 5 to 10, or from 5 to 9, or from 5 to 8, or from 5 to 7, or from 5 to 6, or from 6 to 20, or from 6 to 19, or from 6 to 18, or from 6 to 17, or from 6 to 16, or from 6 to 15, or from 6 to 14, or from 6 to 13, or from 6 to 12, or from 6 to 11, or from 6 to 10, or from 6 to 9, or from 6 to 8, or from 6 to 7, or from 7 to 20, or from 7 to 19, or from 7 to 18, or from 7 to 17, or from 7 to 16, or from 7 to 15, or from 7 to 14, or from 7 to 13, or from 7 to 12, or from 7 to 11, or from 7 to 10, or from 7 to 9, or from 7 to 8. In certain embodiments, the composition exhibits a DAR from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the composition exhibits a DAR of 1 to 10. In certain embodiments, the composition exhibits a DAR of 1 to 5 (e.g., 3 or 3.5 or 4). In certain embodiments, the composition exhibits a DAR of 5 to 10 (e.g., 6, or 6.5, or 7, or 7.5 or 8). In some embodiments, a pharmaceutical composition comprises an ROR1-ADC comprising an ROR1 antibody as described herein, including A27 (see, e.g., Tables 1-2), and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises ROR1-ADC ADC-4 or ADC-8 (see, e.g., Table 3), and a pharmaceutically acceptable excipient. [00558] In some embodiments, provided herein is a pharmaceutical composition comprising an ROR1-ADC of any formula as disclosed herein (such as Formula (I), (II), (XIV-3), (XIV- 4), (XIV-5), (XIV-6), or (XIV-7)) and a pharmaceutically acceptable excipient. In some embodiments, provided herein is a pharmaceutical composition comprising a population of ROR1-ADCs, wherein the ROR1-ADCs are of a same formula as disclosed herein (such as any one of Formula (I), (II), (XIV-3), (XIV-4), (XIV-5), (XIV-6), or (XIV-7)), yet, their s are different. Accordingly, the pharmaceutical composition may exhibit various DAR, such as about 1 to about 10, about 1 to about 8, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 169 NAI-1540479824 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0. [00559] An ROR1-ADC can be formulated in any of a variety of different ways. An ROR1- ADC of the present disclosure can be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and can be formulated for any suitable route of administration, e.g., oral, topical, or parenteral administration. Where an ROR1-ADC is provided as a liquid injectable (such as in those embodiments where they it can be administered intravenously or directly into a tissue), an ROR1-ADC can be provided as a ready-to-use dosage form, or as a storage-stable powder for reconstitution or liquid composed of pharmaceutically acceptable excipients and excipients. [00560] Methods for formulating an ROR1-ADC can be adapted from those available in the art. For example, ROR1-ADCs can be provided in a pharmaceutical composition comprising an effective amount of an ROR1-ADC and a pharmaceutically acceptable excipient (e.g., saline). The pharmaceutical composition can optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like). In some embodiments, the formulations are suitable for administration to a mammal, such as those that are suitable for administration to a human. [00561] Also provided herein are pharmaceutical compositions that contain an effective amount of an ROR1-ADC described herein and a pharmaceutically acceptable excipient. In some embodiments, the ROR1-ADC comprises an ROR1 antibody as described herein, including A27, as described in any one of Tables 1-2. In some embodiments, a pharmaceutical composition comprises an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)) and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises an effective amount of ADC-4 or ADC-8 (see, e.g., Table 3) and a pharmaceutically acceptable excipient. [00562] In some embodiments, a pharmaceutical composition comprises an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)) and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises an effective amount of ADC-4 or ADC-8 (see, e.g., Table 3) and a pharmaceutically acceptable excipient. [00563] The pharmaceutically acceptable excipient can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances that are suitable for administration into a human or veterinary subject (e.g., a physiologically acceptable and/or pharmacologically acceptable). The pharmaceutically acceptable excipient can be co-mingled with one or more of the active components, e.g., a hybrid molecule, and with each other, 170 NAI-1540479824 when more than one pharmaceutically acceptable excipient is present in the pharmaceutical composition, in a manner so as not to substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable materials typically are capable of administration to a subject without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable excipient not to be immunogenic when administered to a human subject for therapeutic purposes. [00564] Pharmaceutical compositions of the invention can additionally contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The pharmaceutical compositions can also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. Pharmaceutical compositions of the invention can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the antibody or antigen-binding fragment of the invention into association with an excipient that constitutes one or more accessory ingredients. In general, the pharmaceutical composition is prepared by uniformly and intimately bringing the active agent into association with a liquid excipient, a finely divided solid excipient, or both, and then, if necessary, shaping the product. [00565] A composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the composition, which preferably is isotonic with the blood of the recipient. This aqueous preparation can be formulated of known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed, such as synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Excipient formulations suitable for oral, subcutaneous, intravenous, intramuscular, and the like, administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. [00566] Preparation of pharmaceutical compositions of the invention and their various routes of administration can be carried out in accordance with methods well known in the art. The delivery systems useful in the context of the invention include time-released, delayed 171 NAI-1540479824 release, and sustained release systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. The composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician, and can be particularly suitable for some compositions of the invention. [00567] Many types of release delivery systems are available and known to those of ordinary skill in the art. Suitable release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are lipids such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides; hydrogel release systems; sylastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include but are not limited to: (a) erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Patents 4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patents 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation. [00568] Generally, an ROR1-ADC or pharmaceutical composition is suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic method. Components can be provided as concentrates (including lyophilized compositions), which can be further diluted prior to use, or they can be provided at the concentration of use. For use of an ROR1-ADC in vivo, single dosages can be provided in sterilized containers having the desired amount and concentration of components. 7.7 METHODS OF TREATMENT [00569] Also provided herein are methods of treating, preventing, or alleviating an ROR1- mediated disease, disorder, or condition, including one or more symptoms of the ROR1- mediated disease, disorder, or condition with an ROR1-ADC comprising an ROR1 antibody, and a drug conjugated directly or indirectly thereto. Also provided herein are methods of killing tumor cells with an ROR1-ADC comprising an ROR1 antibody, and a drug conjugated directly or indirectly thereto. 172 NAI-1540479824 [00570] The antibody that binds to ROR1 can include any as described herein, such as A27, as described in any one of Tables 1-2. In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of Formula (I). In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC comprising a camptothecin analogue (e.g., belotecan), or any derivative thereof. In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)). In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of ADC-4 or ADC-8 (see, e.g., Table 3). In some embodiments, the contacting is in vivo or in vitro. [00571] In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC comprising a camptothecin analogue (e.g., belotecan), or any derivative thereof. In some embodiments, a method of killing a tumor cell comprises contacting the tumor cell with an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)). In some embodiments, the contacting is in vivo or in vitro. [00572] In any embodiment, the method of contacting the tumor cell with an ROR1-ADC comprises contacting the tumor cell with a composition comprising the ROR1-ADC and one or more pharmaceutically acceptable excipients. In some embodiments, the ROR1-ADC in the composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8. [00573] Also provided herein is a method of treating a cancer in a subject in need thereof comprising administering an effective amount of an ROR1-ADC to the subject, wherein the ROR1-ADC comprises an ROR1 antibody and a drug conjugated thereto via a linker, as described herein. In some embodiments, the ROR1 antibody is A27, as described herein, such as in Tables 1-2. In some embodiments, the ROR1 is ADC-4 or ADC-8 (see, e.g., Table 3). [00574] In some embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)). [00575] In some embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)). 