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

Abstract

The present disclosure provides anti-tyrosine protein kinase membrane receptor 1 antibody-drug conjugate structures. The antibody-drug conjugate structure comprises branched linkers, wherein each branched linker links two or more payloads to an antibody. In addition, the present disclosure also encompasses compounds and methods for producing such conjugates. In addition, the present disclosure also encompasses methods of using the conjugates.

Description

[0001] 1. Cross-references to related applications 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 herein by reference in its entirety.

[0002] 2. Sequence List This application contains an electronic sequence list, which has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference in their entirety. The sequence list XML file submitted with this application is titled "14529-152-228_SEQ_LISTING.xml", was created on July 17, 2024, and has a size of 159,402 bytes. 3. Technical Field This disclosure generally relates to antibody-drug conjugates (ADCs) that bind to tyrosine protein kinase membrane receptor 1 (ROR1, such as human ROR1) and methods of using them. 4. Background Technology Many tumors can exhibit cell surface expression of tyrosine protein kinase transmembrane receptor (ROR) antigens, such as those observed by Gentile et al. ( Cancer Res ; 71(8) April 15, 2011), Rebagay et al. ( Front. Oncol (April 18, 2012), Zhang et al. () American Journal of Pathology, Volume 181, Issue 6, December 2012), Henry et al. ( Oncotarget Volume 6, Issue 37, 2015), Zhang et al. ( PLoS ONE 7(3): e31127) and Bainbridge et al. ( PLoS ONE A more detailed description can be found in 9(7): e102695, each of which is incorporated herein by reference in its entirety. Furthermore, ROR expression may not be present in normal tissues (e.g., non-cancerous tissues), or may only show limited expression, as described by Balakrishnan et al. ( Clin Cancer Res The full text of the article described in (June 15, 2017; 23(12): 3061-3071) is incorporated herein by reference. Therefore, the ROR antigen can be used as a tumor-specific marker for certain tumors. Examples of tumors and cancers exhibiting ROR expression include, but are not limited to, pancreatic cancer, ovarian cancer, breast cancer, lung cancer, gastric cancer, melanoma, Ewing's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, and B-ALL, such as Gohil et al. ( OncoimmunologyThe full text of the paper described in 2017; 6(7): e1326437 is incorporated herein by reference. Other cancers include, but are not limited to, hematologic malignancies, prostate cancer, colon cancer, kidney cancer, and uterine cancer. Gohil et al. describe the use of ROR multispecific antibodies constructed on various antibody platforms for targeting tumors in International Applications WO 2017 / 053469, WO 2014 / 167022, U.S. Publication No. 2017 / 0198045, WO 2016 / 094873, WO 2017 / 127499, and WO 2016 / 142768, each of which is incorporated herein by reference in its entirety.

[0005] Therefore, ROR antigen-binding molecules have therapeutic potential in cancer treatment. Multispecific ROR-binding molecules that bind to T cell surface antigens in addition to ROR antigens have the potential to redirect T cells to kill ROR-expressing cancer cells.

[0006] There is still a need in the field for ADCs that can target ROR to treat, prevent or alleviate ROR-mediated diseases, conditions or disorders, such as cancer. 5. Summary of the Invention This disclosure provides an ADC (“ROR1-ADC”) comprising an antibody that binds to tyrosine protein kinase receptor 1. In some embodiments, such a ROR1-ADC binds to the same epitope of human ROR1 as the antibody described herein comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0008] This disclosure also provides pharmaceutical compositions comprising a ROR1-ADC comprising an antibody or a fragment thereof (“ROR1 antibody”) that binds to ROR1 and a drug (directly or indirectly) conjugated thereto. In some embodiments, such pharmaceutical compositions comprise a ROR1-ADC comprising an antibody or a fragment thereof that binds to substantially the same epitope of human ROR1 as the antibodies comprising VH and VL described herein.

[0009] This disclosure also provides methods for treating, preventing, or alleviating ROR1-mediated diseases, conditions, or disorders, such as using ROR1-ADCs to alleviate one or more symptoms of ROR1-mediated diseases, conditions, or disorders.

[0010] More specifically, this disclosure provides a ROR1-ADC comprising (a) a ROR1 antibody and (b) one or more pyridazine-pyrroloconjugated moieties, said moieties comprising a drug conjugated to said pyridazine-pyrroloconjugated moieties via a linker, for example, using hydrazine- different-Pictet-Spengler (HIPS) concatenation method.

[0011] Traditionally, the HIPS conjugation method has been used to generate conjugates in which each HIPS moiety on each aldehyde tag carries one payload, resulting in antibody conjugates with DAR values ​​up to 4. In some embodiments, such as the ROR1-ADC disclosed herein, a branched-chain HIPS linker is included, in which each HIPS moiety carries two (or more) identical or different payload molecules, thus enabling the conjugation of two (or more) small molecule payloads on each aldehyde group of the protein in a single conjugation step. Therefore, the use of such branched linker allows for the generation of conjugates with higher DAR site specificity (e.g., DAR up to 8) with controlled payload placement, which in the context of therapeutic ADCs would result in the delivery of greater amounts of agent to the target tissue.

[0012] This disclosure provides ROR1-ADC structures, each comprising (a) a ROR1 antibody, (b) a branched-chain HIPS linker, and (c) a drug. This disclosure also covers compounds and methods for generating such conjugates, as well as methods for using said conjugates.

[0013] Several aspects of this disclosure include a ROR1-ADC comprising: (a) a ROR1 antibody; and (b) one or more pyridazine-pyrroloconjugated moieties, said moieties comprising one or more drugs conjugated to said pyridazine-pyrroloconjugated moieties via one or more adapters.

[0014] In some implementations, the ROR1-ADC is represented by formula (I), which comprises: a. Antibodies that bind to tyrosine protein kinase receptor 1 (ROR1); and b. Two or more drugs conjugated to pyridazine-pyrrole via a linker. in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4Each is selected from hydrogen and alkyl groups; L A It is the first connector, which includes: -(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 -, in: a, b, c, d, e, and f are each independently 0 or 1, with the constraint that 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 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 1 V 2 V 3 V 4 V5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 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, heterocyclic and substituted heterocyclic; L B It is the second connector, which includes: -(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 -, in: g, h, i, j, k, l, and m are each independently 0 or 1, with the constraint that 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 13Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 7 V 8 V 9 V 10 V 11 V 12 and V 13 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 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, heterocyclic and substituted heterocyclic; s is an integer from 1 to 10; W 1It is the first medicine; and W 2 It is the second drug.

[0015] In some implementation schemes, Z 1 It is CR 4 .

[0016] In some implementation schemes, Z 3 It is CL B -W 2 .

[0017] In some implementation schemes, W 1 and W 2 One or both of them are camptothecin analogues, such as belotecone.

[0018] In some implementations, L A Include: -(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 -, in a, b, c, d, e, and f are each independently 0 or 1, with the constraint that 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 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x- 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 1 V 2 V 3 V 4 V 5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 It 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, heterocyclic and substituted heterocyclic.

[0019] In L A Some implementation schemes: T 1 Selected from (C1-C) 12 )alkyl and substituted (C1-C 12 )alkyl; T 2 T 3 T 4 T 5 and T 6 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, hydrazine, and esters; and V 1 V 2 V 3 V 4 V 5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ;and Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; In other implementations, a, b, c, and d are each 1; and e and f are 0.

[0020] In some implementations, T 1 T 2 T 3 T 4 T 5 and T 6Each can be substituted with a glycoside at its own discretion.

[0021] In some implementations, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally replaced by glycosides.

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

[0023] In some implementations, L A It is a connector, in which: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABC and V 4 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; a, b, c, and d are each 1; and e and f are both 0.

[0024] In other embodiments, PABC is replaced by glycosides, for example, the hydrogen atoms of PABC are replaced by glycosides such as glucuronide, galactoside, glucoside, mannoside, fucoidan, O-GlcNAc and O-GalNAc.

[0025] In some implementations, L B Include: -(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 -V12 ) l -(T 13 -V 13 ) m -, in g, h, i, j, k, l, and m are each independently 0 or 1, with the constraint that 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 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 7 V 8 V 9 V 10 V 11 V 12 and V 13 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15-、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 It 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, heterocyclic and substituted heterocyclic.

[0026] In some implementations, T 7 T 8 T 9 T 10 T 11 T 12 and T 13 Each can be substituted with a glycoside at its own discretion.

[0027] In some implementations, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally replaced by glycosides.

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

[0029] In L B Some implementation schemes: T 7 It is a covalent bond; T 8 T 9 T 10 T 11 and T 12 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, hydrazine, and esters; and V 7 V 8 V9 V 10 V 11 and V 12 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ;and Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; g, h, i, j, and k are each 1; and l and m are both 0.

[0030] In some implementations, 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 Each can be substituted with a glycoside at its own discretion.

[0031] In some implementations, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally replaced by glycosides.

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

[0033] In some implementations, L B It is a connector, in which: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is a substitute (C1-C) 12 )alkyl and V 9 It is -CO-; T 10 Yes (AA) p And V 10 It does not exist (e.g., covalent bonds); T 11 It is PABC and V 11 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; g, h, i, j, and k are each 1; and l and m are both 0.

[0034] In other embodiments, PABC is replaced by glycosides, for example, the hydrogen atoms of PABC are replaced by glycosides such as glucuronide, galactoside, glucoside, mannoside, fucoidan, O-GlcNAc and O-GalNAc.

[0035] In some implementations, the ROR1-ADC is represented by equation (I): in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4 Each is selected from hydrogen and (C1-C) 12 )alkyl; L A It is the first connector, in which: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 Yes (AA) p Where p is an integer from 1 to 20 and V 3 It is a covalent bond; T 4 It is PABC and V 4 It is a covalent bond; a, b, c, and d are each 1; e and f are both 0; and L B It is the second connector, in which: T 7 It is a covalent bond and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is a substitute (C1-C) 12 )alkyl and V 9 It is -CO-; T 10 Yes (AA) p Where p is an integer from 1 to 20 and V 10 It is a covalent bond; T 11 It is PABC and V 11 It is a covalent bond; and g, h, i, j, and k are each 1; and l and m are both 0; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

[0036] In some implementation schemes, W 1 and W 2 One or both of them are camptothecin analogues, such as belotecone.

[0037] In some implementations, the ROR1-ADC is represented by equation (I): in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4 Each is selected from hydrogen and (C1-C) 12 )alkyl; L A It is a connector, in which: T 1 It is a (C1-C6) alkyl group and V 1 It is -CONH-; T 2 It is -NHCO(PEG) t Substituted (C1-C6) alkylene groups, wherein (PEG) t yes And t is an integer from 2 to 10, arbitrarily 8, and V 2 It is -CO-; T 3 It is (AA)2 and V 3 It is a covalent bond; T 4 It is PABC substituted with glycosides and V 4 It is a covalent bond; a, b, c, and d are each 1; and e and f are both 0; and L B It is a connector, in which: T 7 It is a covalent bond and V 7 It is -NHCO-; T 8 It is a (C1-C6) alkyl group and V 8 It is -CONH-; T 9 It is -NHCO(PEG) t Substituted (C1-C6) alkylene groups, wherein (PEG) t yes And t is an integer from 2 to 10, arbitrarily 8, and V9 It is -CO-; T 10 It is (AA)2 and V 10 It is a covalent bond; T 11 It is PABC substituted with glycosides and V 11 It is a covalent bond; g, h, i, j, and k are each 1; and l and m are both 0; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

[0038] In some implementations, T 4 and T 11 One or both of the PABCs are replaced with glucuronide. In some embodiments, T 1 and T 8 One or both of them are ethyl. In some embodiments, T 2 and T 9 One or both of them are -NHCO(PEG) t Substituted C5 alkylene groups, of which (PEG) t yes And t is an integer between 5 and 10, optionally 8. In some implementations, W 1 and W 2 One or both of them are camptothecin analogues, such as belotecone.

[0039] In some implementations, ROR1-ADC is represented by equation (II): in: Ab indicates an antibody that binds to ROR1; and s is an integer from 1 to 10.

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

[0041] Formula (II) can be prepared by conjugating one or more of the linker-loads of Formula (IIa) shown below with an ROR1 antibody: In some embodiments, ROR1-ADC is represented by formula (I) or (II), wherein Ab comprises: VH CDR1, VH CDR2 and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:25; and VL CDR1, VL CDR2 and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:26.

[0042] In some embodiments, ROR1-ADC is represented by formula (I) or (II), wherein Ab comprises: (i) a VH region comprising VH CDR1 containing the amino acid sequence of any one of SEQ ID NO: 1, 2, 3, 4, 5, and 36; VH CDR2 containing the amino acid sequence of any one of SEQ ID NO: 6, 7, 8, 9, and 10; and VH CDR3 containing the amino acid sequence of any one of SEQ ID NO: 11, 12, 13, 14, and 37; and (ii) a VL region comprising VL CDR1 containing the amino acid sequence of any one of SEQ ID NO: 15, 16, 17, and 18; VL CDR2 containing the amino acid sequence of any one of SEQ ID NO: 19, 20, and 21; and VL CDR3 containing the amino acid sequence of any one of SEQ ID NO: 22, 23, and 24.

[0043] In some implementations, the ROR1-ADC is represented by formula (I) or (II), wherein the Ab competes with any ROR1 antibody as disclosed herein for binding to ROR1 (e.g., human ROR1).

[0044] In some implementations, ROR1-ADC is represented by formula (I) or (II), wherein Ab comprises the sequences of frame 1 (FR1), frame 2 (FR2), frame 3 (FR3) and / or frame 4 (FR4), for example, as shown in either SEQ ID NO: 25 and 26.

[0045] In some implementations, ROR1-ADC is represented by formula (I) or (II), where Ab contains a human frame sequence.

[0046] In some embodiments, ROR1-ADC is represented by formula (I) or (II), wherein Ab comprises (i) VH, which contains the amino acid sequence of SEQ ID NO:25; and VL, which contains the amino acid sequence of SEQ ID NO:26.

[0047] In some embodiments, ROR1-ADC is represented by formula (I) or (II), wherein Ab comprises a heavy chain containing the amino acid sequence of SEQ ID NO:41; and a light chain containing the amino acid sequence of SEQ ID NO:28. In other embodiments, s is 4. In some embodiments, ROR1-ADC is represented by formula (I) or (II), wherein Ab comprises a heavy chain containing the amino acid sequence of SEQ ID NO:44; and a light chain containing the amino acid sequence of SEQ ID NO:28. In other embodiments, s is 2.

[0048] In some embodiments, ROR1-ADC is represented by formula (II), wherein s is 4 and wherein Ab comprises a heavy chain containing the amino acid sequence of SEQ ID NO:41; and a light chain containing the amino acid sequence of SEQ ID NO:28. Therefore, the drug-to-antibody ratio (DAR) of ROR1-ADC is 8, and ROR1-ADC is referred to herein as ADC-8. In some embodiments, ROR1-ADC is represented by formula (II), wherein s is 2 and wherein Ab comprises a heavy chain containing the amino acid sequence of SEQ ID NO:44; and a light chain containing the amino acid sequence of SEQ ID NO:28. Therefore, the DAR of ROR1-ADC is 4.

[0049] This disclosure also provides a pharmaceutical composition comprising a 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 of the embodiments herein. In some embodiments, such pharmaceutical compositions exhibit a drug-to-antibody ratio (DAR) of about 1 to about 20 for the ROR1-ADC, such as 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.

[0050] This disclosure also provides a method for treating a subject with cancer or tumor, the method comprising administering a ROR1-ADC to the subject, wherein the ROR1-ADC is represented by formula (I) or (II), or a pharmaceutical composition comprising a ROR1-ADC of formula (I) or (II) and a pharmaceutically acceptable excipient, wherein the ROR1 antibody is as described in any embodiment herein.

[0051] This document provides a kit containing an antibody-drug conjugate or a pharmaceutical composition as disclosed herein, along with instructions for use. 6. Description of the attached drawings Figure 1 Exemplary in vitro cytotoxicity results are provided, as detailed in Example 5.

[0053] Figures 2A-2B Exemplary in vivo efficacy data in the MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model are provided, as detailed in Example 6. Figure 2A The tumor volume was plotted, and Figure 2B The percentage of tumor growth inhibition (TGI%) at day 29 compared to the isotype control was plotted.

[0054] Figures 3A-3B Exemplary in vivo efficacy data in the JEKO-1 metastatic lymphoma (MCL) xenograft model are provided, as detailed in Example 7. Figure 3A The tumor volume was plotted, and Figure 3B The percentage of tumor growth inhibition (TGI%) at day 25 compared to the isotype control was plotted.

[0055] Figure 4 Exemplary pharmacokinetic (PK) results for rats are provided, as detailed in Example 9.

[0056] Figures 5A-5B An exemplary ADC-8 for rats is provided. Figure 5A ) and ADC-4 ( Figure 5B The toxicokinetics (TK) results are detailed in Example 10.

[0057] Figure 6 Exemplary in vivo efficacy data in a non-small cell lung cancer (NSCLC) patient-derived xenograft (PDX) model are provided, as detailed in Example 8. 7. Detailed Implementation This disclosure provides antibody-drug conjugates (ADCs) that bind to ROR1 and to which a drug (directly or indirectly) is conjugated. Such ROR1-ADCs can be used in compositions and in methods of treating, preventing, or alleviating ROR1-mediated diseases, conditions, or disorders (including one or more symptoms of said diseases, conditions, or disorders). ROR1-mediated diseases, conditions, and disorders include a variety of cancers, including but not limited to any cancer in which tumor cells express or overexpress the ROR1 antigen. Furthermore, ROR1-ADCs can be used to kill and / or remove tumor cells. The ROR1-ADCs described herein can be used in compositions and in methods of treating cancer.

[0059] 7.1 Definition The techniques and procedures described or cited herein include those commonly understood and / or used by those skilled in the art using conventional methods, such as the widely used methods described below: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, 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, Volumes 1 and 2 (Kontermann and Dübel, eds., 2nd edition, 2010). Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following description of the terms should apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any description of the terms set forth herein conflicts with any document incorporated herein by reference, the description of the terms set forth herein shall prevail.

[0060] Unless otherwise indicated, the following terms have the following meanings. Any undefined term shall have its meaning as generally accepted in the art.

[0061] "Alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, such as 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. For example, this term includes straight-chain and branched-chain hydrocarbon groups, such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0062] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms (other than the C1 carbon atom) in the alkyl chain have been optionally replaced by heteroatoms, such as -O-, -N-, -S-, -S(O). n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl), and having 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, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclicoxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and -NR a R b , where R ’ and R ” They may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic groups.

[0063] "Alkylene" refers to a divalent aliphatic hydrocarbon group, preferably having 1 to 6, more preferably 1 to 3, carbon atoms, said hydrocarbon group being straight-chain or branched, and optionally interrupted by one or more groups selected from: -O-, -NR 10 -、-NR 10 C(O)-、-C(O)NR 10 - etc. For example, this term includes methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc.

[0064] "Substituted alkylene" means that one to three hydrogens of an alkylene group are replaced by substituents for carbon as defined below for "substituted".

[0065] The term "alkane" refers to alkyl and alkylene compounds as defined herein.

[0066] The terms “alkylaminoalkyl”, “alkylaminoolefin” and “alkylaminoynyl” refer to the group R'NHR”-, where R' is an alkyl group as defined herein and R” is an alkylene group, an olefin group, or an ynylene group as defined herein.

[0067] The terms “alkylaryl” or “aryl” refer to the groups -alkylene-aryl and -substituted alkylene-aryl, wherein alkylene, substituted alkylene and aryl are defined herein.

[0068] "Alkoxy" refers to the -O-alkyl group, where alkyl is as defined herein. For example, alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, etc. 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.

[0069] The term “substituted alkoxy” refers to a substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- group, wherein the substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0070] The term "alkoxyamino" refers to the group -NH-alkoxy, where alkoxy is defined herein.

[0071] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group have been replaced by a halogen group, and includes, for example, groups such as trifluoromethoxy.

[0072] The term "haloalkyl" refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been replaced by a halogen group. Examples of such groups include, but are not limited to, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0073] The term “alkylalkoxy” refers to the group -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, wherein the alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0074] 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 the alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0075] "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and having at least 1, preferably 1 to 2, unsaturated double bond sites. For example, this term includes divinyl, allyl, and but-3-en-1-yl. This term includes cis and trans isomers or mixtures of these isomers.

[0076] The term "substituted alkenyl" refers to an alkenyl group as defined herein, having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0077] "Alynyl" refers to a straight-chain or branched monovalent hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2, triple bond unsaturation sites. Examples of such alkynyl groups include ethynyl (-C≡CH) and propynyl (-CH2C≡CH).

[0078] The term "substituted alkynyl" refers to an alkynyl group as defined herein, having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0079] "Alkyneoxy group" refers to the -O-alkynyl group, where the alkynyl group is as defined herein. Alkyneoxy groups include, for example, acetylenoxy and propynylenoxy.

[0080] "Acyl" refers to the following groups: HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C (O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclic-C(O)-, and substituted heterocyclic-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 groups include "acetyl" CH3C(O)- "Acylamino" refers to the group -NR 20 C(O)alkyl, -NR 20 C(O) substituted alkyl groups, NR 20 C(O)cycloalkyl, -NR 20 C(O) substituted cycloalkyl groups, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O) alkenyl, -NR 20 C(O)-substituted alkenyl groups, -NR 20 C(O) ynyl group, -NR 20 C(O)-substituted alkynyl groups, -NR 20 C(O) aryl, -NR 20 C(O)-substituted aryl, -NR 20 C(O) heteroaryl, -NR 20 C(O)-substituted heteroaryl, -NR 20 C(O) heterocyclic group and -NR 20 C(O)-substituted heterocyclic groups, where R 20 It is hydrogen or alkyl and wherein the 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.

[0081] "Amino carbonyl" or the term "amino acyl" refers to the group -C(O)NR. 21 R 22 , where R 21 and R 22Independently 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 wherein R 21 and R 22 Optionally linked with the nitrogen to which it is attached to form a heterocyclic group or a substituted heterocyclic group, wherein the 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.

[0082] "Amino carbonyl amino" refers to the group -NR 21 C(O)NR 22 R 23 , where R 21 R 22 and R 23 It is independently selected from hydrogen, alkyl, aryl or cycloalkyl, or two of the R groups are linked to form a heterocyclic group.

[0083] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, wherein the alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic groups are as defined herein.

[0084] 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 heterocyclic-C(O)O-, wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0085] "Aminosulfonyl" refers to the group -SO2NR 21 R 22 , where R 21 and R 22 Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and wherein R 21 and R 22Optionally linked with the nitrogen to which it is attached to form a heterocyclic group or a 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.

[0086] "Sulfoamino" refers to the NR group. 21 SO2R 22 , where R 21 and R 22 Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic, and wherein R 21 and R 22 Optionally linked with the atoms to which it is attached to form a heterocyclic group or a substituted heterocyclic group, wherein the 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 groups are as defined herein.

[0087] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group with 6 to 18 carbon atoms, having a monocyclic ring (e.g., present in phenyl) or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthracene, and indenyl), which may or may not be aromatic, the limitation being that the connecting point is through an atom on the aromatic ring. This term includes, for example, phenyl and naphthyl. Unless otherwise limited by the definition of aryl substituents, such aryl groups may optionally be substituted with 1 to 5 or 1 to 3 substituents selected from acyloxy, hydroxyl, 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, alkylaryl, aryl, aryloxy Azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, 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.

[0088] "Aryloxy group" refers to the group -O-aryl, where aryl is as defined herein, including, for example, phenoxy, naphthoxy, etc., including optionally substituted aryl groups as otherwise defined herein.

[0089] "Amino" refers to the group -NH2.

[0090] The term “substituted amino” refers to a group -NRR, wherein each R is independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic, with the restriction that at least one R is not hydrogen.

[0091] The term "azido" refers to the group -N3.

[0092] "Carboxyl" or "carboxylate" refers to -CO2H or its salt.

[0093] "Carboxyl ester" or the term "carboxyalkyl" refers to the group -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-ynyl, -C(O)O-substituted ynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, or -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.

[0094] "(Carboxyl ester)oxy" or "carbonate group" refers to the following groups: -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-ynyl, -OC(O)O-substituted alkenyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl. -OC(O)O-substituted cycloalkenyl, -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic and -OC(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.

[0095] "Cyano" or "nitrile" refers to the -CN group.

[0096] "Cycloalkyl" refers to a cyclic alkyl group with 3 to 10 carbon atoms, having one or more rings, including fused, bridged, and spirocyclic systems. Suitable examples of cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, etc., or polycyclic structures such as adamantyl.

[0097] The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0098] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group with 3 to 10 carbon atoms, which has a monocyclic or polycyclic structure and at least one double bond, preferably 1 to 2 double bonds.

[0099] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, ketone, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0100] "Cycloynyl" refers to a non-aromatic cycloalkyl group with 5 to 10 carbon atoms, which has a monocyclic or polycyclic structure and at least one triple bond.

[0101] “Cycloalkoxy” refers to -O-cycloalkyl.

[0102] "Cycloalkenyl group" refers to -O-cycloalkenyl group.

[0103] "Halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0104] "Hydroxy" refers to the -OH group.

[0105] "Heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, such as 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryls can have a monocyclic ring (such as pyridyl, imidazolyl, or furanyl) or a ring system containing multiple fused rings (for example, groups such as indazinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothiophene), wherein at least one ring in the ring system is aromatic. To satisfy valence requirements, any heteroatom in such a heteroaryl ring may or may not be bonded to H or a substituent (e.g., alkyl or other substituents as described herein). In some embodiments, the nitrogen and / or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide an N-oxide (N→O), sulfinyl, or sulfonyl moiety. This term includes, for example, pyridyl, pyrroleyl, indolyl, thiophene, and furanyl. Unless otherwise limited by the definition of heteroaryl substituents, such heteroaryl groups may optionally be substituted with 1 to 5 substituents or 1 to 3 substituents selected from acyloxy, hydroxyl, 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, alkylaryl, aryl, aryloxy The following groups are included: alkyl, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, 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.

[0106] The term "heteroaryl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are defined herein. This term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, etc.

[0107] "Heteroaryl group" refers to -O-heteroaryl group.

[0108] "Heterocycle," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a monocyclic or multiple fused rings, including fused-bridged and spirocyclic systems, and having 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, wherein in fused-ring systems, one or more rings can be cycloalkyl, aryl, or heteroaryl, with the limitation that the linking point is through a non-aromatic ring. In some embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valence requirements, any heteroatom in such heterocycles may be bonded or unbonded to one or more H atoms or one or more substituents (e.g., alkyl or other substituents as described herein).

[0109] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, azacyclobutane, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, dihydroindole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxaline, isothiazine, phenazine, isoxazine, phenoxazine, phenthiazine, imidazoline, imidazoline, piperazine, etc. Pyridine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazoline, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl / thiamorpholinyl, 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl, etc.

[0110] Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may optionally be substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thionyl, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclicoxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and fused heterocycles.

[0111] "Heterocyclic oxygen group" refers to the -O-heterocyclic group.

[0112] The term "heterocyclic thio" refers to the heterocyclic group -S-.

[0113] The term "heterocyclic alkene" refers to a biradical formed from a heterocycle as defined herein.

[0114] The term "hydroxyamino" refers to the group -NHOH.

[0115] "Nitro" refers to the group -NO2.

[0116] "O-group" refers to an atom (=O).

[0117] "Sulfonyl" refers to the groups -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2-substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cycloalkyl, -SO2-cycloalkenyl, -SO2-substituted cycloalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein the alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl groups include, for example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0118] "Sulfoyloxy" refers to the group -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2-alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cycloalkyl, -OSO2-cycloalkenyl, -OSO2-substituted cycloalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2-heteroaryl, -OSO2-substituted heteroaryl, -OSO2-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.

[0119] "Sulfate group" or "sulfate" refers to the following groups: -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O-substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O-SO2-O-substituted cycloalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cycloalkenyl, -O-SO2-O-aryl, -O-SO2 -O-substituted aryl, -O-SO2-O-heteroaryl, -O-SO2-O-substituted heteroaryl, -O-SO2-O-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.

[0120] The term “aminocarbonyloxy” refers to the group -OC(O)NRR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, wherein the alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic groups are as defined herein.

[0121] "Thiol" refers to the group -SH.

[0122] "Thioxo" or the term "thioketo" refers to an atom (=S).

[0123] "Alkylthio" or the term "thioalkoxy" refers to the group -S-alkyl, where the alkyl group is as defined herein. In some embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may be present as one or more stereoisomers.

[0124] The term "substituted thioalkoxy" refers to an alkyl group that is -S-substituted.

[0125] The term “thioaryloxy” refers to the aryl-S- group, wherein the aryl group is as defined herein, including optionally substituted aryl groups as otherwise defined herein.

[0126] The term “thioheteroaryloxy” refers to the heteroaryl group -S-, wherein the heteroaryl is as defined herein, including optionally substituted aryl groups as otherwise defined herein.

[0127] The term “thioheterocyclic group” refers to a heterocyclic group -S-, wherein the heterocycle is as defined herein, including optionally substituted heterocycles as otherwise defined herein.

[0128] In the disclosure herein, the term “substituted” when used to modify a specified group or radical may also mean that one or more hydrogen atoms of the specified group or radical are each independently replaced by the same or different substituents as defined below.

[0129] Except for the groups disclosed herein for specific terms, unless otherwise stated, substituents used to replace one or more hydrogen atoms on a saturated carbon atom in a designated group or radical (any two hydrogens on a single carbon can be =O, =NR) 70 =N-OR 70 =N2 or =S substitution) is -R 60 , halogen, =O, -OR 70 -SR 70 -NR 80 R 80 Trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 -SO2O - M + -SO2OR 70 -OSO2R 70 -OSO2O - M + -OSO2OR 70 -P(O)(O) - )2(M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -C(O)O - M + -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OC(O)O - M + -OC(O)OR 70 -OC(S)OR 70 -NR 70 C(O)R70 -NR 70 C(S)R 70 -NR 70 CO2 - M + -NR 70 CO2R 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 Choose from the group consisting of: optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each R 70 Independently, it is hydrogen or R 60 ; Each R 80 R is independent 70 Or alternatively, two Rs 80 The nitrogen atoms bonded to them together form 5-, 6-, or 7-membered heterocyclic alkyl groups, which may optionally include 1 to 4 identical or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have -H or C1-C3 alkyl substitutions; and each M + It is a counterion with a single net positive charge. Each M + It can be independently, for example, an alkali metal ion, such as K. + Na + Li + Ammonium ions, such as + N(R 60 )4; or alkaline earth metal ions, 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 of such divalent alkaline earth metal ions can be the ionized form of the compound of the present invention, and the other can be a typical counter ion, such as chloride ions; or the two ionized compounds disclosed herein can be used as counter ions of such divalent alkaline earth metal ions; or the dual ionized compound of the present invention can be used as a counter ion of such divalent alkaline earth metal ions.") As a specific example, -NR 80 R80 Intended to include -NH2, -NH-alkyl, N -pyrrolidinyl, N -piperazinyl, 4 N -methyl-piperazine-1-yl and N -morpholinoyl.