173 NAI-1540479824 [00576] In any embodiment, the method of treating cancer in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising the ROR1- ADC and a pharmaceutically acceptable excipient. In some embodiments, the ROR1-ADC in the pharmaceutical composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8. [00577] In some embodiments, the method of treating cancer in a subject in need thereof is effective to enhance the removal of the cancer cells, for example, tumor cells, in the subject. [00578] “Enhancing” the removal of cancer or tumor cells does not require a 100% enhancement of removal. Any enhancement in the rate of removal is contemplated. Similarly, “modulating” tumor growth refers to reducing the size of the tumor, slowing tumor growth, or inhibiting an increase in the size of an existing tumor. Complete abolition of a tumor is not required; any decrease in tumor size or slowing of tumor growth constitutes a beneficial biological effect in a subject. In this regard, tumor cell removal can be enhanced by, for example, at least about 5%, at least about 10% or at least about 20% compared to levels of removal observed in the absence of the method (e.g., in a biologically matched control subject or specimen that is not exposed to the agent of the method). The effect is detected by, for example, a reduction in tumor size, a decrease or maintenance of the levels of tumor markers, or reduction or maintenance of a tumor cell population. In some embodiments, removal of tumor cells is enhanced by, for example, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more (about 100%) compared to the removal of tumor cells in the absence of an ROR1 antibody of the method. [00579] The present disclosure also provides a method of modulating (e.g., inhibiting, reducing, preventing) tumor growth in a subject in need thereof, the method comprising administering to the subject an ROR1-ADC comprising an ROR1 antibody and a drug linked thereto via a linker, as described herein, in an amount effective to modulate the tumor growth in the subject. In some embodiments, a method of modulating tumor growth in a subject in need thereof comprising administering an effective amount of an ROR1-ADC comprising an ROR1 antibody and a drug conjugated thereto via a linker, as described herein. In some embodiments, the antibody that binds to ROR1 is A27, as described in any one of Tables 1-2. In some embodiments, the ROR1-ADC is ADC-4 or ADC-8 (see, e.g., Table 3). In some embodiments, a method of modulating tumor growth in a subject in need thereof comprises 174 NAI-1540479824 administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)). [00580] In some embodiments, a method of modulating tumor growth in a subject in need thereof comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV- 7), or (II)). [00581] In any embodiment, the method of modulating tumor growth in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising the ROR1-ADC and one or more pharmaceutically acceptable excipients. In some embodiments, the ROR1-ADC in the pharmaceutical composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8. [00582] In some embodiments, the cancer and tumor cells described herein that may be treated and/or killed by the methods described herein express an ROR1 antigen, for example, as on a surface of the cancer or tumor cell. In some embodiments, a tumor or cancer cell may overexpress an ROR1 antigen. As used herein, the term “overexpress” means to transcribe and translate more genetic product than normal (such as in normal cells), the process of which is often a characteristic of cancer cells. [00583] Therefore, the present disclosure also provides a method of alleviating or reducing side effects associated with cancer comprising administering an effective amount of an ROR1-ADC to a subject in need thereof. In some embodiments, a method of alleviating or reducing side effects associated with cancer comprises administering an effective amount of an ROR1-ADC comprising an ROR1 antibody and a drug conjugated thereto via a linker, as described herein. In some embodiments, the antibody that binds to ROR1 is A27, as described in any one of Tables 1-2. In some embodiments, a method of alleviating or reducing side effects associated with cancer comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I) or (II)). In some embodiments, a method of alleviating or reducing side effects associated with cancer comprises administering to the subject an effective amount of ADC-4 or ADC-8 (see, e.g., Table 3). [00584] In some embodiments, a method of alleviating or reducing side effects associated with cancer comprises administering to the subject an effective amount of an ROR1-ADC of any formula as described herein (such as Formula (I), (XIV-3), (XIV-4), (XIV-5), (XIV-6), (XIV-7), or (II)). 175 NAI-1540479824 [00585] In any embodiment, the method of alleviating or reducing side effects associated with cancer in a subject in need thereof comprises administering to the subject a pharmaceutical composition comprising an ROR1-ADC as disclosed herein and one or more pharmaceutically acceptable excipients. In some embodiments, the ROR1-ADC in the pharmaceutical composition is characterized by a DAR of about 1 to about 20, about 1 to about 10, about 2 to about 4, about 3 to about 4, about 5 to about 8, about 6 to about 8, about 6 to about 7, or about 7 to about 8. [00586] Additionally, an ROR1-ADC comprising an ROR1 antibody can be used to treating, preventing, or alleviating an ROR1-mediated disease, disorder, or condition, including one or more symptoms of the ROR1-mediated disease, disorder, or condition with an ROR1-ADC comprising an ROR1 antibody. [00587] In another aspect, provided herein is a method of detecting ROR1 antigen in a subject having or suspected of having a disease or condition, the method comprising: (a) administering to the subject any antibody above; and (b) detecting the presence or the level of ROR1 antigen in the subject. [00588] Also provided is a method for treating a subject with cancer. The method comprises administering a therapeutically effective amount of the ADC as disclosed herein or the pharmaceutical composition as disclosed herein to the subject. In some embodiments, the cancer is an ROR1 antigen expressing cancer. [00589] In some embodiments, the disease or condition is cancer. The cancer may be a cancer from the bladder, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), bone, bone marrow, brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), breast, colon, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), gastrointestinal, gum, head, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia, renal cell carcinoma), liver, lung, nasopharynx, neck, ovary, prostate (adenocarcinoma, sarcoma, castrate resistant prostate cancer), skin, stomach (carcinoma, lymphoma, leiomyosarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma), tongue, or uterus. In some embodiments, the cancer may be a neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal 176 NAI-1540479824 cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma); fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; 177 NAI-1540479824 protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma (reticulum cell sarcoma); Hodgkin’s disease; Hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; myxoma; rhabdomyoma; fibroma; squamous cell carcinomas of the head and neck; laryngeal and hypopharyngeal cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; salivary gland cancer; oral; orppharyngeal cancer; bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, non-small cell lung cancer); alveolar (bronchiolar) carcinoma; bronchial adenoma; chondromatous hamartoma; colorectal cancer; gastrointestinal stromal tumors; carcinoids; Turcot Syndrome; gastric cancer; gastroesophageal junction adenocarcinoma; pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma); small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); metastatic breast cancer; ductal carcinoma in situ; invasive ductal carcinoma; tubular carcinoma; mucinous carcinoma; lobular carcinoma in situ; triple negative breast cancer; bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, urothelial carcinoma); clear cell carcinoma; hepatoma (hepatocellular carcinoma); angiosarcoma; hepatocellular adenoma; hemangioma; osteogenic sarcoma (osteosarcoma); malignant fibrous histiocytoma; malignant giant cell tumor chordoma; osteochrondroma (osteocartilaginous exostoses); benign chondroma; chondromyxofibroma; osteoid osteoma; giant cell tumors; medullary thyroid cancer; differentiated thyroid cancer; papillary thyroid cancer; follicular thyroid cancer; Hürthle cell cancer; anaplastic thyroid cancer; skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans); meninges (meningioma, meningiosarcoma, gliomatosis); spinal cord (neurofibroma, meningioma, glioma, sarcoma); uterus (clear); cervix (cervical carcinoma, pre‑tumor cervical dysplasia); 178 NAI-1540479824 ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa‑thecal cell tumors, Sertoli--Leydig cell tumors, dysgerminoma, malignant teratoma); vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma); vagina (clear cell carcinoma, squamous cell carcinoma); botryoid sarcoma (embryonal rhabdomyosarcoma); fallopian tubes (carcinoma); non‑Hodgkin's lymphoma [malignant lymphoma]; Karposi's sarcoma; moles dysplastic nevi; angioma; dermatofibroma; keloids; psoriasis; neuroblastoma; adrenocortical carcinoma; pheochromocytomas; paragangliomas; Merkel cell carcinoma; pancreatic neuroendocrine and carcinoid tumors; neuroendocrine tumors; carcinoid tumors; pancreatic cancers; gastroesophageal; clear cell renal cell carcinoma; and primary peritoneal cancer. [00590] In some embodiments, the cancer is triple negative breast cancer, non-small cell lung cancer, or mantle cell lymphoma. [00591] An ROR1-ADC comprising an ROR1 antibody of the present disclosure may be administered to a subject per se or in the form of a pharmaceutical composition for the treatment of, e.g., cancer, autoimmunity, transplantation rejection, post-traumatic immune responses, graft-versus-host disease, ischemia, stroke, and infectious diseases (e.g., by targeting viral antigens, such as gp120 of HIV). [00592] In another aspect, an ROR1-ADC as described herein may be used in a method of treating a subject with cancer in combination with one or more additional therapies. The additional therapies that may be used in combination with an ROR1-ADC described herein include but are not limited to: (i) surgery; (ii) radiotherapy; (iii) endocrine therapy; (iv) immunotherapy (including adjuvant therapy and cell therapy such as CAR T-cell therapy); and (v) chemotherapy, including cytotoxic agents and chemotherapeutic agents. [00593] Any therapy that has an activity against a cancer may be used in combination with an ROR1-ADC provided herein. Examples of such agents for cancer treatment can be found, for instance, at www.cancer.gov/about-cancer/treatment/drugs and in publicly available sources such as Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 11th edition (2018), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the type of cancer involved. [00594] In certain embodiments, the additional therapy is a radiotherapy including, for example, gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes. Radiotherapy may comprise 179 NAI-1540479824 radiation or associated administration of radiopharmaceuticals. The source of radiation may be either external or internal to the subject being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Exemplary radioactive elements include, e.g., radium, cesium-137, iridium-192, americium- 241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111. [00595] In