[0130] Except as otherwise stated herein, the substituent for hydrogen on the unsaturated carbon atom in “substituted” alkenes, alkynes, aryls, and heteroaryls is -R. 60 , halogen, -O - M + -OR 70 -SR 70 -S - M + -NR 80 R 80 Trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 -SO3M + -SO3R 70 -OSO2R 70 -OSO3 - M + -OSO3R 70 -PO3 -2 (M + )2、-P(O)(OR 70 )O - M + -P(O)(OR) 70 )2、-C(O)R 70 -C(S)R 70 -C(NR) 70 )R 70 -CO2 - M + -CO2R 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OCO2 - M + -OCO2R 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR70 C(S)R 70 -NR 70 CO2 - M + -NR 70 CO2R 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 R 70 R 80 and M + As previously defined, the limiting condition is that in the case of substituted alkenes or alkynes, the substituent is not -O. - M + -OR 70 -SR 70 or -S - M + .

[0131] Except for the groups disclosed herein for individual terms, unless otherwise stated, the substituents for hydrogen on the nitrogen atom in “substituted” heteroalkyl and cycloalkyl groups are -R 60 -O - M + -OR 70 -SR 70 -S - M + -NR 80 R 80 Trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 -S(O)2O - M + -S(O)2OR 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 -C(S)R 70 -C(NR) 70 )R 70 -C(O)OR 70 -C(S)OR 70 -C(O)NR 80 R 80 -C(NR) 70 )NR 80 R 80 -OC(O)R 70 -OC(S)R 70 -OC(O)OR 70 -OC(S)OR 70 -NR 70 C(O)R 70 -NR 70 C(S)R 70 -NR 70 C(O)OR 70 -NR 70 C(S)OR 70 -NR 70 C(O)NR 80 R 80 -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 , where R 60 R 70 R 80 and M + As previously defined.

[0132] In addition to the disclosure herein, in one embodiment, the substituted group has 1, 2, 3 or 4 substituents, 1, 2 or 3 substituents, 1 or 2 substituents or 1 substituent.

[0133] It should be understood that polymers obtained by defining substituents having other substituents against themselves (e.g., a substituted aryl group having a substituted aryl group as a substituent, the substituent itself being substituted by the substituted aryl group, the substituted aryl group being further substituted by the substituted aryl group, etc.) among all the substituted groups defined above are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of a substituted aryl group that is particularly considered herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0134] Unless otherwise indicated, the naming of substituents not explicitly defined herein is accomplished by naming the terminal portion of the functional group, followed by naming the adjacent functional group toward the junction. For example, the substituent “arylalkoxycarbonyl” refers to the group (aryl)-(alkyl)-OC(O)-.

[0135] For any group containing one or more substituents disclosed herein, it should be understood that such group does not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. Furthermore, the subject compounds also include all stereochemical isomers arising from the substitution of these compounds.

[0136] The term "pharmaceutically acceptable salt" means a salt (a salt in which the counterion has acceptable mammalian safety under a given dosing regimen) that is acceptable to administer to patients (such as mammals). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound derived from various organic and inorganic counterions well known in the art, and by way of example only, includes sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; and when the molecule contains a basic functional group, it refers to a salt of an organic or inorganic acid, such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, etc.

[0137] The term "its salt" refers to a compound formed when the proton of an acid is replaced by a cation (such as a metal cation or an organic cation). Where applicable, the salt is a pharmaceutically acceptable salt, except for salts of intermediate compounds not intended for administration to a patient. For example, salts of the compounds of the present invention include those where the compound is protonated by an inorganic or organic acid to form a cation and the conjugate base of the inorganic or organic acid is the anionic component of the salt.

[0138] A solvate is a complex formed by the combination of solvent molecules and solute molecules or ions. Solvents can be organic compounds, inorganic compounds, or mixtures of both. Some examples of solvents include, but are not limited to, methanol. N , N - Dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0139] Stereoisomers are compounds that have the same atomic bonds but different spatial arrangements of atoms. Stereoisomers include cis-trans isomers, E and Z Isomers, enantiomers and diastereomers.

[0140] "Tautomers" refer to alternating forms of molecules that differ only in the electronic bonding and / or proton positions of atoms, such as enol-ketone and imine-enamine tautomers, or tautomers of heteroaryl groups containing a -N=C(H)-NH- ring arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetraazoles. Those skilled in the art will recognize that other tautomeristic ring arrangements are also possible.

[0141] It should be understood that the term "or its salt or solvate or stereoisomer" is intended to include all arrangements of salts, solvates and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compound.

[0142] "Pharmaceutical effective amount" and "therapeutic effective amount" refer to the amount of a compound sufficient to treat a specified condition or disease or one or more of its symptoms and / or prevent the occurrence of said disease or condition. In the case of tumorigenic proliferative diseases, a pharmaceutically or therapeutically effective amount includes, in particular, an amount sufficient to shrink a tumor or reduce its growth rate.

[0143] "Prophylactic effective amount" is the amount of a pharmaceutical composition that, when administered to a subject, produces the intended preventive effect (e.g., preventing or delaying the onset (or recurrence) of a disease, condition, or disorder, or reducing the likelihood of the onset (or recurrence) of a disease, condition, or disorder or related symptoms).

[0144] A complete therapeutic or preventative effect may not occur with the administration of a single dose, but may only occur after a series of doses. Therefore, an effective dose for treatment or prevention may be administered in one or more administrations.

[0145] "Patients" refers to human and non-human subjects, especially mammalian subjects.

[0146] As used herein, the term “treating / treatment” means treatment of a disease or medical condition in a patient (e.g., a mammal, particularly a human), including: (a) preventing the occurrence of said disease or medical condition, such as by administering preventive treatment to a subject; (b) improving said disease or medical condition, such as by eliminating or resolving said disease or medical condition in the patient; (c) inhibiting said disease or medical condition, such as by slowing or preventing the development of said disease or medical condition in the patient; or (d) alleviating the symptoms of said disease or medical condition in the patient.

[0147] In some implementation schemes, the term "treatment" excludes preventative treatment.

[0148] "Reactive complex" refers to a molecule or molecular moiety that undergoes a specific reaction with another reactive complex to produce a reaction product. Exemplary reactive complexes include cysteine ​​or serine in a sulfatase motif and formylglycine synthase (FGE), which react to form a reaction product with a converted aldehyde tag containing formylglycine (fGly) in place of cysteine ​​or serine in the motif. Other exemplary reactive complexes include aldehydes (e.g., reactive aldehyde groups) of fGly residues in a converted aldehyde tag; and "aldehyde reactive reactive complexes" that contain an aldehyde reactive group and a moiety of interest, and react to form a reaction product with a polypeptide having a moiety of interest conjugated to the polypeptide via an fGly residue.

[0149] "N-terminus" refers to the terminal amino acid residue in a polypeptide that has a free amine group, which is usually part of the covalent backbone of the polypeptide in non-N-terminal amino acid residues.

[0150] "C-terminus" refers to the terminal amino acid residue of a polypeptide that has a free carboxyl group, which is usually part of the covalent backbone of the polypeptide in non-C-terminal amino acid residues.

[0151] As used with respect to polypeptides or the amino acid sequence of polypeptides, "internal site" means a region in the polypeptide that is neither at the N-terminus nor the C-terminus.

[0152] The term "subject" refers to both human and non-human subjects, especially mammalian subjects.

[0153] As used herein, the term “natural amino acid sequence” refers to the amino acid sequence of a polypeptide prior to its modification to include modified amino acid residues.

[0154] The terms "amino acid analog," "non-natural amino acid," etc., are used interchangeably and include amino acid compounds that are structurally and / or in overall shape similar 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 having modified side chains or backbones. Amino acid analogs also include amino acid analogs having the same stereochemistry as naturally occurring D-type amino acids, as well as L-type amino acid analogs. In some embodiments, amino acid analogs share the backbone and / or side chain structure with one or more natural amino acids, differing only in one or more modified groups in the molecule. Such modifications can include, but are not limited to, substituting an atom (e.g., S) with an atom (e.g., N), adding a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g., Cl or Br, etc.), deleting a group, substituting a covalent bond (single bond substituting for double bond, etc.), or combinations thereof. For example, amino acid analogs can include α-hydroxy acids and α-amino acids, etc.

[0155] The term "amino acid side chain" is used to refer to a substituent attached to the α-carbon of an amino acid residue, including natural amino acids, non-natural amino acids, and amino acid analogs. Amino acid side chains may also include those described in the background of modified amino acids and / or conjugates as described herein.

[0156] The term "carbohydrate" is used to refer to monomeric units and / or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term "sugar" is used to refer to smaller carbohydrates, such as monosaccharides and disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical method), or absent (e.g., eliminated or substituted with H). A variety of carbohydrates and carbohydrate derivatives are available and can be adapted to subject compounds and conjugates.

[0157] The term "glycoside" or "glycosyl group" refers to a sugar molecule or group that is bound to a moiety via a glycosidic bond. For example, the moiety to which a glycoside is bound can be a cleavable linker as described herein. Glycosidic bonds can connect a glycoside to another moiety via various types of bonds, such as, but not limited to, O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyl groups). In some cases, glycosides can be cleaved from the group to which they are bound, for example, through chemically mediated or enzyme-mediated hydrolysis.

[0158] As used herein, “ROR1 antigen” refers to a member of the tyrosine protein kinase transmembrane receptor (ROR) family. ROR2 is another member of the same family. However, in some embodiments, the ROR1-ADC and ROR1 antibody disclosed herein do not bind to ROR2 (e.g., human ROR2). In some embodiments, the ROR1-ADC and ROR1 antibody disclosed herein do not bind to human ROR2 or cyno ROR2. In other embodiments, the ROR1-ADC and ROR1 antibody disclosed herein have a higher affinity for ROR1 (e.g., human ROR1) than for ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADC and ROR1 antibody disclosed herein to ROR1 (e.g., human ROR1) is at least twice that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADC and ROR1 antibody, as disclosed herein, to ROR1 (e.g., human ROR1) is at least 5 times greater than that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADC and ROR1 antibody, as disclosed herein, to ROR1 (e.g., human ROR1) is at least 10 times greater than that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADC and ROR1 antibody, as disclosed herein, to ROR1 (e.g., human ROR1) is at least 100 times greater than that to ROR2 (e.g., human ROR2). In some embodiments, the binding affinity of the ROR1-ADC and ROR1 antibody, as disclosed herein, to ROR1 (e.g., human ROR1) is at least 1000 times greater than that to ROR2 (e.g., human ROR2).

[0159] Therefore, in one implementation, the term “ROR1” or “ROR1 antigen” as used herein refers to ROR1 (e.g., human ROR1).

[0160] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably and in the broadest sense herein, and specifically encompass, for example, polyclonal antibodies, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, and full-length monoclonal antibodies), antibody compositions having multiple or single epitope specificity, recombinant antibodies, single-domain antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), synthetic antibodies, chimeric antibodies, humanized antibodies, or human-form antibodies having full-length heavy and / or light chains. Antibodies also include single antibody domains and antibody fragments (and / or polypeptides containing antibody fragments) that retain ROR1 binding characteristics. Non-limiting examples of antibody fragments include antigen-binding and / or effector regions of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-chain antibody molecules, bivariate domain antibodies, monovariate domain antibodies, linear antibodies, V regions, multispecific antibodies formed from antibody fragments, F(ab)2, Fd, Fc, bifunctional antibodies, di-bifunctional antibodies, disulfide-linked Fv (dsFv), monovariate antibodies (e.g., nanobodies), or other fragments (e.g., fragments composed of non-covalently coupled heavy and light chain variable regions). Generally, the variable (V) region can be any suitable arrangement of immunoglobulin heavy chain (VH) and / or light chain (VL) variable regions. For example, antibodies also include tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, and antibody heavy chain monomers. Thus, by way of example, the V region can be a dimer and contain a VH-VH, VH-VL, or VL-VL dimer that binds ROR1. In any implementation, the VH and VL regions can be covalently coupled directly or via a linker to form a single-chain Fv (scFv). For ease of reference, the scFv protein is referred to herein as included in the category “antibody fragments.” Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of an antibody. CDRs (also known as “minimum recognition units” or “hypervariates”) can be obtained by constructing a polynucleotide encoding one or more CDRs of interest.For example, such polynucleotides can be prepared by using polymerase chain reaction to synthesize variable regions from the mRNA of antibody-generating cells as templates (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology, 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” Monoclonal Antibodies Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166, Cambridge University Press (1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), p. 137, Wiley-Liss, Inc. (1995)). Antibody fragments may be incorporated into, for example, single-domain antibodies, large antibodies, small antibodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, neoantigen receptor variable regions (v-NAR), and double-single-chain Fv regions (see, for example, Hollinger and Hudson, Nature Biotechnology, 23(9):1126-1136, 2005). In some embodiments, antibodies containing VH and / or VL contain light chain and / or heavy chain constant regions, such as one or more constant regions, including one or more IgG1, IgG2, IgG3, and / or IgG4 constant regions. In some embodiments, antibodies may include epitope-binding fragments of any of the above. The antibodies described herein may belong to any class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules.

[0161] The terms "humanized antibody" or "humanized immunoglobulin" refer to non-human (e.g., mouse or rabbit) antibodies in which one or more amino acids (e.g., in the frame region, constant region, or CDR) have been replaced by amino acids from human antibodies at corresponding locations. Generally, humanized antibodies produce a reduced immune response in a human host compared to the non-humanized form of the same antibody. Antibodies can be humanized using a variety of techniques known in the art, such as CDR transplantation (EP 239,400; PCT publication WO 91 / 09967; US Patent Nos. 5,225,539; 5,530,101; and 5,585,089), surface remodeling or remodeling (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)) and chain truncation (US Patent No. 5,565,332). In some embodiments, framework substitutions are identified by modeling the interaction between the CDR and framework residues to identify framework residues important for antigen binding and by performing sequence comparisons to identify uncommon framework residues at specific locations (see, for example, U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies intended for use in this invention are described in U.S. Patent 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, as well as PCT Publications WO 98 / 45331 and WO 98 / 45332. In a specific embodiment, the test rabbit antibody can be humanized according to the methods described in US20040086979 and US20050033031. Therefore, the above-mentioned antibody can be humanized using methods well known in the art.

[0162] The term "chimeric antibody" refers to an antibody whose light and heavy chain genes are typically constructed from variable and constant region genes of antibodies belonging to different species through genetic engineering. For example, a variable region from a mouse monoclonal antibody gene can be linked to a human constant region (such as γ1 and γ3). One example of a therapeutic chimeric antibody is a hybrid protein consisting of a variable or antigen-binding domain from a mouse antibody and a constant or effector domain from a human antibody; however, domains from other mammalian species can also be used.

[0163] As used herein, the term “monospecific” refers to an antibody having one or more binding sites, each binding to the same epitope of the same antigen.

[0164] The term "multispecific" when used to refer to an antibody means that the antibody can specifically bind to at least two different epitopes. For example, the two binding sites may each form a variable domain (VH) of the antibody heavy chain and a variable domain (VL) of the antibody light chain, or a pair of VHH domains each binding to different epitopes on different antigens or the same antigen. Such bispecific antibodies can have a 1+1 form (containing one binding site against a first antigen or epitope and one binding site against a second antigen or epitope). Other forms of bispecific antibodies may be 2+1 or 1+2 (containing two binding sites against a first antigen or epitope and one binding site against a second antigen or epitope) or 2+2 (containing two binding sites against a first antigen or epitope and two binding sites against a second antigen or epitope). When a bispecific antibody contains two antigen-binding sites, each site can bind to a different epitope. Such bispecific antibodies can bind to two different epitopes on the same antigen (e.g., epitopes on the ROR).