certain embodiments, the additional therapy is an immunotherapy. Immunotherapy (also called biological response modifier therapy, biologic therapy, biotherapy, immune therapy, or biological therapy) is treatment that uses parts of the immune system to fight disease. Immunotherapy can help the immune system recognize cancer cells or enhance a response against cancer cells. Immunotherapies include active and passive immunotherapies. Active immunotherapies, including immunotherapeutic agents, stimulate the body's own immune system (e.g., vaccines) while passive immunotherapies, including immunotherapeutic agents, generally use immune system components created outside of the body (e.g., antibodies), antibodies conjugated with drugs, toxins, or radionuclides, and targeted therapeutics. [00596] Exemplary immunotherapeutic agents include immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor used in methods of treatment can totally or partially reduce, inhibit, interfere with, or modulate one or more checkpoint proteins which regulate T-cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with its ligands PD-Ll and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). Immune checkpoint inhibitors include antibodies or are derived from antibodies. [00597] In certain embodiments, the checkpoint inhibitor is an OX40 (CD134) agonist. In some embodiments, the checkpoint inhibitor is an anti-OX40 antibody. In some embodiments, the anti-OX40 antibody is anti-OX-40. In some embodiments, the anti-OX40 antibody is MEDI6469. [00598] In certain embodiments, the checkpoint inhibitor is a CD40 agonist. In some embodiments, the checkpoint inhibitor is an anti-CD40 antibody. In some embodiments, the anti-CD40 antibody is CF-870,893. [00599] In certain embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti CTLA 4 antibodies include, but are not limited to, those described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. In some embodiments, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP- 180 NAI-1540479824 675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the trade name Yervoy™. [00600] In certain embodiments, the checkpoint inhibitor is a PD-1/PD-L1 inhibitor. Examples of PD-l/PD-L1 inhibitors include, but are not limited to, those described in US Patent Nos.7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Patent Application Publication Nos. WO2003042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699. [00601] In certain embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti- PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106) or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is marketed under the trade name Opdivo™. In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is marketed under the trade name Keytruda™. In some embodiments, the anti-PD-1 antibody is CT-011, a humanized antibody. CT-011 administered alone has failed to show response in treating acute myeloid leukemia (AML) at relapse. In some embodiments, the anti-PD-1 antibody is AMP-224, a fusion protein. In some embodiments, the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody in which the ability to bind Fc gamma receptor I is specifically engineered out, and which has a unique binding signature to PD-1 with high affinity and superior target specificity. [00602] In certain embodiments, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti- PD-L1 antibody is MEDI4736 (durvalumab). In some embodiments, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In some embodiments, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A, and Tecentriq®). [00603] In certain embodiments, the checkpoint inhibitor is a PD-L2 inhibitor. In some embodiments, the PD-L2 inhibitor is an anti-PD-L2 antibody. In some embodiments, the anti- PD-L2 antibody is rHIgM12B7A. [00604] In certain embodiments, the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor. In some embodiments, the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007, 179, 4202-4211). In some embodiments, the LAG-3 inhibitor is BMS-986016. 181 NAI-1540479824 [00605] In certain embodiments, the checkpoint inhibitor is a B7 inhibitor. In some embodiments, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In some embodiments, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834). [00606] In certain embodiments, the checkpoint inhibitor is a TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94). [00607] In certain embodiments, the checkpoint inhibitor is a GITR agonist. In some embodiments, the checkpoint inhibitor is an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518. [00608] In certain embodiments, the checkpoint inhibitor is a CD137 agonist. In some embodiments, the checkpoint inhibitor is an anti-CD137 antibody. In some embodiments, the anti-CD137 antibody is urelumab. In some embodiments, the anti-CD137 antibody is PF- 05082566. [00609] In certain embodiments, the checkpoint inhibitor is recombinant human interleukin- 15 (rhIL-15). [00610] In certain embodiments, the checkpoint inhibitor is an IDO inhibitor. In some embodiments, the IDO inhibitor is INCB024360. In some embodiments, the IDO inhibitor is indoximod. [00611] Other exemplary immunotherapies include adjuvant therapies, including immunotherapeutic agents such as cytokines, chemokines, interferons, interleukins, or lymphokines. Examples include cytokines, such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1-alpha, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL- 12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors (including TNF-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide (alum); Bacille Calmette-Guerin (BCG); Keyhole limpet hemocyanin (KLH); Incomplete Freund's adjuvant (IFA); QS-21; DETOX; Levamisole; and Dinitrophenyl (DNP), and combinations thereof, such as, for example, combinations of, interleukins, for example, IL-2 with other cytokines, such as IFN-alpha. [00612] Other exemplary immunotherapies include cell therapies, for example, a population of immune cells, such as leukocytes (nucleated white blood cells), comprising (e.g., expressing) a receptor that binds to an antigen of interest. A leukocyte of the present disclosure may be, for example, a neutrophil, eosinophil, basophil, lymphocyte, or a 182 NAI-1540479824 monocyte. In some embodiments, a leukocyte is a lymphocyte. Examples of lymphocytes include T cells, B cells, Natural Killer (NK) cells or NKT cells. In some embodiments, a T- cell is a CD4+ Th (T helper) cell, a CD8+ cytotoxic T cell, a γδT cell or a regulatory (suppressor) T cell. In some embodiments, an immune cell is a dendritic cell. In some embodiments, the cell therapies are CAR-T cell therapies. In some embodiments, a bispecific CAR is comprised of two distinct antigen recognition domains present in tandem on a single transgenic receptor (referred to as a TanCAR; see, e.g., Grada Z et al. Molecular Therapy Nucleic Acids 2013; 2:e105, incorporated herein by reference in its entirety). Thus, methods, in some embodiments, comprise delivering to a tumor a combination comprising an ROR1- ADC and an immunotherapeutic agent, wherein the immunotherapeutic agent is an engineered nucleic acid that encodes an antigen, or delivering to a tumor an engineered nucleic acid that induces expression of a self-antigen, and delivering to the tumor an immune cell expressing a bispecific CAR that binds to two antigens, one of which is encoded by the engineered nucleic acid. [00613] Other exemplary immunotherapies include immunotherapeutic agents such as cancer vaccines, which can be used to elicit an immune response in a subject against a cancer antigen. An exemplary method involves administering to a subject a RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide or an immunogenic fragment thereof, thereby inducing in the subject an immune response specific to the antigenic polypeptide or an immunogenic fragment thereof, in combination with administering an ROR1-ADC either in the same composition or a separate composition, administered at the same time, or sequentially dosed, wherein the anti-antigenic polypeptide antibody titer in the subject is increased following vaccination relative to anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the cancer. [00614] In certain embodiments, the additional therapies include chemotherapy such as one or more cytotoxic agents or one or more chemotherapeutic agents. A cytotoxic agent can inhibit or prevent a cellular function and/or cause cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. 183 NAI-1540479824 [00615] In certain embodiments, the additional therapy includes one or more chemotherapeutic agents. Chemotherapeutic agents include chemical compounds useful in the treatment of cancer. Chemotherapeutic agents include (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iii) anti-androgens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, including those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation; (viii) vaccines such as gene therapy vaccines. Chemotherapeutic agents can also include antibodies. [00616] Exemplary kinase inhibitors include erlotinib (Tarceva®), gefitinib (Iressa®), dasatinib (Sprycel®), nilotinib (Tasigna®), crizotinib (Xalkori®), ruxolitinib (Jakafi®), vemurafenib (Zelboraf®), vandetanib (Caprelsa®), pazopanib (Votrient®), afatinib, alisertib, amuvatinib, axitinib, baricitinib, bosutinib, brivanib, canertinib, cabozantinib (Cabometyx®), cediranib, ceritinib, crenolanib, dabrafenib, dacomitinib, danusertib, dovitinib, foretinib, ganetespib, ibrutinib, idelalisib, imatinib, iniparib, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, nintedanib, niraparib, oprozomib, olaparib, palbociclib, pictilisib, pirfenidone, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, saracatinib, saridegib, sorafenib, sunitinib, tandutinib, tasocitinib, telatinib, tivantinib, tivozanib, tofacitinib, trametinib, veliparib, vismodegib, volasertib, cobimetinib (Cotellic®), XL-147, XL-765, XL-499, XL-880, and others. In some embodiments, an ROR1-ADC is used in combination with a HSP90 inhibitor (e.g., XL888), liver X receptor (LXR) modulators, retinoid-related orphan receptor gamma (RORy) modulators, a CK1 inhibitor, a CK1-α inhibitor, a Wnt pathway inhibitor (e.g., SST-215), or a mineralocorticoid receptor inhibitor, (e.g., esaxerenone or XL-550) for the treatment of cancer. [00617] Kinase inhibitors can be tyrosine kinase inhibitors, such as the EGFR inhibitors; small molecule HER2 tyrosine kinase inhibitor such as Mubritonib (TAK165, Takeda); CP- 724.714, (Axon Medchem BV, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI- 166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate 184 NAI-1540479824 (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase 1 inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; antisense molecules (e.g., those that bind to HER-encoding nucleic acid); quinoxalines (U.S. Pat. No.5,804,396); tryphostins (U.S. Pat. No.5,804,396); Affinitac (ISIS 3521; Isis/Lilly); PKI166 (Novartis); Semaxinib (Pfizer); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: U.S. Pat. No.5,804,396; WO 1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca). [00618] Combined treatment with the ROR1-ADC provided herein and the additional therapy, such as a therapeutic agent, may be simultaneous, separate, or sequential, in any order. For combinations of therapeutic agents such as an ROR1-ADC and another therapeutic agent such as an immunotherapeutic agent or a chemotherapeutic agent, simultaneous administration, the therapeutics agents may be administered as one composition or as separate compositions, as appropriate. 