[0165] In the context of two or more nucleic acids or peptides, the term "identity" or "percentage of identity" refers to two or more sequences or subsequences being identical when compared and aligned (with gaps introduced where necessary) to achieve maximum correspondence, or having a specified percentage of identical nucleotide or amino acid residues, regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are well known in the art for obtaining amino acid or nucleotide sequence alignments. These include, but are not limited to, the Basic Local Alignment Search Tool (BLAST), ALIGN, MegAlign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or peptides are substantially identical, meaning that when compared and aligned to achieve maximum correspondence, 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, as measured using sequence comparison algorithms or by visual inspection. In some embodiments, identity exists in regions of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value between these lengths in the amino acid sequence. In some embodiments, identity exists in regions longer than 60-80 residues, for example, at least about 80-100 residues; and in some embodiments, the sequences are substantially identical across the full length of the compared sequence (e.g., the coding region of a target protein or antibody). In some embodiments, identity exists in regions of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 residues, or any integer value between these lengths in the nucleotide sequence. In some embodiments, identity exists in regions longer than 60-80 bases, for example, at least about 80-1000 bases or more; and in some embodiments, the sequences are substantially identical across the full length of the compared sequence (e.g., the nucleotide sequence encoding the protein of interest).

[0166] "Conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a similar chemical signature in its side chain. Families of amino acid residues with similar side chains are 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 (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, phenylalanine replacing tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of peptides, soluble proteins, and / or antibodies of this disclosure do not eliminate the binding of the peptide, soluble protein, or antibody containing the said amino acid sequence to the target binding site. Methods for identifying conserved substitutions of amino acids that do not eliminate binding are well known in the art.

[0167] The term "peptide" refers to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, and may include non-amino acids (e.g., substituted for them). The term also covers amino acid polymers that are naturally or through intervention; for example, disulfide bond formation, glycosylation, lipoylation, acetylation, phosphorylation, or any other operation or modification, such as direct or indirect linkage or conjugation to a moiety (e.g., a labeled component or a drug (e.g., a toxin)). The definition also includes, for example, peptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art. It should be understood that, since the peptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, the peptides may exist as single chains or single-chain dimers.

[0168] As used herein, an "antigen" is a portion or molecule containing an epitope that an antibody can bind to. Therefore, an antigen can be bound by an antibody. In some embodiments, the antigen bound by the antibody described herein is a ROR1 antigen (e.g., human ROR1 antigen) or a fragment thereof.

[0169] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen that an antibody can bind to. An epitope can be a linear epitope or a conformational, non-linear, or discontinuous epitope. For example, in the case of a peptide antigen, an epitope can be a continuous amino acid of the peptide ("linear" epitope), or an epitope can comprise amino acids from two or more discontinuous regions of the peptide ("conformational," "non-linear," or "discontinuous" epitope), such as human ROR1. Those skilled in the art will understand that, in general, a linear epitope may or may not depend on secondary, tertiary, or quaternary structures. For example, in some embodiments, an antibody binds to a set of amino acids regardless of whether they fold into a native three-dimensional protein structure. In other embodiments, the antibody requires the amino acid residues constituting the epitope to exhibit a specific conformation (e.g., bending, twisting, rotating, or folding) in order to recognize and bind to the epitope.

[0170] Antibodies bind to “epitopes,” “substantially identical epitopes,” or “identical epitopes” with reference antibodies. The most widely used and rapid method for determining whether two antibodies bind to the same, overlapping, or adjacent epitopes in three-dimensional space is a competition assay, which can be configured in many different ways, for example, using labeled antigens or labeled antibodies. In some assays, the antigen is immobilized on a 96-well plate or expressed on a cell surface, and radioactive, fluorescent, or enzyme labeling is used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody.

[0171] As used herein, the terms “specific binding,” “specific recognition,” “immune-specific binding,” “selective binding,” “immune-specific recognition,” and “immune specificity” are similar terms in the context of antibodies and refer to molecules that bind to antigens (e.g., epitopes) as understood by those skilled in the art.

[0172] In some embodiments, "specific binding" means, for example, that the interaction between a peptide or molecule and an epitope, protein, or target molecule is more frequent, faster, longer-lasting, has greater affinity, or a combination of the above characteristics compared to alternative substances (including related and unrelated proteins). For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with typically lower affinity, as determined by, for example, immunoassays, BIACORE™, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), Octet QK384 systems (ForteBio, Menlo Park, CA), or other assays known in the art. In some embodiments, the antibody or antigen-binding region binds to or specifically binds to the antigen when the affinity of the antibody or antigen-binding region to the antigen is higher than the affinity to any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction will be at least twice the background signal or noise, and possibly more than ten times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd ed., 1989). In some embodiments, the antibody or antigen-binding region binds to less than about 10% of its binding to a “non-target” protein, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. In some embodiments, the Kk of a molecule that specifically binds to an antigen is determined when it binds to the antigen. A Compared to the K value when the molecule binds to another antigen A The binding intensity is at least 2 log, 2.5 log, 3 log, 4 log, or higher. In some embodiments, the molecule that specifically binds to the antigen does not cross-react with other proteins. In another specific embodiment, the molecule that specifically binds to the antigen does not cross-react with other non-ROR1 antigens. In some embodiments, "specific binding" means, for example, that a peptide or molecule binds to the K of a protein or target. D It is approximately 0.1 mM or lower, but more typically less than approximately 1 µM. In some embodiments, "specific binding" means that the peptide or molecule binds to the K+ of the target. DThe specific binding concentration is at least about 0.1 µM or less, at least about 0.01 µM or less, or at least about 1 nM or less. Due to sequence identity between homologous proteins of different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to identity within certain regions of the peptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, a peptide or molecule that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding, e.g., binding to a single target. Therefore, in some embodiments, a peptide or molecule may specifically bind to more than one target. In some embodiments, multiple targets may be bound by the same antigen-binding site on the peptide or molecule. For example, in some embodiments, an antibody may contain two identical antigen-binding sites, each specifically binding to the same epitope on two or more proteins. In alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities. Generally, but not necessarily, the term “binding” means “specific binding.”

[0173] The term "binding affinity" generally refers to the aggregate strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding complex (e.g., an antigen, such as a ROR). Unless otherwise indicated, as used herein, "binding affinity" refers to the 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 complex Y can typically be expressed using the dissociation constant (K). D () indicates. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate rapidly, while high-affinity antibodies generally bind to antigens more quickly and tend to remain bound for longer. Various methods for measuring binding affinity are known in the art, any of which can be used to achieve the purposes of this disclosure. In one embodiment, “K” D "or "K D The "value" can be measured using biological layer interferometry (BLI) with a system such as the Octet QK384 (ForteBio, Menlo Park, CA). Alternatively, K... DIt can also be measured in radiolabeled antigen binding assays (RIA), for example, with the Fab form of the antibody of interest and its antigen (Chen et al., (1999) J. Mol Biol 293:865-881) or using surface plasmon resonance (SPR) assays via BIACORE™, using, for example, BIACORE™-2000 or BIACORE™-3000 (BIAcore, Inc., Piscataway, NJ). The "on-rate of association / association rate" or "k" is used. 缔合 "and dissociation rate (off-rate / rate of dissociation / dissociation rate)" or "k 解离 "The same SPR or BLI techniques described herein can also be used to determine this, for example, the Octet QK384 system (ForteBio, Menlo Park, CA) or the BIACORE™-2000 or BIACORE™-3000 (BIACORE™, Inc., Piscataway, NJ).

[0174] The term "competition," when used in the context of ROR1 antibodies, 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 the binding of that 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). Various types of competitive binding assays can be used to determine whether a test antibody competes with a reference molecule for binding to ROR1 (e.g., human ROR1). Examples of assays that may be used include solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see, for example, Stahli et al., (1983) Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin-EIA (see, for example, Kirkland et al., (1986) J. Immunol. 137:3614-3619 or Cheung et al., (1990) Virology 176:546-552); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 label (see, for example, Morel et al., (1988) Molec. Immunol. 25:7-15); and directly labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such assays involve using a purified antigen (e.g., ROR1, such as human ROR1) bound to a solid surface or cell carrying an unlabeled test antigen-binding protein (e.g., a test ROR1 antibody or ADC) or a labeled reference antigen-binding protein (e.g., a reference ROR1 antibody or ADC). Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, an excess of the test antigen-binding protein is present. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and / or antibodies that bind to adjacent epitopes (e.g., similar epitopes or overlapping epitopes) that are sufficiently close to the epitope bound to the reference antibody to cause steric hindrance.Typically, when an excess of competitive antibody is present, it inhibits the specific binding of the reference antibody to the 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.

[0175] The terms “constant region” and “constant domain” are used interchangeably herein and are well-known antibody terms in the art, referring to antibody portions, such as the carboxyl-terminal portions of the light and / or heavy chains, that do not directly participate in antibody-antigen binding but can exhibit various effector functions, such as interacting with Fc receptors. The terminology also includes portions of immunoglobulin molecules that have a generally more conserved amino acid sequence relative to the immunoglobulin variable region.

[0176] Antibody "effective function" refers to those biological activities attributable to the Fc region of the antibody (e.g., the native Fc region or the Fc region with amino acid sequence variations), and varies with antibody isotype. Examples of antibody effector functions include: C1q and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0177] In this document, the term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 (EU numbering system) or from Pro230 (EU numbering system) to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 in EU numbering system) may be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. An exemplary Fc region sequence is provided below (CH2 domain = bold text; CH3 domain = underlined text): CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:38).

[0178] The “functional Fc fragment” possesses an “effective function” of the native Fc region. Exemplary “effective functions” include C1q; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require a combination of the Fc region with a binding region or binding domain (e.g., antibody variable region or domain) and can be assessed using a variety of disclosed assays.

[0179] The “native sequence Fc region” contains the same amino acid sequence as the Fc region found in nature and has not been manipulated, modified, and / or altered by humans (e.g., isolated, purified, selected, included, or combined with other sequences, such as variable region sequences). The native sequence human Fc region includes the native sequence human IgG1 Fc region (non-A and A allotypes); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as their naturally occurring variants.

[0180] A “variant Fc region” comprises an amino acid sequence that differs from the amino acid sequence of the native Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion), preferably one or more amino acid substitutions. In some embodiments, the variant Fc region has at least one amino acid substitution in the native Fc region or the Fc region of the parent peptide compared to the native Fc region or the Fc region of the parent peptide, for example, about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions. The variant Fc region described herein may have at least about 80% homology with the native Fc region and / or the Fc region of the parent peptide, or at least about 90% homology with it, for example, at least about 95% homology with it. The variant Fc region described herein may lose its effector function (e.g., silence the Fc (also referred to herein as “sFc”)).

[0181] In some embodiments, sFc contains 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").

[0182] The following provides an example variant Fc region (“silent Fc”) sequence (CH2 domain = bold text and amino acid changes are underlined; CH3 domain = underlined text): CPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL K APIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO:39).

[0183] Alternatively, the variant Fc region has reduced potential immunogenicity. In other embodiments, the variant Fc region includes 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 at position Leu358 (L358) according to the EU numbering system (also referred to herein as “EEM” or “D356E / E357E / L358M”). When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, wherein the amino-terminal portion includes a variable region of approximately 120 to 130 or more amino acids, and the carboxyl-terminal portion includes one or more constant regions. "Heavy chain" can refer to any different type, such as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu) based on the amino acid sequence of the constant regions, which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0184] As used herein, the term "light chain," when used to refer to an antibody, can refer to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion comprises a variable region of about 100 to about 110 or more amino acids, and the carboxyl-terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant region, two different types exist, such as κ (kappa) or λ (lambda). The amino acid sequences of light chains are well known in the art. In one embodiment, the "chain" (e.g., heavy chain or light chain) is itself a molecule (e.g., a polypeptide). In another embodiment, the "chain" (e.g., heavy chain or light chain) is part of a molecule (e.g., a polypeptide), for example, directly or indirectly conjugated to the rest of the molecule (e.g., a polypeptide).

[0185] The terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms refer to a portion of an antibody that contains amino acid residues that interact with an antigen and confers specificity and affinity of the binding fragment or region for the antigen (e.g., a CDR). As used herein, “antigen-binding fragment” includes “antibody fragment,” which contains a portion of an antibody, including one or more CDRs, such as an antigen-binding region or variable region of the antibody.

[0186] The antibodies described herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (e.g., including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelified antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0187] In some embodiments, the antibodies described herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing one or more antigen-binding sites that bind to ROR1.

[0188] The antibodies described herein can belong to 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 molecules. In some embodiments, the ROR1 antibody described herein is an IgG antibody (e.g., human IgG) or its class (e.g., human IgG1, IgG2, IgG3, or IgG4) or subclass.

[0189] In some embodiments, the antibody is a 4-chain antibody unit comprising two heavy (H) chain / light (L) chain pairs. In other 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. Or, additionally, the amino acid sequences of the L chains are different from each other. For example, the antibody comprises a first H / L chain pair and a second H / L chain pair, wherein the first H / L chain pair binds to a ROR1 antigen and the second H / L chain pair binds to another ROR1 antigen or a non-ROR1 antigen. In some embodiments, the antibody is a 2-chain antibody unit comprising a VHH-VHH pair. In other embodiments, the amino acid sequences of the VHHs are identical. In other embodiments, the amino acid sequences of the VHHs are different from each other. For example, the antibody comprises a first VHH and a second VHH, wherein the first VHH binds to a 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, such as human constant regions. In some embodiments, the L-chain constant region of such antibodies is a κ or λ light chain constant region, such as the human κ or λ light chain constant region. In some embodiments, the H-chain constant region of such antibodies comprises a γ heavy chain constant region, such as the human γ heavy chain constant region. In some embodiments, such antibodies comprise an IgG constant region, such as the human IgG constant region (e.g., IgG1, IgG2, IgG3, and / or IgG4 constant regions).

[0190] 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 may preferentially bind to ROR1 (e.g., human ROR1), meaning that the antibody or fragment thereof has a higher affinity for ROR1 (e.g., human ROR1) than it does for an unrelated control protein. For example, an antibody or fragment thereof may specifically recognize and bind to ROR1 or a portion thereof. "Specific binding" means that the affinity of the ROR1 antibody or fragment thereof for ROR1 is at least 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or 10,000 times higher than that for an unrelated control protein (e.g., chicken oocyte lysozyme). In some embodiments, the ROR1 antibody or fragment thereof may bind substantially specifically to ROR1 (e.g., capable of distinguishing ROR1 from other known peptides, for example, by measurable differences in binding affinity). In some implementations, the ROR1 antibody can react with ROR1 sequences other than the human ROR1 sequence (e.g., the cynomolgus monkey ROR1 sequence).

[0191] The terms “variable region” and “variable domain” are used interchangeably to refer to portions of the antibody light and heavy chains, typically located at the amino terminus of the light and heavy chains, having a length of approximately 120 to 130 amino acids in the heavy chain and approximately 100 to 110 amino acids in the light chain, and serving as the binding and specificity of each antibody to 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, within an antibody, certain segments of the variable region have widely different sequences. The V region mediates antigen binding and determines the specificity of a particular antibody to its antigen. However, this variability is not uniformly distributed across the 110-amino acid span of the variable region. In practice, the V region consists of segments with less variability (e.g., relatively invariant), referred to as frame regions (FRs), having approximately 15–30 amino acids, separated by shorter regions with greater variability (e.g., extreme variability), referred to as “hypervariable regions” or alternatively, “complementarity-determining regions.” The variable regions of both the heavy and light chains each contain four frames (FR1, FR2, FR3, and FR4), which are predominantly β-sheet configurations connected by three hypervariable regions. These hypervariable regions form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structures. The hypervariable regions in each chain are tightly held together by the frames and, together with the hypervariable regions of the other chain, participate in the formation of the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, (1991)). Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions, such as enabling antibodies to participate in antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequences of the variable regions vary widely among different antibodies. Sequence variations are concentrated in the CDRs, while less variable portions of the variable regions are called frame regions (FRs). The CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions. In certain embodiments, the variable regions are human variable regions.