5.8 KITS [00619] Also provided herein are kits comprising an ROR1-ADC provided herein, or a composition (e.g., a pharmaceutical composition) provided herein, packaged into suitable packaging material. A kit optionally includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. [00620] The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.). [00621] Kits provided herein can include labels or inserts. Labels or inserts include “printed matter,” e.g., paper or cardboard, separate or affixed to a component, a kit or packing 185 NAI-1540479824 material (e.g., a box), or attached to, for example, an ampoule, tube, or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media, or memory type cards. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location, and date. [00622] Kits provided herein can additionally include other components. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Kits can also be designed for cold storage. A kit can further be designed to contain antibodies provided herein, or cells that contain nucleic acids encoding the antibodies provided herein. The cells in the kit can be maintained under appropriate storage conditions until ready to use. [00623] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described herein. [00624] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a range of 90-100% includes 91-99%, 92-98%, 93-95%, 91-98%, 91-97%, 91-96%, 91-95%, 91-94%, 91-93%, and so forth. Reference to a range of 90-100% also includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. [00625] In addition, reference to a range of 1-3, 3-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50- 60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-225, 225-250 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 186 NAI-1540479824 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. In a further example, reference to a range of 25- 250, 250-500, 500-1,000, 1,000-2,500, 2,500-5,000, 5,000-25,000, 25,000-50,000 includes any numerical value or range within or encompassing such values, e.g., 25, 26, 27, 28, 29…250, 251, 252, 253, 254…500, 501, 502, 503, 504…, etc. [00626] As also used herein a series of ranges are disclosed throughout this document. The use of a series of ranges includes combinations of the upper and lower ranges to provide another range. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document. Thus, for example, reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, includes ranges such as 5- 20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, and 20-40, 20-50, 20-75, 20-100, 20-150, and so forth. [00627] It is understood that modifications which do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Accordingly, the following examples are intended to illustrate but not limit the present disclosure. [00628] It is understood that modifications that do not substantially affect the activity of the various embodiments described herein are also provided within the definition of the subject matter described herein. Accordingly, the following examples are intended to illustrate but not limit the present disclosure. 8. EXAMPLES [00629] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. By “average” is meant the arithmetic mean. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. [00630] General Synthetic Procedures 187 NAI-1540479824 [00631] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). [00632] Compounds as described herein can be purified by any purification protocol known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969. [00633] During any of the processes for preparation of the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, London and New York 1973, in T. W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Third edition, Wiley, New York 1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in “Methoden der organischen Chemie”, Houben-Weyl, 4th edition, Vol.15/l, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide and Derivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known from the art. [00634] The subject compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. A variety of examples of synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below. [00635] Example 1. Antibody: Binding Affinity 188 NAI-1540479824 [00636] Surface plasmon resonance (SPR) study was performed to access the monovalent KD values of the binding between the parental anti-ROR1 antibody (VH SEQ ID NO:27 and VL SEQ ID NO:28) and ROR1/2 of various species. [00637] Briefly, a Carterra monovalent KD method was used. For each chip location, the intrinsic on-rate (kon) and off-rates (koff) were obtained by measuring the association and dissociation events using optimal conditions in which the time constant for the process exceeded 30 seconds, such that it is within the instrument resolution. This generally resulted in analyzing sensorgrams employing [A] of 100 nM or less. koff was determined directly from sensorgrams as the observed dissociation rate constant, while kon was determined from combined analysis of multiple association process according to the reversible pseudo-first order process rate equation kon = kobs[A] + koff. KD could then be calculated from the rate constants according to koff/kon. For processes with rapid dissociations, kinetics could not be determined beyond a lower limit for koff of approximately 0.2 s-1. For such binding interactions, KD was calculated using the equilibrium Response Units (RU) value during the association phase to report total binding according to RU = RUmax[A]/(KD + [A]). [00638] Reported uncertainties for all kinetics and binding parameters reflect the standard deviation across determinations at different chip locations. Curves plotted on top of the sensorgrams in the slides were generated by simulating from the optimal averaged parameters, and the faded bands surrounding these curves display the impact of toggling these parameters by their respective standard deviations. [00639] Binding interactions with maximum RU < 100 were ranked as nonbinders, since this was not significantly above some baseline/bulk shifts. [00640] R2 values were determined from averaging values from the top 3 antigen concentrations used in the determination of the KD. The significance and thresholds for R2 varied with signal amplitude and intrinsic noise, but for this dataset, R2 values less than 0.75 denoted increasingly inadequate description of the binding process by one-step kinetic model and therefore the obtained parameters were highly uncertain well outside of the listed standard deviations. [00641] Experiments on the Carterra LSA were performed by chemically linking the antibodies to an HC30M chip using standard amine chemistry with running buffer 25 mM MES, pH 5.5. The tested antibody was printed to the chip at four separate locations. For kinetics and epitope binning experiments, all materials were prepared using running buffer 1X HBSTE (10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.01% Tween-20, pH 7.4) with 189 NAI-1540479824 0.5 mg/mL BSA added. Between each condition, regeneration using pH 2.8 Glycine buffer was performed to deplete residually-bound antigen. [00642] Kinetics were determined for human, cynomolgus, and mouse ROR1 and ROR2 using antigen concentrations ([A]) of 0.4, 1.2, 3.7, 11.1, 33.3, 100, 300 and 900 nM. For each concentration, association kinetics were observed by flowing antigen across the chip surface for a 10-minute association interval; subsequently, dissociation was observed from a 25- minute buffer-only interval. Binding data were recovered by double-referencing to subtract the closest reference cell and blank injection. [00643] The results are provided in Table E1 below, showing that the parental anti-ROR1 antibody demonstrated an about 10 nM affinity for ROR1 receptor across species, while no binding was observed for ROR2 receptor at human ROR2 receptor. Similar results at cyno ROR2 receptor were presumed in view of the similarity between the two proteins. Table E1. Binding affinities (KD) of the parental anti-ROR1 antibody to ROR1 and ROR2 of various species.
Figure imgf000192_0001
* n/a means the experiments were not conducted since the antigens were not available. [00644] Example 2. Aldehyde-Tagged Antibodies: Preparation [00645] Tagged antibodies were prepared according to methods known in the art, such as those described in Rabuka D. et al., “Site-specific chemical protein conjugation using 190 NAI-1540479824 genetically encoded aldehyde tags,” Nat Protoc., 2012; 7(6):1052-1067 and in U.S. Patent No.7,985,783 B2, each of which is incorporated in its entirety herein by reference. Briefly, an antibody containing a heterologous sulfatase motif was contacted with a formylglycine- generating enzyme to convert a cysteine or serine in the motif to a formylglycine having an aldehyde moiety, thereby generating an “aldehyde-tagged antibody.” Any formylglycine- generating enzyme can be used, such as Mycobacterium tuberculosis or Mycobacterium tuberculosis. [00646] Example 3. Linker-Payload: Preparation [00647] Synthetic reagents were purchased from Sigma-Aldrich, Acros, AK Scientific, or other commercial sources and used as received. Anhydrous solvents were obtained from commercial sources in sealed bottles. Compound 12 (previously reported in Chuprakov et al. Bioconjugate Chem.2021, 32, 746-754) was obtained commercially from Shanghai Medicilon and used without purification. Belotecan 13 as well as compounds 16 and 17 were obtained from commercial sources and used as received. Column chromatography was performed using a Biotage Isolera or Biotage Selekt chromatography system. Low-resolution mass spectra (LRMS) were acquired on Agilent Technology 6120 Quadrupole LC/MS, equipped with Agilent 1260 Infinity HPLC system, G1314 variable wavelength detector, and Agilent Poroshell 120 SB C18, 4.6 mm x 50 mm column at room temperature using 10-100% gradient of water and acetonitrile containing 0.1% formic acid. HPLCs were monitored at 254 or 205 nm.