[0192] The terms “hypervariant region,” “HVR,” “HV,” “complementarity-determining region,” and “CDR,” as used herein, refer to regions within the antibody variable region that exhibit sequence hypervariability and / or form structurally defined loops. Generally, antibodies contain six hypervariant regions: three in the VH region (H1 or VH CDR1, H2 or VH CDR2, and H3 or VH CDR3) and three in the VL region (L1 or VL CDR1, L2 or VLCDR2, and L3 or VL CDR3). Several hypervariant region descriptions are in use and are covered herein. Kabat CDR is based on sequence variability and is the most commonly used (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia, on the other hand, refers to the location of structural loops (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the ChothiaCDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme inserts at H35A and H35B; if neither 35A nor 35B is present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; if both 35A and 35B are present, the loop terminates at 34). AbM hypervariable regions represent a compromise between the Kabat CDR and the Chothia structural loop and are used in Oxford Molecular's AbM antibody modeling software (see, for example, Martin, Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). “Contact” hypervariable regions are based on analysis of available complex crystal structures. The residues of each of these hypervariable regions, or CDRs, are listed below.

[0193] A universal numbering system has been developed and widely adopted, namely ImMunoGeneTics (IMGT). ® Information systems (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT ®This is a comprehensive information system focusing on immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDRs are referred to based on their amino acid sequence and their position within the light or heavy chain. Because the “position” of CDRs within the variable region structure of immunoglobulins is conserved across species and exists in structures called loops, CDRs and framework residues can be easily identified using a numbering system that compares the variable region sequences of structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into the receptor framework, typically derived from human antibodies. An additional numbering system (AHon) has been developed: Honegger and Plückthun, J. Mol. Biol 309:657-670 (2001). Numbering systems (including, for example, Kabat numbering and IMGT) ® The correspondence between unique numbering systems is well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.), and is also described below. Various systems known in the art or described herein represent different ways of depicting a CDR, and are generally considered equivalent when used to define the same antibody. The exemplary system shown herein combines Kabat and Chothia. The high-variability region may include the following "extended high-variability regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. As used herein, the terms "high-variability region," "HVR," "HV," "complementary determinant region," or "CDR" are used interchangeably.

[0194] As used herein, the term "isolated" is intended to describe a compound of interest in an environment different from that in which the compound naturally occurs. "Isolated" also includes compounds that are substantially enriched within a sample and / or partially or substantially purified from the compound of interest.

[0195] The terms “ROR1-mediated disease,” “ROR1-mediated symptom,” and “ROR1-mediated disorder” are used interchangeably and refer to any disease, symptom, or disorder that is associated with or characterized by cells expressing ROR1, such as tumor cells expressing ROR1. ROR1-mediated diseases include cancers, including but not limited to cancers that express or overexpress ROR1.

[0196] In any implementation thereof, the term “tumor” refers to any proliferative cell growth or proliferation, whether malignant or benign, and refers to all precancerous and cancerous cells and tissues.

[0197] The terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals, which is typically characterized by disordered cell growth.

[0198] The term "ADC" refers to an antibody-drug conjugate, and in the context of this invention, it refers to the conjugation of a ROR1 antibody as described herein with another component comprising a drug.

[0199] As used in this article, "drug" refers to a biologically active compound, such as a cytotoxic compound (e.g., cytotoxic small molecules, cytotoxic synthetic peptides, etc.).

[0200] Examples of pharmaceuticals include small molecule drugs, such as cancer chemotherapeutic agents. For example, in the case where the polypeptide is an antibody (or fragment thereof) specific to tumor cells, the antibody may be modified as described herein to include modified amino acids, which may then be conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptide (e.g., non-protein) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cell inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, phytoalkaloids (catharanthus vinca), and steroid hormones. Peptide compounds may also be used.

[0201] Suitable cancer chemotherapy agents include saliton toxin and its active analogs and derivatives; and aurestatin and its active analogs and derivatives (e.g., monomethylaurestatin D (MMAD), monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), etc.). See, for example, WO 96 / 33212, WO 96 / 14856 and US 6,323,315. For example, saliton toxin 10 or aurestatin PE may be included in the ROR1-ADC of this disclosure. Suitable cancer chemotherapy agents also include maytansines and their active analogs and derivatives (see, for example, EP 1391213; and Liu et al. (1996)). Proc. Natl. Acad. Sci. USA 93:8618-8623); Ducamycin and its active analogs and derivatives (e.g., including synthetic analogs KW-2189 and CB 1-TM1); and benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBD)).

[0202] Agents for reducing cell proliferation are known in the art and are widely used. Such agents include alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazines, including but not limited to dichloromethyldiethylamine, cyclophosphamide (CYNOTAN™), melphalan (L-sarcomain), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozotocin, chloruramycin, uracil nitrogen mustard, chlormethine, ifosfamide, chlorbutazone nitrogen mustard, piperobromidine, triethylene melamine, triethylene thiophosphamide, busulfan, dacarbazine, and temozolomide.

[0203] Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including but not limited to cytarabine (CYTOSAR-U). ® ), cytosine cytarabine, fluorouracil (5-FU), fluorouracil (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostine, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-didezafolate (PDDF, CB3717), 5,8-didezatetrahydrofolate (DDATHF), formyltetrahydrofolate, fludarabine phosphate, pentostatin, and gemcitabine.

[0204] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxin) include, but are not limited to, Ara-C and paclitaxel (TAXOL). ® ), Taxotere ® Deoxycofromycin, mitomycin-C,L-asparaginase, azathioprine; boraquarl; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesin, etc.; podophyllotoxins, such as etoposide, teniposide, etc.; antibiotics, such as anthracyclines, daunorubicin hydrochloride (daunorubicin, erythromycin, doxorubicin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives, etc.; phenoxizone biscyclopeptides, such as actinomycin D; basic glycopeptides, such as bleomycin; anthraquinone glycosides, such as purcamycin; anthraquinones, such as mitoxaquinone; aziridine pyrroloindole diones, such as mitomycin; macrocyclic immunosuppressants, such as cyclosporine, FK-506 (tacrolimus, prazosin), rapamycin, etc.; etc.

[0205] Other anti-cytotoxic agents include navelbene, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, cyclophosphamide, ifosfamide, and droloxafine.

[0206] Microtubule-influencing agents with antiproliferative activity are also suitable for use, including but not limited to allocolchicine (NSC406042), squalene B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC33410), sea haretoxin 10 (NSC 376128), maytansine (NSC 153858), rhizomycin (NSC 332598), and paclitaxel (TAXOL). ® ), TAXOL ® Derivatives, Taxostatin ® Colchicine thiocarbazone (NSC 361792), triphenylmethylcysteine, vincristine sulfate, vinblastine sulfate, natural and synthetic epothilones (including but not limited to epothilone A, epothilone B, and spongioside); estradiol, nocodazole, etc.

[0207] Suitable hormone modulators and steroids (including synthetic analogs) include, but are not limited to, corticosteroids such as prednisone and dexamethasone; estrogens and progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, and tamoxifen; adrenocortical inhibitors such as aminoglutethimide; 17α-ethinyl estradiol; diethylstilbestrol, testosterone, fluoromethesterone, drotaldoline, testosterone lactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorestradiol ether, hydroxyprogesterone, aminoglutethimide, estradiol, medroxyprogesterone acetate, leuprorelin, and flutamide. ® ), Toremifen (FARESTON) ® ) and ZOLADEX ® Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

[0208] Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, etc.; ureas, such as hydroxyurea; hydrazine, such as N-methylhydrazine; epipodophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxanone; folate; tegafur; etc. Other antiproliferative agents of interest include immunosuppressants such as mycophenolate mofetil, thalidomide, deoxyguanidin, azazopyr, leflunomide, imidazolidin, azaspirane (SKF 105685); gefitinib (IRESSA). ® ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazolin; etc.

[0209] Taxanes are suitable for use. "Taxanes" includes paclitaxel, as well as any active taxane derivatives or prodrugs. "Pacific paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as docetaxel, TAXOL) ® TAXOTERE ® (Formulations of docetaxel), 10-deacetylated analogs of paclitaxel and 3'N-debenzoyl-3'N-tert-butoxycarbonyl analogs of paclitaxel) can be readily prepared using techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO93 / 10076; U.S. Patent 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 various commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402, from Taxus chinensis). (Taxus brevifolia) Or T-1912, from Yunnan yew. (Taxus yannanensis) It should be understood that paclitaxel refers not only to the common chemically available form of paclitaxel, but also to its analogues and derivatives (e.g., TAXOTERE). ® Docetaxel (as described herein) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextrose, or paclitaxel-xylose).

[0210] The term "taxane" also includes a variety of known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazine and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfinamide derivatives described in U.S. Patent No. 5,821,263; and paclitaxel derivatives described in U.S. Patent No. 5,415,869. It also includes prodrugs of paclitaxel, including but not limited to those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0211] Suitable bioresponse modifiers include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) angiogenesis inhibitors.

[0212] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the occurrence of symptoms and / or their underlying causes, and / or improve or repair damage caused by or related to a disease, condition, or disorder. In some implementations, the effective amount is a therapeutically effective amount.

[0213] As used herein, the term "therapeuticly effective amount" refers to an amount of antibody or ADC described herein that is sufficient to reduce and / or improve the severity and / or duration of a given disease, condition, or disorder and / or associated symptoms. A therapeutically effective amount of an agent, including therapeutic agents, may be an amount necessary to: (i) reduce or improve the development or progression of a given disease, condition, or disorder; (ii) reduce or improve the recurrence, development, or onset of a given disease, condition, or disorder; and / or (iii) improve or enhance the therapeutic effect of another therapy (e.g., a therapy other than the administration of the antibody or ADC described herein). The "therapeuticly effective amount" of the substances / molecules / agents disclosed herein (e.g., ROR1 antibodies or ADCs) may vary based on a variety of factors, such as an individual's disease state, age, sex, and weight, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutically effective amount encompasses any toxic or adverse effects of the substance / molecule / agent that are less than the amount of the therapeutically beneficial effect. In some implementations, the term "therapeutic effective amount" refers to the amount of antibody or other agent (e.g., or drug) that effectively "treats" a disease, condition, or ailment of a subject or mammal.

[0214] In some embodiments, the drug is a microtubule-influencing agent with antiproliferative activity, such as maytansine. In some embodiments, the drug is an antimitotic agent, such as oligristatin or an active oligristatin analog or derivative thereof. In some embodiments, the drug is a DNA alkylating agent.

[0215] As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeias for use in animals and, more particularly, for use in humans.

[0216] "Excipients" include carriers, excipients, preservatives, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used, and may be included, for example, to affect stability, cause swelling of the formulation, or impart therapeutic enhancement to the active ingredient in the final dosage form (e.g., to promote absorption, reduce viscosity, or enhance solubility). "Excipients" can be natural or synthetic organic or inorganic components that are combined with the active ingredient to facilitate the use of the active ingredient, for example, by administering the active ingredient to a subject. Examples of excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) peptides; 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 dextrin; 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 catalyst administered with a therapeutic agent. Such excipients can be sterile liquids such as water and oils such as petroleum, animal, plant, or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition is administered intravenously (e.g., a pharmaceutical composition), water is an exemplary excipient. Saline solutions, dextran solutions, and glycerol solutions can also be used as liquid excipients, especially for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. In any embodiment, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. The composition can be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc. Oral compositions (such as formulations) may include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable excipients are described in Remington: The Science and Practice of Pharmacy (2020) (Elsevier Science, Amsterdam, Netherlands), including pharmaceutical compounds that may contain an effective or therapeutically effective amount of ROR1-ADC (e.g., in isolated or purified form), together with an appropriate amount of excipient to provide a form suitable for administration to a subject. The formulation should be adapted to the mode of administration.

[0217] The terms “about” and “approximately” refer to a variation of 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of a given value or range.

[0218] As used herein, comparative terms (such as decrease, reduction, increase, or any grammatical variation thereof) may refer to a change relative to a reference value. In some embodiments, such a change may refer to a value that is 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, or about 2, or about 3, or about 4, or about 5, or about 10, or about 20, or about 30, or about 40, or about 100 times or more. In some implementations, such changes may refer to approximately 1%, or approximately 2%, or approximately 3%, or approximately 4%, or approximately 5%, or approximately 6%, or approximately 7%, or approximately 8%, or approximately 9%, or approximately 10%, or approximately 20%, or approximately 30%, or approximately 40%, or approximately 50%, or approximately 60%, or approximately 70%, or approximately 80%, or approximately 90%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99% of the reference value.

[0219] Unless the context clearly specifies otherwise, as used in this disclosure and claims, the singular forms “a / an” and “the” include the plural forms.

[0220] In some implementations, the terms "first," "second," "third," "fourth," etc., in the component names are used to distinguish and identify more than one component that shares a certain identity in its name. For example, "first antibody" and "second antibody" are used to distinguish two antibodies.

[0221] It should be understood that in any instance where the implementation is described herein using the term "comprising," other similar implementations described as "consisting of" and / or "substantially consisting of" are also provided.

[0222] As used in phrases such as "between A and B" or "between A and B," the term "between" refers to a range that includes both A and B.

[0223] As used in this document in phrases such as “A and / or B”, the term “and / or” is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as “A, B, and / or C”, the term “and / or” is intended to cover 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).

[0224] The terms “optional” or “optionally” mean that the situation described below may or may not occur, such that the description includes both the scenario in which the situation occurs and the scenario in which the situation does not occur.

[0225] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, and therefore variations are naturally possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0226] When providing ranges of values, it should be understood that every intermediate value between the upper and lower limits of that range (unless otherwise clearly stated herein, such intermediate value is one-tenth of the lower limit unit) and any other stated value or intermediate value within that stated range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also included within the scope of this invention, limited by any explicitly excluded limit values ​​within the stated range. When a stated range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included within the scope of this invention.

[0227] It should be understood that certain features of the invention described in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to the invention are specifically covered by the invention and are disclosed herein as if each combination were individually and explicitly disclosed, provided that the subject matter covered by such combinations is, for example, compounds as stable compounds (e.g., compounds that can be prepared, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of various embodiments and their elements (e.g., elements of chemical groups listed in embodiments describing such variables) are also specifically covered by the invention and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0228] 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 pertains. While any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are described hereafter. All publications referenced herein are incorporated by way of citation to disclose and describe methods and / or materials associated with the cited publications.

[0229] It should be understood that certain features of the invention described in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually or in any suitable sub-combination.

[0230] The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to a prior art prior to such publications. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0231] 7.2 ROR1-ADC An antibody that binds to tyrosine protein kinase receptor 1 (ROR1) (also referred to herein as a “ROR1 antibody,” “anti-ROR1 antibody,” “ROR1 Ab,” “Ab,” or “antibody”) and a drug can be directly or indirectly linked together via a pyridazine-pyrrole conjugate to form a ROR1-ADC as described herein. In some embodiments, the ROR1 antibody and two or more drugs or active agents are linked together via one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds may include a branched linker as described herein.

[0232] The part of interest (e.g., a drug or active agent) can be conjugated to the ROR1 antibody at any desired site. Thus, for example, this disclosure provides a ROR1 antibody having portions conjugated at two or more sites on the antibody, such as sites on or near the C-terminus of the antibody, sites on or near the N-terminus of the antibody, and sites between the C-terminus and N-terminus of the antibody (e.g., at internal sites on the antibody). Combinations of the above conjugation sites are also possible.

[0233] In some embodiments, the conjugates of this disclosure comprise two (or more) pharmaceutical or active agents conjugated to amino acid residues of a ROR1 antibody at the α-carbon of an amino acid residue. In other words, the conjugate comprises a ROR1 antibody wherein the side chains of the amino acid residues in the antibody have been modified and linked to two (or more) pharmaceutical or active agents (e.g., linked to the two pharmaceutical or active agents via branched linkers as described herein). For example, the conjugate comprises a ROR1 antibody wherein the α-carbon of the amino acid residues in the antibody has been modified and linked to two pharmaceutical or active agents (e.g., linked to the two pharmaceutical or active agents via branched linkers as described herein).

[0234] Embodiments of this disclosure include conjugates in which a ROR1 antibody is conjugated to two or more moieties, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more moieties. The moieties may be conjugated to the antibody at multiple sites in the ROR1 antibody. In some embodiments, two moieties may be conjugated to a single amino acid residue of the ROR1 antibody. For example, 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, the ROR1 antibody may be conjugated to the first and second moieties at the first amino acid residue, and to the third and fourth moieties at the 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 portions (e.g., two portions), with each pair of portions conjugated to the ROR1 antibody via a branched linker as described herein. In some cases, one amino acid residue in the ROR1 antibody is conjugated to a pair of portions via a branched linker as described herein. In other cases, two or more amino acid residues (e.g., 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 portions via a branched linker as described herein.

[0235] One or more amino acid residues in the ROR1 antibody conjugated to the moiety of interest can be naturally occurring amino acids, non-natural amino acids, or combinations thereof. For example, the conjugate may include the moiety of interest (e.g., a drug or active agent) conjugated to naturally occurring amino acid residues of the ROR1 antibody. In other cases, the conjugate may include the moiety of interest conjugated to non-natural amino acid residues of the ROR1 antibody. The moiety of interest may be conjugated to the ROR1 antibody at a single natural or non-natural amino acid residue as described above. One or more natural or non-natural amino acid residues in the ROR1 antibody may be conjugated to the moiety of interest as described herein. For example, two (or more) amino acid residues (e.g., natural or non-natural amino acid residues) in the ROR1 antibody may each be conjugated to two moieties via branched linkers, such that multiple sites in the ROR1 antibody are conjugated to the moiety of interest.

[0236] As described herein, ROR1 antibodies can be conjugated to two or more parts of interest. In some embodiments, the part of interest is a payload, such as a chemical entity, like a drug, an active agent, or a detectable label. For example, a drug (or an active agent, such as a cytokine) can be conjugated to a ROR1 antibody, or in other embodiments, a detectable label can be conjugated to a ROR1 antibody. In other embodiments, combinations of different payloads can be conjugated to a ROR1 antibody. Thus, embodiments of this disclosure include, for example, including but not limited to: conjugates of ROR1 antibodies with two or more drugs; conjugates of ROR1 antibodies with two or more active agents (e.g., cytokines); conjugates of ROR1 antibodies with two or more detectable labels; and combinations thereof.

[0237] In some embodiments, the ROR1 antibody and the moiety of interest (e.g., a drug or active agent) are conjugated via a conjugation motif. For example, the ROR1 antibody and the moiety of interest may each be bound to the conjugation motif (e.g., covalently), thereby indirectly conjugating the ROR1 antibody and the moiety of interest via the conjugation motif. In some cases, the conjugation motif comprises a hydrazyl-indolyl compound or a hydrazyl-pyrrolo-pyridyl compound, or a derivative thereof. For example, the following general reaction scheme illustrates a general scheme for coupling the moiety of interest to the ROR1 antibody via a hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugation motif. The hydrazyl-indolyl and hydrazyl-pyrrolo-pyridyl conjugation motifs are also referred to herein as hydrazyl- different Pictet-Spengler (HIPS) conjugate and aza-hydrazine- different Pictet-Spengler (azaHIPS) suture. In the above reaction scheme, each R independently includes a portion of interest (e.g., a drug or active agent) conjugated to a ROR1 antibody (e.g., conjugated to a ROR1 antibody via a linker as described herein), where n is an integer from 1 to 4. As shown in the above reaction scheme, the conjugated portion (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugated portion) is linked to two or more drugs or active agents R. The ROR1 antibody including a 2-formylglycine residue (fGly) reacts with the conjugated portion to generate a ROR1 antibody conjugate, thereby linking the two or more drugs or active agents to the ROR1 antibody via the conjugated portion.

[0238] As described herein, the portion can be any of a variety of portions, such as, but not limited to, chemical entities, such as detectable labels or drugs or active agents. R' and R" can each independently be any desired substituent, such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. Z can be CR 21 NR 22 , N, O or S, where R 21 and R 22 Each is independently selected from any of the substituents described above for R' and R”.

[0239] Other hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugates are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate can be linked (e.g., covalently linked) to two or more connectors. Therefore, embodiments of this disclosure include hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugates, each of which is connected to two or more pharmaceutical or active agents via a corresponding connector. Thus, the conjugates of this disclosure may include two or more connectors, each connector linking a corresponding pharmaceutical or active agent to the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate. Therefore, the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate and the two or more connectors can be collectively considered as a “branched link head,” wherein the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate is connected to two or more “branched chains,” each of which includes a connector connected to a pharmaceutical or active agent.

[0240] Combinations of identical or different payloads can be conjugated to ROR1 antibodies via branched linkers. In some embodiments, the two payloads (e.g., drugs, active agents, or detectable markers) linked to the branched linker are identical payloads (e.g., drugs, active agents, or detectable markers). For example, a first branch of the branched linker can be linked to a payload (e.g., a drug, active agent, or detectable marker), and a second branch of the branched linker can be linked to the same payload (e.g., a drug, active agent, or detectable marker) as the first branch.

[0241] In other embodiments, the two payloads (e.g., a drug, an active agent, or a detectable marker) connected to the branch link head are different payloads (e.g., a drug, an active agent, or a detectable marker). For example, a first branch chain of the branch link head may be connected to a first payload (e.g., a first drug, an active agent, or a detectable marker), and a second branch chain of the branch link head may be connected to a second payload (e.g., a second drug, an active agent, or a detectable marker) that is different from the first payload (e.g., a first drug, an active agent, or a detectable marker) connected to the first branch chain.

[0242] In some implementations, when two different drugs or active agents are attached to the branch linker, the drugs or active agents may be selected from those with synergistic therapeutic effects. "Synergistic" or "synergism / synergy" means that the therapeutic effect is greater than the sum of the effects of the drugs or active agents used alone. For example, in some cases, using two different drugs or active agents attached to the branch linker can provide a lower therapeutically effective concentration, allowing both payloads to function, thereby increasing the overall potency of the ADC.

[0243] In some embodiments, when two different drugs or active agents are attached to the branch linker, the drugs or active agents may be selected from those that provide enhanced therapeutic benefits compared to using the drugs or active agents alone. For example, the drugs or active agents may increase the drug delivery effect of the ADC (e.g., some payloads, such as iRGD peptides, may increase extravasation into tissues and enhance tumor penetration).

[0244] In some implementations, when two different drugs or active agents are attached to the branch linker, the drugs or active agents may be selected from drugs and active agents using different mechanisms of action. In some cases, this can reduce tumor resistance by targeting multiple pathways. Examples of payload combinations include, but are not limited to, cytotoxic drugs, immunomodulatory molecules for activating or inhibiting immune cell populations, cytokines, hormones, chelating agents loaded with radioisotopes, etc.

[0245] In some implementations, when two different payloads are connected to the branch connector, the payloads can be selected from a combination of a drug or active agent and a detectable marker. For example, the first payload can be a detectable marker used as an imaging agent or tracer to detect the location of the ADC in vivo, while the second payload can be a drug or active agent that provides therapeutic activity.

[0246] This document describes in detail several embodiments of a connector that can couple a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate to a drug or active agent. For example, in some cases, the connector is a cleavable connector, such as the cleavable connector described herein.

[0247] In some embodiments, the ROR1 antibody may be conjugated to two or more moieties of interest, wherein one or more amino acids of the ROR1 antibody are modified prior to conjugation to the moieties of interest. Modification of one or more amino acids of the ROR1 antibody can produce a ROR1 antibody containing 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 linked to the conjugation moieties, such as the hydrazino-indolyl or hydrazino-pyrrolo-pyridyl conjugation moieties described above). For example, the amino acids of the ROR1 antibody may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). The reactive aldehyde may be included in an “aldehyde tag” or “ald tag,” as used herein, referring 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 the action of a formylglycine synthase (FGE) to contain a 2-formylglycine residue (referred to herein as “fGly”). The fGly residue generated by FGE can also be referred to as "formylglycine". In other words, 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 cysteine ​​or serine residues have been converted to fGly by the action of FGE, such as L(fGly)TPSR, SEQ ID NO:123). A converted sulfatase motif can be generated from an amino acid sequence that includes an "unconverted" sulfatase motif (e.g., a sulfatase motif in which cysteine ​​or serine residues have not yet been converted to fGly by FGE but are capable of conversion, such as an unconverted sulfatase motif having the following sequence: LCTPSR, SEQ ID NO:100). As used in the context of the action of formylglycine synthase (FGE) on sulfatase motifs, "conversion" refers to the biochemical modification of cysteine ​​or serine residues in a sulfatase motif to a formylglycine (fGly) residue (e.g., Cys to fGly or Ser to fGly). U.S. Patent Nos. 7,985,783 and 8,729,232 describe additional aspects of aldehyde tags and their use in site-specific protein modifications, the disclosures of each of which are incorporated herein by reference.

[0248] In some cases, to generate conjugates, ROR1 antibodies containing fGly residues can be conjugated to the moiety of interest by reacting fGly with a compound (e.g., a compound containing a hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate moiety as described above). For example, a ROR1 antibody containing fGly can be contacted with a reaction conjugate under conditions suitable for conjugating two or more drugs to a ROR1 antibody. In some cases, the reaction conjugate may include a hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate moiety as described above. For example, two or more drugs or active agents may be linked to a hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate moiety. In some cases, the drug or active agent is linked to the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate moiety, for example, covalently linked to a hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl group, wherein each drug or active agent is linked to the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate moiety via a corresponding linker. Therefore, the fGly residues that are conjugated with the part of interest after the reaction are referred to as fGly' in this paper.

[0249] In some embodiments, the conjugates of this disclosure comprise ROR1 antibodies having at least one amino acid residue linked to two or more moieties of interest (e.g., pharmaceuticals or active agents). To prepare the conjugates, the amino acid residues of the ROR1 antibody may be modified and then conjugated with two or more pharmaceuticals or active agents linked to the hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate moieties as described above. In some embodiments, the amino acid residues of the ROR1 antibody are cysteine ​​or serine residues modified as fGly residues as described above. In some embodiments, the modified amino acid residues (e.g., fGly residues) are conjugated with two or more pharmaceuticals or active agents containing the hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate moieties as described above to provide the conjugates of this disclosure, wherein the two or more pharmaceuticals or active agents are conjugated to the ROR1 antibody via the hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate moieties. As used herein, the term fGly' refers to the amino acid residue in a ROR1 antibody that is conjugated to the part of interest (e.g., a drug or active agent).

[0250] In some embodiments, the conjugate comprises a ROR1 antibody having at least one amino acid residue linked to a branched linker as described herein, which in turn is linked to two or more drugs or active agents. For example, the conjugate may comprise a ROR1 antibody having at least one amino acid residue (fGly') conjugated to a moiety of interest (e.g., a drug or active agent) as described above.

[0251] Several aspects of this disclosure include a conjugate of formula (I): in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 Z 3 and Z 4 Each independently selected from CR 4 N and CL B -W 2 At least one of Z 1 Z 2 Z 3 and Z 4 It is CL B -W 2 ; R 1 Selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic; R 2 and R 3 Each is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, or R 2 and R 3 Optionally linked in a ring to form a 5- or 6-membered heterocyclic group; Each R 4 Independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic; L A It is the first connector; L B It is the second connector; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

[0252] The substituents associated with the conjugate of formula (I) are described in more detail below.

[0253] In some implementations, Z 1 Z 2 Z 3 and Z 4 Each independently selected from CR 4 N and CL B -W 2 At least one of Z 1 Z 2 Z 3 and Z 4 It is CL B -W 2 In some implementations, Z 1 It is CR 4 In some implementations, Z 1 It is N. In some implementations, Z 1 It is CL B -W 2 In some implementations, Z 2 It is CR 4 In some implementations, Z 2 It is N. In some implementations, Z 2 It is CL B -W 2 In some implementations, Z 3 It is CR 4 In some implementations, Z 3 It is N. In some implementations, Z 3 It is CL B -W 2 In some implementations, Z 4 It is CR 4 In some implementations, Z 4 It is N. In some implementations, Z 4 It is CL B -W 2 In some implementation schemes, Z 1 Z 3 and Z 4 Each of them is CR 4 In some implementation schemes, Z 3 It is CL B -W 2 .

[0254] Multiple Z 1 Z 2 Z 3 and Z 4Combinations of Z are possible. For example, in some cases, Z 1 It is CL B -W 2 Z 2 It is CR 4 Z 3 It is CR 4 And Z 4 It is CR 4 In some cases, Z 1 It is CR 4 Z 2 It is CL B -W 2 Z 3 It is CR 4 And Z 4 It is CR 4 In some cases, Z 1 It is CR 4 Z 2 It is CR 4 Z 3 It is CL B -W 2 And Z 4 It is CR 4 In some cases, Z 1 It is CR 4 Z 2 It is CR 4 Z 3 It is CR 4 And Z 4 It is CL B -W 2 .

[0255] In some implementations, R 1 The group is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, R 1 It is hydrogen. In some implementations, R 1 It is an alkyl or substituted alkyl group, such as C 1-6 Alkyl or C 1-6 Substituted alkyl groups, or C 1-4 Alkyl or C 1-4 Substituted alkyl, or C 1-3 Alkyl or C 1-3 Substituted alkyl groups. In some embodiments, R 1 It is an alkenyl or substituted alkenyl group, such as C 2-6 alkenyl or C 2-6 Substituted alkenyl, or C 2-4 alkenyl or C2-4 Substituted alkenyl, or C 2-3 alkenyl or C 2-3 Substituted alkenyl groups. In some embodiments, R 1 It is an alkynyl or substituted alkynyl group, 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 groups. In some embodiments, R 1 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups. In some embodiments, R 1 It is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R 1 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 Substituted cycloalkyl groups. In some embodiments, R 1 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic group or C 3-8 Substituted heterocyclic groups, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0256] In some implementations, R 2 and R 3 Each is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, or R 2 and R 3 Optionally, they are linked in a ring to form 5- or 6-membered heterocyclic groups.