Figure imgf000193_0001
SCHEME 1 191 NAI-1540479824 [00648] Preparation of (5-nitro-1H-indol-2-yl)methanol (2) [00649] To an oven-dried round-bottom flask were added ethyl 5-nitro-1H-indole-2- carboxylate (1, 25 g, 107 mmol) and 250 mL of anhydrous THF. The resulting solution was cooled down to 0 °C and treated with lithium aluminum hydride (4.9 g, 129 mmol) in small portions over 30 minutes with vigorous stirring under nitrogen. Reaction mixture was stirred at room temperature for 2 h, poured into 100 mL of water, and extracted with ethyl acetate (400 mL). Organic layer was concentrated under vacuum and purified on silica gel (75% v/v EtOAc/hexane) to afford 14 g (73 mmol, 68% yield) of product 2 as a brown solid. LRMS (ESI): m/z 192.9 [M+H]+, calculated for C9H8N2O3 m/z 193.1. [00650] Preparation of tert-butyl 3-(2-(hydroxymethyl)-5-nitro-1H-indol-1-yl)propanoate (3) [00651] In an oven-dried round-bottom flask were combined 5-nitro-1H-indol-2- yl)methanol (2, 14 g, 73 mmol), 140 mL of acetonitrile, and 12 g (94 mmol) of tert-butyl acrylate. The resulting solution was treated with DBU (16.6 g, 109 mmol) at room temperature over 30 minutes. Reaction mixture was stirred at 80 °C for 3 h, then poured into 100 mL of water and concentrated under reduced pressure to remove acetonitrile. Aqueous residue was then extracted with dichloromethane (300 mL). Organic layer was washed with brine, dried over sodium sulfate, concentrated under vacuum, and purified on silica gel (25% v/v EtOAc-hexane) to give 18 g (56 mmol, 77 % yield) of product 3 as a yellow solid. LRMS (ESI): m/z 320.9 [M+H]+, calculated for C16H20N2O5 m/z 321.1. [00652] Preparation of tert-butyl 3-(2-formyl-5-nitro-1H-indol-1-yl)propanoate (4) [00653] To a stirred solution of compound 3 (18 g, 56 mmol) in 200 mL of anhydrous DCM were added Dess-Martin periodinane (28 g, 66 mmol) in small portions over 15 minutes at room temperature. The resulting mixture was stirred for 1 h at RT and quenched by adding 100 mL of water. Aqueous layer was extracted with DCM (200 mL), combined organic layers were washed with brined and dried over sodium sulfate. After removal of solvents, the residue was purified on silica gel (0-25% v/v EtOAc-hexanes) to give 14 g (41 mmol, 73% yield) of aldehyde 4 as a yellow solid. LRMS (ESI): m/z 340.9 [M+Na]+, calculated for C16H18N2O5 m/z 341.1. [00654] Preparation of (9H-fluoren-9-yl)methyl 1,2-dimethylhydrazine-1-carboxylate (5) [00655] To a solution of 1,2-dimethylhydrazine dihydrochloride (20 g, 150 mmol) in DCM (200 mL) at 25 °C were added triethylamine (45.6 g, 452 mmol) slowly over 10 min. The mixture was stirred at 25°C for 30 min and treated with (9H-fluoren-9-yl)methyl 192 NAI-1540479824 carbonochloridate (Fmoc chloride, 19.4 g, 75 mmol) at room temperature and stirred for 3 h. Reaction mixture was poured into water (300 mL) and extracted with ethyl acetate (200 mL). Combined organic phase was dried over sodium sulfate, concentrated under vacuum, and purified by silica-gel chromatography (eluting with 30% v/v EtOAc-hexanes) to give compound 5 (20 g, 71 mmol, 95% yield) as a yellow low-melting solid. LRMS (ESI): m/z 283.2 [M+H]+, calculated for C17H18N2O2 m/z 283.1. [00656] Preparation of (9H-fluoren-9-yl)methyl 2-((1-(3-(tert-butoxy)-3-oxopropyl)-5- nitro-1H-indol-2-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate (6) [00657] In an oven-dried round-bottom flask were combined aldehyde 4 (14 g, 41 mmol) and hydrazine 5 (11 g, 39 mmol) in 140 mL of anhydrous DCE at ambient temperature. The mixture was stirred for 1 h and then treated with STAB (17 g, 80 mmol) in portions over 30 minutes. Stirring continued for 2 h at room temperature, then reaction mixture was quenched with 100 mL of water, aqueous layer was extracted with DCM (300 mL). Combined organic layer was washed with brine and dried over sodium sulfate. After removal of solvents, the residue was purified on silica gel (0-25% v/v EtOAc-hexane) to obtain 18 g (31 mmol, 80 % yield) of compound 6 as a brown solid. LRMS (ESI): m/z 584.9 [M+H]+, calculated for C33H36N4O6 m/z 585.3. [00658] Preparation of (9H-fluoren-9-yl)methyl 2-((5-amino-1-(3-(tert-butoxy)-3- oxopropyl)-1H-indol-2-yl)methyl)-1,2-dimethylhydrazine-1-carboxylate (7) [00659] A solution of nitro compound 6 (18 g, 31 mmol) in 180 mL of THF was combined with a solution of ammonium chloride (13 g, 243 mmol) in 180 mL of water, and 90 mL of methanol at ambient temperature. The resulting mixture was treated with zinc powder (16 g, 245 mmol) in several portions at room temperature. Reaction mixture was stirred vigorously for 2 hours, solids were filtered off, filtrate was concentrated under vacuum and partitioned between saturated aqueous ammonium chloride (100 mL) and ethyl acetate (100 mL). Aqueous layer was separated and extracted with ethyl acetate (100 mL). Combined organic layer was washed with brine and dried over sodium sulfate. Removal of solvents under vacuum afforded 15 g of crude product 7 as a brown oil which was used further without purification. LRMS (ESI): m/z 555.3 [M+H]+, calculated for C33H38N4O4 m/z 555.3. [00660] Preparation of 4-((2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2- dimethylhydrazineyl)methyl)-1-(3-(tert-butoxy)-3-oxopropyl)-1H-indol-5-yl)amino)-4- oxobutanoic acid (9) [00661] To a solution of compound 7 (1.0 g, 1.8 mmol) in MeCN (10 mL) were added succinic anhydride (905 mg, 5.0 equiv., 9 mmol) and triethylamine (1.2 mL, 9 mmol). 193 NAI-1540479824 Reaction mixture was stirred for 3 hours at room temperature and purified by reversed-phase chromatography (C18 column, 0-100% v/v MeCN/H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure until murky, and lyophilized to give 0.95 g of product 9 (1.5 mmol, 83% yield) as an off-white solid. LRMS (ESI): m/z 677.3 [M+Na]+, calculated for C37H42N4O7 m/z 677.3. [00662] Preparation of 4-((2-((2-(((9H-fluoren-9-yl)methoxy)carbonyl)-1,2- dimethylhydrazinyl)methyl)-1-(2-carboxyethyl)-1H-indol-5-yl)amino)-4-oxobutanoic acid (10) [00663] Compound 9 (9 g, 14 mmol) was dissolved in 90 mL of hexafluoro isopropanol and treated with 10 mL of concentrated HCl at room temperature. Reaction mixture was stirred for 1 h at room temperature, then solvent was removed in vacuum, and the residue was purified by reversed-phase flash chromatography (C18, 0-100% v/v CH3CN-H2O with 0.05% TFA). Fractions containing product were combined and lyophilized to give diacid 10 (6.5 g, 11 mmol, 79% yield) as a tan solid. LRMS (ESI): m/z 621.3 [M+Na]+, calculated for C33H34N4O7 m/z 621.2. [00664] Preparation of (9H-fluoren-9-yl)methyl 1,2-dimethyl-2-((1-(3-oxo-3- (perfluorophenoxy)propyl)-5-(4-oxo-4-(perfluorophenoxy)butanamido)-1H-indol-2- yl)methyl)hydrazine-1-carboxylate (11) [00665] A solution of diacid 10 (2.0 g, 3.34 mmol) in 40 mL of anhydrous THF was combined with 6.2 g (33.4 mmol) of pentafluorophenol. The mixture was stirred and treated with DCC (2.0 g, 10 mmol) in a few small portions at room temperature. Reaction mixture was stirred for 48 hours at RT, then all solids were removed by filtration and washed with THF on filter. Combined filtrates were concentrated under vacuum and purified by silica gel chromatography (0-25-35% gradient of EtOAc-Hexanes) to give 2.4 g of bis-PFP ester 11 (2.6 mmol, 77% yield) as a white foaming solid. LRMS (ESI): m/z 931.2 [M+H]+, calculated for C45H32F10N4O7 m/z 931.2. [00666] Synthesis of linker-payload (IIa) [00667] Preparation of (2S,3S,4S,5R,6S)-6-(2-((S)-2-((S)-2-amino-3- methylbutanamido)propanamido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14- tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (15) 194 NAI-1540479824
[00668] Belotecan hydrochloride 13 (2.35 g, 5.0 mmol) was suspended in a mixture of 30 mL of anhydrous DMF and 1.75 mL of DIPEA (10 mmol). The resulting mixture was stirred and treated with HOAt (0.68 g, 5 mmol), followed by PNP-carbonate 12 (5.1 g, 5 mmol) in small portions at room temperature. Reaction mixture was stirred at RT for 8 h until starting materials were judged fully consumed based on HPLC analysis. The mixture was poured onto 300 mL of ice with vigorous stirring, the resulting yellowish precipitate was collected by filtration, washed with 30 mL of water twice, dried on air overnight to give 6.7 g of crude coupling product 14 as a light-yellow powder. LRMS (ESI): m/z 1307.5 [M+H]+, calculated for C69H74N6O20 m/z 1307.5. [00669] A solution of crude intermediate 14 (6.7 g) in 30 mL of THF was cooled down to 0 °C in an ice bath and treated slowly with 2 M aqueous lithium hydroxide solution (10 mL). Reaction mixture was stirred at 0 °C for 1 h, then another 10 mL of 2 M LiOH solution was added and stirring continued for 15 minutes before warming the reaction mixture to room temperature and adding another 10 mL of 2 M lithium hydroxide and 5 mL of methanol. The resulting mixture was stirred for 1 h at room temperature, then quenched by adding 2 M aqueous HCl solution to pH 2-3 and let stir for 30 minutes. The mixture was transferred to a separatory funnel and washed with MTBE (2x50 mL). Aqueous layer was separated and directly purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure, and lyophilized to give 3.4 g of product 15 (3.6 mmol, 72% yield over 2 steps) as a bright-yellow fluffy powder. LRMS (ESI): m/z 945.4 [M+H]+, calculated for C47H56N6O15 m/z 945.4. 195 NAI-1540479824