[0257] In some implementations, R 2 The group is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, R 2 It is hydrogen. In some implementations, R 2 It is an alkyl or substituted alkyl group, 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 groups. In some embodiments, R 2 It is methyl. In some embodiments, R 2 It is an alkenyl or substituted alkenyl group, 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 groups. In some embodiments, R 2 It is an alkynyl or substituted alkynyl group. In some embodiments, R 2 It is an alkoxy or a substituted alkoxy group. In some embodiments, R 2 It is an amino or substituted amino group. In some embodiments, R 2 It is a carboxyl group or a carboxyl ester. In some embodiments, R 2 It is an acyl or acyloxy group. In some embodiments, R 2 It is an acylamino or aminoacyl group. In some embodiments, R 2 It is an alkylamide or a substituted alkylamide. In some embodiments, R 2 It is a sulfonyl group. In some embodiments, R 2 It is a thioalkoxy or a substituted thioalkoxy. In some embodiments, R 2 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups. In some embodiments, R 2 It is a heteroaryl or substituted heteroaryl, such as C 5-8heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R 2 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 Substituted cycloalkyl groups. In some embodiments, R 2 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0258] In some implementations, R 3 The group is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, R 3 It is hydrogen. In some implementations, R 3 It is an alkyl or substituted alkyl group, 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 groups. In some embodiments, R 3 It is methyl. In some embodiments, R 3 It is an alkenyl or substituted alkenyl group, 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 groups. In some embodiments, R 3 It is an alkynyl or substituted alkynyl group. In some embodiments, R 3 It is an alkoxy or a substituted alkoxy group. In some embodiments, R 3 It is an amino or substituted amino group. In some embodiments, R 3It is a carboxyl group or a carboxyl ester. In some embodiments, R 3 It is an acyl or acyloxy group. In some embodiments, R 3 It is an acylamino or aminoacyl group. In some embodiments, R 3 It is an alkylamide or a substituted alkylamide. In some embodiments, R 3 It is a sulfonyl group. In some embodiments, R 3 It is a thioalkoxy or a substituted thioalkoxy. In some embodiments, R 3 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups. In some embodiments, R 3 It is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R 3 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 Substituted cycloalkyl groups. In some embodiments, R 3 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic group or C 3-8 Substituted heterocyclic groups, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0259] In some implementations, R 2 and R 3 Both are methyl groups.

[0260] In some implementations, R 2 and R 3 Optionally, they are linked in a ring to form a 5- or 6-membered heterocyclic group. In some embodiments, R 2 and R 3 They are linked in a ring to form 5- or 6-membered heterocyclic groups. In some embodiments, R 2 and R 3 The rings are linked to form a 5-membered heterocyclic group. In some embodiments, R 2 and R 3They are linked in a ring to form a 6-membered heterocyclic group.

[0261] In some implementations, each R 4 Independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic.

[0262] The following describes each R in more detail. 4 Various possibilities. In some implementations, R 4 It is hydrogen. In some implementations, each R 4 It is hydrogen. In some implementations, R 4 It is a halogen, such as F, Cl, Br, or I. In some embodiments, R 4 It is F. In some implementations, R 4 It is Cl. In some implementations, R 4 It is Br. In some implementations, R 4 It is I. In some implementations, R 4 It is an alkyl or substituted alkyl group, 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 groups. In some embodiments, R 4 It is methyl. In some embodiments, R 4 It is an alkenyl or substituted alkenyl group, 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 groups. In some embodiments, R 4 It is an alkynyl or substituted alkynyl group. In some embodiments, R 4 It is an alkoxy or a substituted alkoxy group. In some embodiments, R 4 It is an amino or substituted amino group. In some embodiments, R 4 It is a carboxyl group or a carboxyl ester. In some embodiments, R 4 It is an acyl or acyloxy group. In some embodiments, R 4It is an acylamino or aminoacyl group. In some embodiments, R 4 It is an alkylamide or a substituted alkylamide. In some embodiments, R 4 It is a sulfonyl group. In some embodiments, R 4 It is a thioalkoxy or a substituted thioalkoxy. In some embodiments, R 4 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups (e.g., phenyl or substituted phenyl). In some embodiments, R 4 It is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R 4 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 Substituted cycloalkyl groups. In some embodiments, R 4 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic group or C 3-8 Substituted heterocyclic groups, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0263] In some implementations, L A This is the first joint. Examples of joints that can be used in the conjugates of this disclosure are described in more detail below.

[0264] In some implementations, L B This is the second connector. Examples of connectors that can be used in the conjugates of this disclosure are described in more detail below.

[0265] In some implementations, W 1 It is the first drug (or the first active agent). Examples of drugs and active agents that can be used in the conjugates of this disclosure are described in more detail below.

[0266] In some implementations, W 2It is a second drug (or a second active agent). Examples of drugs and active agents that can be used in the conjugates of this disclosure are described in more detail below.

[0267] In some embodiments, Ab represents an antibody that binds to ROR1 (“ROR1 antibody”). In some embodiments, the antibody Ab contains one or more fGly' residues as described herein. In some embodiments, the ROR1 antibody is linked to the remainder of the conjugate via the fGly' residues as described herein. Examples of ROR1 antibodies that can be used in the conjugates of this disclosure are described in more detail below.

[0268] In some embodiments, the conjugate of formula (I) includes a first connector L. A First connector L A This can be used to bind the first part of interest (e.g., a first drug or active agent) to a ROR1 antibody via a conjugation site. First linker L A It can be coupled (e.g., covalently bonded) to a joining portion (e.g., as described herein). For example, the first connector L A The hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate can be attached to the first drug. The hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate can be used to attach the first connector L. A (And therefore the first drug) was conjugated with the ROR1 antibody.

[0269] For example, as shown in equation (I) above, L A The antibody Ab is linked to the conjugated portion via a hydrazino-indole or hydrazino-pyrrolo-pyridyl conjugated portion, and thus the antibody Ab is indirectly linked to the linker L via a hydrazino-indole or hydrazino-pyrrolo-pyridyl conjugated portion. A Bonding. As mentioned above, antibody Ab is a ROR1 antibody, and therefore L A Linked to the ROR1 antibody via a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate, for example, the linker L A Indirectly bound to ROR1 antibody via hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugates.

[0270] Any convenient connector can be used as the first connector in the subject conjugate and compound. A In some implementations, the first connector L A It may include groups selected from the following: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, the first connector L AIt may include alkyl or substituted alkyl groups. In some embodiments, the first connector L A It may include alkenyl or substituted alkenyl groups. In some embodiments, the first connector L A It may include an alkynyl group or a substituted alkynyl group. In some embodiments, the first connector L A It may include alkoxy or substituted alkoxy groups. In some embodiments, the first connector L A It may include amino or substituted amino groups. In some embodiments, the first connector L A It may include a carboxyl group or a carboxyl ester group. In some embodiments, the first connector L A It may include an acylamino group. In some embodiments, the first connector L A It may include alkylamides or substituted alkylamide groups. In some embodiments, the first connector L A It may include aryl or substituted aryl groups. In some embodiments, the first connector L A It may include heteroaryl or substituted heteroaryl groups. In some embodiments, the first connector L A It may include cycloalkyl or substituted cycloalkyl groups. In some embodiments, the first connector L A It may include heterocyclic groups or substituted heterocyclic groups.

[0271] In some implementations, the first connector L A The polymer may be included. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxy polyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., wherein the homopolymers and copolymers are unsubstituted or alkyl-substituted at one end), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, etc. In some embodiments, the polymer is a polyalkylene glycol. In some embodiments, the polymer is polyethylene glycol. Other connectors are also possible, as shown in the conjugates and compounds described in more detail below.

[0272] In some implementations, L A The first connector is described by the following formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, Where L 1L 2 L 3 L 4 L 5 and L 6 Each of them is an independent connector subunit, and a, b, c, d, e, and f are each independently 0 or 1, with the constraint that at least one of a, b, c, d, e, and f is 1.

[0273] In some embodiments, the sum of a, b, c, d, e, and f is 1 to 6. In some embodiments, the sum of a, b, c, d, e, and f is 1. In some embodiments, the sum of a, b, c, d, e, and f is 2. In some embodiments, the sum of a, b, c, d, e, and f is 3. In some embodiments, the sum of a, b, c, d, e, and f is 4. In some embodiments, the sum of a, b, c, d, e, and f is 5. In some embodiments, the sum of a, b, c, d, e, and f is 6. In some embodiments, a, b, c, d, e, and f are each 1. In some embodiments, a, b, c, d, and e are each 1 and f is 0. In some embodiments, a, b, c, and d are each 1 and e and f are each 0. In some embodiments, a, b, c are each 1 and d, e, and f are each 0. In some implementations, a and b are each 1 and c, d, e, and f are each 0.

[0274] In some implementations, the connector subunit L 1 Connected to a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate (e.g., as shown in formula (I) above). In some embodiments, the connector subunit L 2 (If present) Connected to the first drug or active agent W 1 In some implementations, the connector subunit L 3 (If present) Connected to the first drug or active agent W 1 In some implementations, the connector subunit L 4 (If present) Connected to the first drug or active agent W 1 In some implementations, the connector subunit L 5 (If present) Connected to the first drug or active agent W 1 In some implementations, the connector subunit L 6 (If present) Connected to the first drug or active agent W 1 .

[0275] Any convenient connector subunit can be used for the first connector L AThe linker subunits of interest include, but are not limited to, polymer units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues and their derivatives, polynucleotides, alkyl, aryl, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, L 1 L 2 L 3 L 4 L 5 and L 6 Each of (if present) contains one or more groups independently selected from: polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl, substituted alkyl, aryl, substituted aryl and diamine (e.g., including the linking group of alkylene diamine).

[0276] In some implementations, L 1 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 1 Contains polyethylene glycol. In some embodiments, L 1 Contains modified polyethylene glycol. In some embodiments, L 1 It contains amino acid residues. In some embodiments, L 1 Contains alkyl or substituted alkyl groups. In some embodiments, L 1 Contains an aryl or substituted aryl group. In some embodiments, L 1 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0277] In some implementations, L 2 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 2 Contains polyethylene glycol. In some embodiments, L 2 Contains modified polyethylene glycol. In some embodiments, L 2 It contains amino acid residues. In some embodiments, L 2 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 2 Contains an aryl or substituted aryl group. In some embodiments, L 2 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0278] In some implementations, L 3(If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 3 Contains polyethylene glycol. In some embodiments, L 3 Contains modified polyethylene glycol. In some embodiments, L 3 It contains amino acid residues. In some embodiments, L 3 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 3 Contains an aryl or substituted aryl group. In some embodiments, L 3 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0279] In some implementations, L 4 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 4 Contains polyethylene glycol. In some embodiments, L 4 Contains modified polyethylene glycol. In some embodiments, L 4 It contains amino acid residues. In some embodiments, L 4 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 4 Contains an aryl or substituted aryl group. In some embodiments, L 4 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0280] In some implementations, L 5 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 5 Contains polyethylene glycol. In some embodiments, L 5 Contains modified polyethylene glycol. In some embodiments, L 5 It contains amino acid residues. In some embodiments, L 5 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 5 Contains an aryl or substituted aryl group. In some embodiments, L 5 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0281] In some implementations, L 6 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 6Contains polyethylene glycol. In some embodiments, L 6 Contains modified polyethylene glycol. In some embodiments, L 6 It contains amino acid residues. In some embodiments, L 6 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 6 Contains an aryl or substituted aryl group. In some embodiments, L 6 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0282] In some implementations, L A It is the first connector, which includes: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, in: -(L 1 ) a -is-(T) 1 -V 1 ) a -; -(L 2 ) b -is-(T) 2 -V 2 ) b -; -(L 3 ) c -is-(T) 3 -V 3 ) c -; -(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 -, in: T 1 T 2 T 3 T 4 T 5 and T 6 (If present) is a chain group; V 1 V 2 V 3 V 4 V 5 and V 6 (If present) is a covalent bond or a linking functional group; and a, b, c, d, e, and f are each independently 0 or 1, with the constraint that at least one of a, b, c, d, e, and f is 1.

[0283] In some embodiments, the sum of a, b, c, d, e, and f is 1 to 6. In some embodiments, the sum of a, b, c, d, e, and f is 1. In some embodiments, the sum of a, b, c, d, e, and f is 2. In some embodiments, the sum of a, b, c, d, e, and f is 3. In some embodiments, the sum of a, b, c, d, e, and f is 4. In some embodiments, the sum of a, b, c, d, e, and f is 5. In some embodiments, the sum of a, b, c, d, e, and f is 6. In some embodiments, a, b, c, d, e, and f are each 1. In some embodiments, a, b, c, d, and e are each 1 and f is 0. In some embodiments, a, b, c, and d are each 1 and e and f are each 0. In some embodiments, a, b, c are each 1 and d, e, and f are each 0. In some implementations, a and b are each 1 and c, d, e, and f are each 0.

[0284] As described above, in some implementations, L 1 It is attached to a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate (e.g., as shown in formula (I) above). Therefore, in some embodiments, T 1 Connected to a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate (e.g., as shown in formula (I) above). In some embodiments, V 1 Connected to a first drug or active agent. In some embodiments, L 2 (If present) linked to a first drug or active agent. Therefore, in some embodiments, T 2 (If present) linked to the first drug or active agent, or V 2(If present) linked to a first drug or active agent. In some embodiments, L 3 (If present) linked to a first drug or active agent. Therefore, in some embodiments, T 3 (If present) linked to the first drug or active agent, or V 3 (If present) linked to a first drug or active agent. In some embodiments, L 4 (If present) linked to a first drug or active agent. Therefore, in some embodiments, T 4 (If present) linked to the first drug or active agent, or V 4 (If present) linked to a first drug or active agent. In some embodiments, L 5 (If present) linked to a first drug or active agent. Therefore, in some embodiments, T 5 (If present) linked to the first drug or active agent, or V 5 (If present) linked to a first drug or active agent. In some embodiments, L 6 (If present) linked to a first drug or active agent. Therefore, in some embodiments, T 6 (If present) linked to the first drug or active agent, or V 6 (If present) Connected to the first drug or active agent.

[0285] In some embodiments, the conjugate of formula (I) includes a second connector L. B Second connector L B This can be used to bind a second part of interest (e.g., a second drug or active agent) to a ROR1 antibody via a conjugation site. Second linker L B It can be combined (e.g., covalently bonded) with a joining portion (e.g., as described herein). For example, the second connector L B The hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate can be attached to a second drug. The hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate can be used to attach a second connector L. B (And therefore the second drug) is conjugated with the ROR1 antibody.

[0286] For example, as shown in equation (I) above, L B The antibody Ab is linked to the conjugation site via a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugation site, and thus the antibody Ab is indirectly linked to the second linker L via a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugation site. B Bonding. As mentioned above, antibody Ab is a ROR1 antibody, and therefore L B Linked to the ROR1 antibody via a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate, for example, the linker L BIndirectly bound to ROR1 antibody via hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugates.

[0287] Any convenient connector can be used as a second connector L in the subject conjugate and compound. B In some implementations, the second connector L B It may include groups selected from the following: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acylamino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, the second connector L B It may include alkyl or substituted alkyl groups. In some embodiments, the second connector L B It may include alkenyl or substituted alkenyl groups. In some embodiments, the second connector L B It may include an alkynyl group or a substituted alkynyl group. In some embodiments, the second connector L B It may include alkoxy or substituted alkoxy groups. In some embodiments, the second connector L B It may include amino or substituted amino groups. In some embodiments, the second connector L B It may include carboxyl groups or carboxyl ester groups. In some embodiments, the second connector L B It may include an acylamino group. In some embodiments, the second connector L B It may include alkylamides or substituted alkylamide groups. In some embodiments, the second connector L B It may include aryl or substituted aryl groups. In some embodiments, the second connector L B It may include heteroaryl or substituted heteroaryl groups. In some embodiments, the second connector L B It may include cycloalkyl or substituted cycloalkyl groups. In some embodiments, the second connector L B It may include heterocyclic groups or substituted heterocyclic groups.

[0288] In some implementations, the second connector L B The polymer may be included. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxy polyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol and propylene glycol (e.g., wherein the homopolymers and copolymers are unsubstituted or alkyl-substituted at one end), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, combinations thereof, etc. In some embodiments, the polymer is a polyalkylene glycol. In some embodiments, the polymer is polyethylene glycol. Other connectors are also possible, as shown in the conjugates and compounds described in more detail below.