SCHEME 2 [00670] Preparation of N6-(((9H-fluoren-9-yl)methoxy)carbonyl)-N2-(3-(2-(2- methoxyethoxy)ethoxy)propanoyl)-L-lysine (18) [00671] Carboxylic acid 16 (5.0 g, 12 mmol) was dissolved in anhydrous DMF (10 mL) and treated with DIPEA (2.1 mL, 12 mmol), followed by HATU (4.6 g, 12 mmol) at ambient temperature. The resulting mixture was stirred for 30 minutes and then combined with solid H-Lys(Fmoc)-OH 17 (4.5 g, 12 mmol). Reaction mixture was stirred for 1 h at RT and then directly purified by reversed-phase chromatography (C18 column, 0-70% CH3CN-H2O with 0.05% TFA). Fractions containing product were combined and solvents were removed in vacuum. The residue was dried under high vacuum overnight to give product 18 (5.5 g, 7.2 mmol, 60% yield) as a clear colorless oil. LRMS (ESI): m/z 763.4 [M+H]+, calculated for C39H58N2O13 m/z 763.4. [00672] Preparation of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-aminobutyl)-31- isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33- triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14- tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (19) 196 NAI-1540479824 [00673] Carboxylic acid 18 (2.9 g, 3.8 mmol) was combined with DIPEA (1.3 mL, 7.6 mmol) and HATU (1.45 g, 3.8 mmol) in 12 mL of anhydrous DMF at room temperature. The mixture was stirred for 20 minutes before combining with a solution of compound 15 (3.3 g, 3.5 mmol) in 15 mL of DMF. The resulting mixture was stirred at room temperature for 1 hour, then treated with 13 mL of triethylamine and stirred at room temperature for 7 h until reaction was judged complete by LCMS analysis. Reaction mixture was then concentrated under reduced pressure to remove triethylamine and purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure to ~100 mL final volume, and lyophilized to give 3.5 g of product 19 as a bright-yellow light powder (2.4 mmol, 69% yield over 2 steps). LRMS (ESI): m/z 1467.7 [M+H]+, calculated for C71H102N8O25 m/z 1467.7. [00674] Preparation of (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)-28-(((S)-1- (((S)-1-((2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-4- ((((2-((S)-4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H- pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3- methyl-1-oxobutan-2-yl)carbamoyl)-26,34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27,33- diazaheptatriacontan-37-amido)-2-((1,2-dimethylhydrazineyl)methyl)-1H-indol-1- yl)propanamido)butyl)-31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23- octaoxa-27,30,33-triazapentatriacontan-35-amido)-5-((((2-((S)-4-ethyl-4-hydroxy-3,14- dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11- yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid (IIa) [00675] To a stirred solution of compound 19 (3.5 g, 2.4 mmol) in 16 mL of anhydrous DMA were added DIPEA (0.84 mL, 4.8 mmol) and HOAt (0.33 g, 2.4 mmol) at room temperature. The resulting mixture was treated with a separately prepared solution of bis-PFP ester 11 ester (1.0 g, 1.1 mmol in 2 mL of DMA) in a few small portions with 10 minutes intervals between additions. After the addition was complete, reaction mixture was stirred for 15 minutes at room temperature and treated with 2.1 mL of piperidine (22 mmol). After 20 minutes, reaction mixture was directly purified by reversed-phase chromatography (C18 column, 0-40% CH3CN-H2O with 0.05% TFA). Pure fractions were combined, concentrated under reduced pressure at 30 °C, and lyophilized to obtain 3.2 g of linker-payload (IIa) as a bright-yellow fluffy powder (0.98 mmol, 89% yield of two steps based on 11). LRMS (ESI): m/z 1638.3 [M+H]2+, calculated for C160H224N20O53 m/z 1638.8. 197 NAI-1540479824 [00676] Example 4. ADC: Preparation [00677] Aldehyde-tagged antibodies (15 mg/mL) were conjugated to synthesized linker- drugs (17.5 mol. equivalents drug:antibody for DAR8) by reacting the linker-drug and aldehyde-tagged antibody for 72 hours at 37 °C in 67 mM sodium citrate, pH 5.5, 43 mM sodium chloride containing 3.5 or 4.5% DMA. [00678] After conjugation, residual free drug was removed by diafiltration using a 115V Labscale Tangential Flow Filtration (TFF) system (Millipore, Cat. No. XX42LSS11) with a 30 kDa TFF cassette (Sigma-Aldrich, Cat. No. P3C030C00, filtration area 88 cm2, NMWCO 30 kDa) and exchanging for 12 diavolumes (600 mL) into 20 mM sodium citrate, pH 5.5, 50 mM NaCl. [00679] In some embodiments, residual free drug was further removed using SEC-FPLC (HiLoad 26/600 Superdex® 200) followed by concentration using AMICON® 0.5 mL 30 kD molecular weight cut off (MWCO) centrifugal filters (Millipore Sigma, Cat. No. #UFC5030BK). After the FPLC purification, the ROR1-ADC samples were measured as having about 1.4% free payload. [00680] In some embodiments, ion exchange chromatography is further used to purify the ROR1-ADC samples. [00681] To determine the drug-to-antibody ratio (DAR) of the final product, antibody-drug conjugates were examined by analytical hydrophobic interaction chromatography (HIC) or polymeric reverse phase chromatography (PLRP). [00682] The HIC column (Tosoh Bioscience, Cat. No.14947) was run with 1.5 M ammonium sulfate and 25 mM sodium phosphate (pH 7.0) as mobile phase A and 25% isopropanol and 18.75 mM sodium phosphate (pH 7.0) as mobile phase B. The PLRP column (Agilent, Cat. No. PL1912-1802) was run with 0.1% trifluoroacetic acid in H2O as mobile phase A and 0.1% trifluoroacetic acid in CH3CN for mobile phase B, with the column heated to 80 °C. [00683] In some embodiments, analytical Hydrophobic Interaction Chromatography (HIC) was performed as described below: Samples were analyzed on an Agilent 1100 Instrument equipped with a 4.6 mm x 35 mm TSK gel butyl-NPR column from Tosoh (#14947) held at 25 °C. Samples were prepared by adding 20 µL of 50 mM NaCl (pH 5.5) and 20 µL of Mobile Phase A to 20 µg of sample and were injected at room temperature. Gradient elution utilized mobile phase A (0.1% trifluoroacetic acid in H2O) and Mobile Phase B (0.1% trifluoroacetic acid in CH3CN) in the program reported in Table E2 below at a flow rate of 1.0 mL/minute. Detection was via UV at 215 nm, 252 nm, and 280 nm. 198 NAI-1540479824 [00684] In some embodiments, analytical Hydrophobic Interaction Chromatography (HIC) (Tosoh #14947) with UV/Vis detection was performed. The reduced form of the final product was analyzed by reverse phase liquid chromatography (RP-HPLC) (Agilent Cat. No. PL1912-1802) with UV/Vis detection to verify DAR. Table E2. HIC Gradient Elution Program
Figure imgf000201_0001
[00685] In some embodiments, Reverse Phase HPLC was performed as described below: Samples were analyzed on an Agilent 1100 Instrument equipped with a 50 x 2.1 mm PL1912- 1802 (8 µm, 1000Å) column from Agilent held at 80 °C. Samples were prepared by adding 10 µL of a mixture of 0.5 mM DTT, 50 µL of 8 M guanidine HCl, 130 mM tris, and 1 mM EDTA (pH 7.6) to a 20 µg sample and adding PBS to a final volume of 100 µL. Samples were incubated at 37 °C for 30 minutes after preparation. Gradient elution utilized mobile phase A (25 mM Na3PO4, 1.5 M (NH4)2SO4, pH 7.0) and Mobile Phase B (18.75 mM Na3PO4, pH 7.0, 25% isopropyl alcohol) in the program reported in Table E3 below at a flow rate of 2.0 mL/minute. Detection was via UV at 215 nm, 252 nm, and 280 nm. Table E3. RPLC Gradient Elution Program
Figure imgf000201_0002
[00686] To determine aggregation, samples were analyzed using analytical size exclusion chromatography (Tosoh Bioscience, Cat. No.08541) using a mobile phase of 300 mM NaCl, 25 mM sodium phosphate, and 5% isopropanol (pH 6.8). 199 NAI-1540479824 [00687] Example 5. ADC: In Vitro Cytotoxicity [00688] In vitro cytotoxicity of the ADC prepared as described in Examples 1-4 was further assessed. [00689] hROR1 expressing HEK cells (1500 cells/well in 100 µL of media) were plated in Costar (Corning) 3610 plate (individually packed, sterile, white wall clear bottom, TC treated, VWR# 29444-010) and incubated overnight. [00690] Dilution series of the tested articles (such as, ADC-8) were made. Belotecan and isotype control (FITC antibody conjugated to the ADC-8 linker payload) were tested in parallel and served as controls. The final top concentration was 100 nM for free drugs and 10 nM for ADC-8. To get a good sigmoidal curve, the free drug was serially diluted 1:6 and ADC-8 was serially diluted 1:4. Starting with a top concentration of 600 nM free drug or 60 nM ADC (6x the final top concentration), each of the samples was serially diluted 5 times (at 1:6 for free drug and 1:4 for ADC).20 µL of each sample were added to 100 µL cells. The assay plate with each sample was prepared in duplicate. The treated cells were incubated for 5 days. The cell viability reading was taken using Cell Titer-Glo (Promega, cat# G7573) and normalized to the untreated cells. [00691] The results are provided in FIG.1 and Table E4 below, showing good in vitro cytotoxicity of ADC-8. Table E4. In vitro cytotoxicity in HEK293-huROR1 cells.
Figure imgf000202_0001
[00692] Example 6. ADC: In Vivo Efficacy in MDA-MB-231 TNBC Xenograft Model [00693] CB.17 female SCID mice were implanted (flank) with 1.0 x 107 of MDA-MB-231 tumor cells in 50% MATRIGEL® (Corning Life Sciences) matrix (1:1 cell to Matrigel) on Day -13. When the average tumor volume reached about 200 mm3 on Day 1, mice were randomized into respective treatment groups (10 mice per group). The mice then received intravenous injections of the tested ADC (at 7.5 mg/kg) on Day 1 and Day 8 (Q7D x 2). PBS vehicle, Control FITC ADC (FITC conjugated to the ADC-8 linker-payload), and a benchmark ADC (which comprises a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1 (CAS # 1643432-38-5), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE)) were tested in parallel, serving as controls. 200 NAI-1540479824 Body weight and tumor volume were measured twice per week. The study was ended on Day 45. [00694] Tumor volumes were measured and %TGI was calculated. The tumor growth inhibition rate TGITV was calculated by the following equation: TGITV% =[1-(Ti-T0)/(Vi-V0)] ×100%. (Ti: means tumor volume of the treated groups at day i following treatment; T0: means tumor volume of the treated groups at day 0; Vi: means tumor volume of the vehicle groups at day i following treatment; V0: means tumor volume of the vehicle groups at day 0). Further, the %TGI result was analyzed with One Way ANOVA with Dunnett’s post hoc test as appropriate, while **** indicated p < 0.0001 and NS indicated p > 0.05. [00695] The result is provided in FIGs.2A-2B, showing ADC-8 exhibited improved efficacy compared to the benchmark ADC. [00696] Example 7. ADC: In Vivo Efficacy in JEKO-1 MCL Xenograft Model [00697] CB.17 female SCID mice were inoculated subcutaneously in the right up flank region with Jeko-1 tumor cells (5× 106) in 0.1 ml of PBS with Matrigel (1:1) for tumor development. When the tumor volumes reached about 150~250 mm3 on Day 1, mice were randomized into respective treatment groups (8 mice per group). The mice then received intravenous injections of vehicle or the tested ADC (7.5 mg/kg) on Day 1 and Day 8. PBS vehicle, Control FITC ADC (FITC conjugated to the ADC-8 linker-payload), and a benchmark ADC (which comprises a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1 (CAS # 1643432-38-5), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE)) were tested in parallel, serving as controls. [00698] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (Body weights were measured twice per week after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. [00699] Tumor volumes were measured twice per week after randomization in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: “V = (L x W x W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). Dosing as well as tumor and body weight measurements were conducted in a Laminar Flow Cabinet. 201 NAI-1540479824 [00700] Tumor volumes were measured and %TGI was calculated. The tumor growth inhibition rate TGITV was calculated by the following equation: TGITV% =[1-(Ti-T0)/(Vi-V0)] ×100%. (Ti: means tumor volume of the treated groups at day i following treatment; T0: means tumor volume of the treated groups at day 0; Vi: means tumor volume of the vehicle groups at day i following treatment; V0: means tumor volume of the vehicle groups at day 0). Further, the %TGI result was analyzed with One Way ANOVA with Dunnett’s post hoc test as appropriate, while **** indicated p < 0.0001. [00701] The result is provided in FIGs.3A-3B, showing ADC-8 demonstrated comparable efficacy to the benchmark ADC. [00702] Example 8. ADC: In Vivo Efficacy in Patient-Derived Xenograft (PDX) Models [00703] CB.17 female SCID mice are implanted (flank) with patient tumor cells in 50% MATRIGEL® (Corning Life Sciences) matrix (1:1 cell to Matrigel). When the average tumor volume reaches about 200 mm3 on Day 1, mice are randomized into respective treatment groups and receive intravenous injections of vehicle or the tested ADC (10 mg/kg) every three weeks (Q3W). Tumor volumes are measured and %TGI is calculated. [00704] The tumor growth inhibition rate TGITV is calculated by the following equation: TGITV% =[1-(Ti-T0)/(Vi-V0)] ×100%. (Ti: means tumor volume of the treated groups at day i following treatment; T0: means tumor volume of the treated groups at Day 0; Vi: means tumor volume of the vehicle groups at day i following treatment; V0: means tumor volume of the vehicle groups at day 0). Further, the %TGI result is analyzed with One Way ANOVA with Dunnett’s post hoc test as appropriate. [00705] Additionally, female athymic nude-Foxn1nu mice were used. When a sufficient number of stock animal exhibiting non-small cell lung cancer (NSCLC) tumors having a volume of 1000-1500 mm³, the tumors were harvested for re-implantation into pre-study animals. Pre-study animals were implanted unilaterally on the left flank with tumor fragments harvested from stock animals. When the average tumor volume reached 150-300 mm3, mice were matched by tumor volume into treatment or control groups to be used for dosing (6 mice per group) and dosing initiated on Day 0. The animals received intravenous injections of vehicle or the tested ADCs (10 mg/kg) once per week for 2 weeks (Q7D x 2). Body weights and tumor volumes (TV) were measured twice per week until the end of the study. Any animal exhibiting >20% net weight loss for a period lasting 7 days or mice displaying >30% net weight loss when compared to Day 0 was considered moribund and euthanized. 202 NAI-1540479824 [00706] Beginning Day 0, tumor dimensions were measured twice weekly by digital caliper, and data including individual and mean estimated tumor volumes (Mean TV ± SEM) was recorded for each group; tumor volume (TV) was calculated using the following formula: TV= width2 x length x 0.52. [00707] Results are provided in FIG.6, which demonstrates that ADC-8 significantly inhibited the tumor growth. Additionally, there was no evidence of body weight loss greater than 5% across the study. [00708] Example 9. ADC: Pharmacokinetics Study in Rats [00709] Sprague-Dawley rats were dosed intravenously with a single dose of 5 mg/kg of the tested ADC on Day 0 after 16 hours of fasting. The rats’ body weights were measured on Day -3. Day 0, Day 7, Day 14, and Day21.100 µL plasma was collected at 30 min (on Day 0), 6 h (on Day 0), 24 h (on Day 1), 48 h (on Day 2), Day 4, Day 7, Day 10, and Day14, saved in bullet tubes with K2EDTA, and stored at -20 °C until end of the study. [00710] Total antibody and total ADC concentrations (referred to herein as tAb and tADC, respectively) were quantified using MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (having an assay MRD of 1:100 and a quantitation range of 10 ng/mL (LLOQ) – 10240 ng/mL (ULOQ)). A 1000X dilution linearity was used, while the assay controls included 34 ng/mL (LQC), 800 ng/mL (MQC), 8000 ng/mL (HQC). [00711] For total antibody measurements, conjugates were captured with an anti-human IgG (Fc specific), F(ab′)2 fragment, highly cross adsorbed-Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 µg/mL and detected with a Sulfo TAG-labeled goat anti- human antibody (R32AJ) at 0.15 µg/mL. For total belotecan ADC measurements, conjugates were captured with an anti-human IgG (Fc specific), F(ab′)2 fragment, highly cross adsorbed- Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 µg/mL and detected with a Sulfo TAG-labeled anti-Belotecan (Clone 1H11, Catalent) at 1 µg/mL. Pooled Sprague Dawley rat plasma was used as a negative control (NC). The assay buffer used was 1xPBS+1%BSA+0.1%Tween, while the read buffer was 1X MSD Read Buffer T (MSD R92TC-1). Data were analyzed and plotted using GraphPad Prism. [00712] As shown in FIG.4 and Table E5 below, ADC-8 displayed good in vivo stability when administered at a single dose of 5 mg/kg. 203 NAI-1540479824 Table E5. Rat PK results.
Figure imgf000206_0001
[00713] Example 10. ADC: Toxicity and Toxicokinetics (TK) Study in Rats [00714] Female Sprague-Dawley rats were dosed intravenously with the tested ADC via i.v. bolus once per week for four weeks (Q1W x 4) as detailed in Table E6 below.100 µL plasma was collected at various time points post-dose, saved in bullet tubes with EDTA and stored at -20 °C until end of the study. In addition, clinical observation, body weights (BW), clinical pathology, and anatomical pathology were also monitored. Toxicokinetic (TK) profiles were analyzed for the 1st and last dose cycles. Table E6. Rat toxicity study design.
Figure imgf000206_0002
* Based on the most recent body weight measurement. [00715] Total antibody and total ADC concentrations (referred to herein as tAb and tADC respectively) were quantified using MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (having an assay MRD of 1:100 and a quantitation range of 10 ng/mL (LLOQ) – 10240 ng/mL (ULOQ)). A 1000X dilution linearity was used, while the assay controls included 34 ng/mL (LQC), 800 ng/mL (MQC), 8000 ng/mL (HQC). [00716] For total antibody measurements, conjugates were captured with an anti-human IgG (Fc specific), F(ab′)2 fragment, highly cross adsorbed-Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 µg/mL and detected with a Sulfo TAG-labeled goat anti- human antibody (R32AJ) at 0.15 µg/mL. For total belotecan ADC measurements, conjugates were captured with an anti-human IgG (Fc specific), F(ab′)2 fragment, highly cross adsorbed- Biotin antibody produced in goat (Sigma Aldrich, SAB3701268) at 1 µg/mL and detected 204 NAI-1540479824 with a Sulfo TAG-labeled anti-Belotecan (Clone 1H11, Catalent) at 1 µg/mL. Pooled Sprague Dawley rat plasma was used as a negative control (NC). The assay buffer used was 1xPBS+1%BSA+0.1%Tween, while the read buffer was 1X MSD Read Buffer T (MSD R92TC-1). Data were analyzed and plotted using GraphPad Prism. [00717] Briefly, ADC-8 was well-tolerated and having a projected maximum tolerated dose (MTD) of ≥ 60 mg/kg, while ADC-4 was also well-tolerated and having an MTD of > 90 mg/kg. [00718] As shown in FIG.5A, ADC-8 exhibited a linear PK profile, while the total mAb and total ADC curves overlapped, suggesting good in vivo stability. This TK analysis further showed that following the Q1W x 4 administration, there was accumulation with AR (accumulation ratios) of 1.94 to 2.0. [00719] As further shown in FIG.5B, ADC-4 also showed that total ADC and total antibody were overlapped, suggesting stable ADC. The mean ADC accumulation ratio ranged from 1.88 to 2.09. ***** [00720] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. 205 NAI-1540479824

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 linker
Figure imgf000208_0001
wherein: Ab represents the antibody that binds to ROR1; Z1, Z2, and Z4 are each independently CR4; Z3 is C-LB-W2; R1, R2, R3, and R4 are each selected from hydrogen and alkyl; LA is 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 T6 are 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-1540479824 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 V6 are 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 R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; LB is 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 T13 are 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 V13 are 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-1540479824 each R13 is independently selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl; and each R15 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; s is an integer from 1 to 10; W1 is a first drug; and W2 is a second drug. 2. The ADC of claim 1, wherein: T1 is selected from a (C1-C12)alkyl and a substituted (C1-C12)alkyl; T2, T3, T4, T5, and T6 are 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 V6 are 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-;
Figure imgf000210_0002
4-amino-piperidine
Figure imgf000210_0001
each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups may be cyclically linked to form a piperazinyl ring; q is an integer from 1 to 6; 208 NAI-1540479824 r is 0 or 1; and y is an integer from 1 to 6. 3. The ADC of claims 1 or 2, wherein: T1 is (C1-C12)alkyl and V1 is -CONH-; T2 is substituted (C1-C12)alkyl and V2 is -CO-; T3 is (AA)p and V3 is absent; T4 is PABC and V4 is 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: T7 is a covalent bond; T8, T9, T10, T11, and T12 are 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 V12 are 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:
Figure imgf000211_0001
integer from 1 to 30; EDA is an ethylene diamine moiety having the following structure:
Figure imgf000211_0002
209 NAI-1540479824 each R12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl, and a substituted aryl, wherein any two adjacent R12 groups 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: T7 is absent and V7 is -NHCO-; T8 is (C1-C12)alkyl and V8 is -CONH-; T9 is substituted (C1-C12)alkyl and V9 is -CO-; T10 is (AA)p and V10 is absent; T11 is PABC and V11 is 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 T2 and T9 is (C1- C6)alkylene substituted with -NHCO(PEG)t, wherein
Figure imgf000212_0001
and t is an integer from 2 to 10, optionally 8. 7. The ADC of any one of claims 1-6, wherein one or both T3 and T10 has 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 W1 and W2 is a camptothecin analog, optionally wherein the camptothecin analog is belotecan. 11. The ADC of any one of claim 1-10, wherein each of W1 and W2 is belotecan. 210 NAI-1540479824
12. An ADC represented by Formula (II):
Figure imgf000213_0001
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-1540479824
17. 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-1540479824 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; (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-1540479824
20. 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; Z20 is either a proline (P) or alanine (A) residue; Z30 is 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); X1 is 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, X1 is present; and X2 and X3 independently 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-1540479824 further 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-1540479824
31. 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
PCT/US2024/038778 2023-07-20 2024-07-19 Tyrosine-protein kinase membrane receptor 1 (ror1) antibody-drug conjugates and uses thereof Pending WO2025019787A1 (en)

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AU2024292307A AU2024292307A1 (en) 2023-07-20 2024-07-19 Tyrosine-protein kinase membrane receptor 1 (ror1) antibody-drug conjugates and uses thereof
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019118411A2 (en) * 2017-12-11 2019-06-20 Triphase Accelerator U.S. Corporation Anti-cd22 antibody-maytansine conjugates, combinations, and methods of use thereof
US20210155692A1 (en) * 2018-04-18 2021-05-27 Exelixis, Inc. Anti-ror antibody constructs
US20220241423A1 (en) * 2021-01-15 2022-08-04 R.P. Scherer Technologies, Llc Camptothecine antibody-drug conjugates and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019118411A2 (en) * 2017-12-11 2019-06-20 Triphase Accelerator U.S. Corporation Anti-cd22 antibody-maytansine conjugates, combinations, and methods of use thereof
US20210155692A1 (en) * 2018-04-18 2021-05-27 Exelixis, Inc. Anti-ror antibody constructs
US20220241423A1 (en) * 2021-01-15 2022-08-04 R.P. Scherer Technologies, Llc Camptothecine antibody-drug conjugates and methods of use thereof

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