[0289] In some implementations, L B The second connector is described by the following formula: -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m , Where L 7 L 8 L 9 L 10 L 11 L 12 and L 13 Each is an independent connector subunit, and g, h, i, j, k, l, and m are each independently 0 or 1, with the constraint that at least one of g, h, i, j, k, l, and m is 1.

[0290] In some embodiments, the sum of g, h, i, j, k, l, and m is 1 to 7. In some embodiments, the sum of g, h, i, j, k, l, and m is 1. In some embodiments, the sum of g, h, i, j, k, l, and m is 2. In some embodiments, the sum of g, h, i, j, k, l, and m is 3. In some embodiments, the sum of g, h, i, j, k, l, and m is 4. In some embodiments, the sum of g, h, i, j, k, l, and m is 5. In some embodiments, the sum of g, h, i, j, k, l, and m is 6. In some embodiments, the sum of g, h, i, j, k, l, and m is 7. In some embodiments, g, h, i, j, k, l, and m are each 1. In some embodiments, g, h, i, j, k, l, and m are each 1, and m is 0. In some implementations, g, h, i, j, and k are each 1, and l and m are each 0. In some implementations, g, h, i, and j are each 1, and k, l, and m are each 0. In some implementations, g, h, and i are each 1, and j, k, l, and m are each 0. In some implementations, g and h are each 1, and i, j, k, l, and m are each 0. In some implementations, g is 1, and h, i, j, k, l, and m are each 0. In some implementations, g, h, i, j, k, l, and m are each 0.

[0291] In some implementations, the connector subunit L 7 Connected to a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate (e.g., as shown in formula (I) above). In some embodiments, the connector subunit L 8 (If present) Connected to a second drug or active agent W 2 In some implementations, the connector subunit L 9 (If present) Connected to a second drug or active agent W 2 In some implementations, the connector subunit L 10 (If present) Connected to a second drug or active agent W 2 In some implementations, the connector subunit L 11 (If present) Connected to a second drug or active agent W 2 In some implementations, the connector subunit L 12 (If present) Connected to a second drug or active agent W 2 In some implementations, the connector subunit L 13 (If present) Connected to a second drug or active agent W 2 .

[0292] Any convenient connector subunit can be used for the second connector L B The linker subunits of interest include, but are not limited to, polymer units such as polyethylene glycol, polyethylene and polyacrylate, amino acid residues, carbohydrate-based polymers or carbohydrate residues and their derivatives, polynucleotides, alkyl, aryl, heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, L 7 L 8 L 9 L 10 L 11 L 12 and L 13 Each of (if present) contains one or more groups independently selected from: polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl, substituted alkyl, aryl, substituted aryl and diamine (e.g., including the linking group of alkylene diamine).

[0293] In some implementations, L 7 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 7 Contains polyethylene glycol. In some embodiments, L 7 Contains modified polyethylene glycol. In some embodiments, L 7 It contains amino acid residues. In some embodiments, L 7Contains an alkyl group or a substituted alkyl group. In some embodiments, L 7 Contains an aryl or substituted aryl group. In some embodiments, L 7 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0294] In some implementations, L 8 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 8 Contains polyethylene glycol. In some embodiments, L 8 Contains modified polyethylene glycol. In some embodiments, L 8 It contains amino acid residues. In some embodiments, L 8 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 8 Contains an aryl or substituted aryl group. In some embodiments, L 8 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0295] In some implementations, L 9 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 9 Contains polyethylene glycol. In some embodiments, L 9 Contains modified polyethylene glycol. In some embodiments, L 9 It contains amino acid residues. In some embodiments, L 9 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 9 Contains an aryl or substituted aryl group. In some embodiments, L 9 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0296] In some implementations, L 10 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 10 Contains polyethylene glycol. In some embodiments, L 10 Contains modified polyethylene glycol. In some embodiments, L 10 It contains amino acid residues. In some embodiments, L 10 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 10 Contains an aryl or substituted aryl group. In some embodiments, L 10Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0297] In some implementations, L 11 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 11 Contains polyethylene glycol. In some embodiments, L 11 Contains modified polyethylene glycol. In some embodiments, L 11 It contains amino acid residues. In some embodiments, L 11 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 11 Contains an aryl or substituted aryl group. In some embodiments, L 11 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0298] In some implementations, L 12 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 12 Contains polyethylene glycol. In some embodiments, L 12 Contains modified polyethylene glycol. In some embodiments, L 12 It contains amino acid residues. In some embodiments, L 12 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 12 Contains an aryl or substituted aryl group. In some embodiments, L 12 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0299] In some implementations, L 13 (If present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 13 Contains polyethylene glycol. In some embodiments, L 13 Contains modified polyethylene glycol. In some embodiments, L 13 It contains amino acid residues. In some embodiments, L 13 Contains an alkyl group or a substituted alkyl group. In some embodiments, L 13 Contains an aryl or substituted aryl group. In some embodiments, L 13 Contains a diamine (e.g., a linking group containing an alkylene diamine).

[0300] In some implementations, L B It is the second connector, which includes: -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l -(L 13 ) m -, in: -(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 -; -(L 11 ) k -is-(T) 11 -V 11 ) k -; -(L 12 ) l -is-(T) 12 -V 12 ) l -;and -(L 13 ) m -is-(T) 13 -V 13 ) m -, in: T 7 T 8 T 9 T 10 T 11 T 12and T 13 (If present) is a chain group; V 7 V 8 V 9 V 10 V 11 V 12 and V 13 (If present) is a covalent bond or a linking functional group; and g, h, i, j, k, l, and m are each independently 0 or 1, with the constraint that at least one of g, h, i, j, k, l, and m is 1.

[0301] In some embodiments, the sum of g, h, i, j, k, l, and m is 1 to 7. In some embodiments, the sum of g, h, i, j, k, l, and m is 1. In some embodiments, the sum of g, h, i, j, k, l, and m is 2. In some embodiments, the sum of g, h, i, j, k, l, and m is 3. In some embodiments, the sum of g, h, i, j, k, l, and m is 4. In some embodiments, the sum of g, h, i, j, k, l, and m is 5. In some embodiments, the sum of g, h, i, j, k, l, and m is 6. In some embodiments, the sum of g, h, i, j, k, l, and m is 7. In some embodiments, g, h, i, j, k, l, and m are each 1. In some embodiments, g, h, i, j, k, l, and m are each 1, and m is 0. In some implementations, g, h, i, j, and k are each 1, and l and m are each 0. In some implementations, g, h, i, and j are each 1, and k, l, and m are each 0. In some implementations, g, h, and i are each 1, and j, k, l, and m are each 0. In some implementations, g and h are each 1, and i, j, k, l, and m are each 0. In some implementations, g is 1, and h, i, j, k, l, and m are each 0. In some implementations, g, h, i, j, k, l, and m are each 0.

[0302] As described above, in some implementations, L 7 It is attached to a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate (e.g., as shown in formula (I) above). Therefore, in some embodiments, T 7 Connected to a hydrazine-indolyl or hydrazine-pyrrolo-pyridyl conjugate (e.g., as shown in formula (I) above). In some embodiments, V 7 Connected to a second drug or active agent. In some embodiments, L 8 (If present) linked to a second drug or active agent. Therefore, in some embodiments, T 8(If present) linked to a second drug or active agent, or V 8 (If present) linked to a second drug or active agent. In some embodiments, L 9 (If present) linked to a second drug or active agent. Therefore, in some embodiments, T 9 (If present) linked to a second drug or active agent, or V 9 (If present) linked to a second drug or active agent. In some embodiments, L 10 (If present) linked to a second drug or active agent. Therefore, in some embodiments, T 10 (If present) linked to a second drug or active agent, or V 10 (If present) linked to a second drug or active agent. In some embodiments, L 11 (If present) linked to a second drug or active agent. Therefore, in some embodiments, T 11 (If present) linked to a second drug or active agent, or V 11 (If present) linked to a second drug or active agent. In some embodiments, L 12 (If present) linked to a second drug or active agent. Therefore, in some embodiments, T 12 (If present) linked to a second drug or active agent, or V 12 (If present) linked to a second drug or active agent. In some embodiments, L 13 (If present) linked to a second drug or active agent. Therefore, in some embodiments, T 13 (If present) linked to a second drug or active agent, or V 13 (If present) Connect to a second drug or active agent.

[0303] Regarding the chain group 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 linker group can be used for the subject connector. In some embodiments, T 1 T 2 T 3 T 4 T 5 T 6T 7 T 8 T 9 T 10 T 11 T 12 and T 13 Each contains one or more groups independently selected from the following: covalent bonds, (C1-C2) groups, and (C1-C2) groups. 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, 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.

[0304] In some embodiments, the chain 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 ) including (C1-C 12 )alkyl or substituted (C1-C 12 )alkyl. In some embodiments, (C1-C 12 Alkyl groups are straight-chain or branched alkyl groups comprising 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, (C1-C...) 12 Alkyl groups can be alkyl or substituted alkyl groups, such as C1-C1 alkyl groups. 12 Alkyl, or C1-C 10Alkyl, or C1-C6 alkyl or C1-C3 alkyl. In some cases, (C1-C6 alkyl, C1-C3 alkyl) 12 Alkyl groups are C2-alkyl groups. For example, (C1-C2) alkyl groups are C2-alkyl groups. 12 Alkyl groups can be alkylene groups or substituted alkylene groups, such as C1-C1. 12 Alkylene, or C1-C 10 Alkylene, or C1-C6 alkylene or C1-C3 alkylene. In some cases, (C1-C6 alkylene) 12 Alkyl groups are C1-alkylene groups (e.g., CH2). In some cases, (C1-C...) 12 Alkyl groups are C2-alkylene groups (e.g., CH2CH2). In some cases, (C1-C...) 12 Alkyl groups are C3-alkylene groups (e.g., CH2CH2CH2).

[0305] In some implementations, the replacement (C1-C) 12 Alkyl groups are straight-chain or branched substituted alkyl groups comprising 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, the substituted (C1-C2) 12 Alkyl groups can be substituted alkyl groups, such as substituted C1-C alkyl groups. 12 Alkyl or substituted C1-C 10 Alkyl, or substituted C1-C6 alkyl, or substituted C1-C3 alkyl. In some cases, substituted (C1-C6) alkyl... 12 Alkyl groups are substituted C2-alkyl groups. For example, substituted (C1-C2-alkyl groups) 12 Alkyl groups can be substituted alkylene groups, such as substituted C1-C alkylene groups. 12 Alkylene or substituted C1-C 10 Alkylene, or substituted C1-C6 alkylene or substituted C1-C3 alkylene. In some cases, substituted (C1-C6) alkylene... 12 Alkyl groups are substituted C1-alkylene groups (e.g., C1-alkylene groups substituted with -SO3H). In some cases, substituted (C1-C... 12 Alkyl groups are substituted C2-alkylene groups. In some cases, substituted (C1-C2-alkylene groups) are also substituted. 12 Alkyl groups are substituted C3-alkylene groups. For example, substituted (C1-C2) alkylene groups are substituted C3-alkylene groups. 12 Alkyl groups may include those described herein as (PEG). k Groups (e.g., -CONH (PEG)) t Such as -CONH(PEG)3 or -CONH(PEG)5; or -NHCO(PEG) kSuch as C1-C substituted with -NHCO(PEG)7) 12 Alkylenes (e.g., C3-alkylenes or C5-alkylenes), or may include C1-C groups substituted with -CONHCH2CH2SO3H groups. 12 Alkylenes (e.g., C3-alkylenes), or may include C1-C groups substituted with -NHCOCH2SO3H groups. 12 Alkylene (e.g., C5-alkylene).

[0306] In some implementations, the replacement (C1-C) 12 Alkyl groups may include those described herein as (PEG). t Groups (e.g., -NHCO(PEG)) t , of which (PEG) t yes . The C1-C group substituted with the carbonyl group of -NHCO- (where t is an integer) indicates the connection point with the carbonyl group of -NHCO-. 12 Alkylenes (e.g., C3-alkylenes or C5-alkylenes), such as -NHCO(CH2CH2O)3CH3 or -NHCO(CH2CH2O)5CH3 or -NHCO(CH2CH2O)8CH3.

[0307] In some embodiments, the chain 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 This includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, or substituted heterocyclic groups. In some cases, the chain 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 13This includes aryl or substituted aryl groups. For example, the aryl group can be phenyl. In some cases, the substituted aryl group is a substituted phenyl group. The substituted phenyl group can be substituted by one or more substituents selected from the following: (C1-C2). 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic. In some cases, the substituted aryl is a substituted phenyl, wherein the substituent includes a cleavable moiety as described herein (e.g., an enzyme-cleavable moiety, such as a glycoside or glycoside derivative).

[0308] In some cases, chain groups (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 This includes heteroaryl or substituted heteroaryl groups, such as triazolyl (e.g., 1,2,3-triazolyl). In some cases, chain groups (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 This includes cycloalkyl or substituted cycloalkyl groups. In some cases, the chain 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 This includes heterocyclic groups or substituted heterocyclic groups. In some cases, the substituents on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclic groups include cleavable moieties as described herein (e.g., enzyme-cleavable moieties, such as glycosides or glycoside derivatives).

[0309] In some embodiments, the chain 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 This includes an ethylenediamine (EDA) moiety, such as a chain group containing EDA. In some embodiments, (EDA) w It includes one or more EDA portions, such as where w is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, or 6. The linked ethylenediamine (EDA) portion may optionally be substituted at one or more convenient positions with any convenient substituent (e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl). In some embodiments, the EDA portion is described by the following structure: , Where y is an integer from 1 to 6, r is 0 or 1, and each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic. In some embodiments, y is 1, 2, 3, 4, 5 or 6. In some embodiments, y is 1 and r is 0. In some embodiments, y is 1 and r is 1. In some embodiments, y is 2 and r is 0. In some embodiments, y is 2 and r is 1. In some embodiments, each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In some embodiments, any two adjacent R of EDA 12 The groups can be cyclically linked, for example, to form a piperazine ring. In some embodiments, y is 1 and two adjacent R groups... 12 The group is an alkyl group, which is cyclically linked to form a piperazine ring. In some embodiments, y is 1 and adjacent R 12The group is selected from hydrogen, alkyl (e.g., methyl) and substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

[0310] In some embodiments, the chain 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 4AP moiety comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more convenient positions with any convenient substituent (e.g., alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl). In some embodiments, the 4AP moiety is described by the following structure: Where R 12 The group is selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, R 12 It is the polyethylene glycol portion. In some embodiments, R 12 It is carboxyl-modified polyethylene glycol.

[0311] In some implementations, R 12 Includes the polyethylene glycol portion described by the following formula: (PEG) k It can be represented by the following structure: , Where k is an integer from 1 to 20, such as 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, k is 2. In some implementations, R 17The group is selected from OH, COOH, OR, or COOR, wherein R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, R... 17 It is COOH. In some implementations, R 17 It is OH. In some implementations, R 17 It is OCH3.

[0312] In some implementations, (PEG) k It has the following structure (PEG). t : , Where t is an integer from 2 to 10. In some implementations, t is 8.

[0313] In some embodiments, the chain 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 (including PEG) n , of which (PEG) n It is a polyethylene glycol or modified polyethylene glycol linking unit. In some embodiments, (PEG) n It is described by the following structure: , Where n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

[0314] In some embodiments, the chain group (e.g., T) 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T9 T 10 T 11 T 12 and / or T 13 (Including (AA)) p AA represents an amino acid residue. Any convenient amino acid can be used. Amino acids of interest include, but are not limited to, L- and D-amino acids, naturally occurring amino acids (such as any one of the 20 major α-amino acids and β-alanine), and non-naturally occurring amino acids (e.g., amino acid analogs), such as non-naturally occurring α-amino acids or non-naturally occurring β-amino acids. In some embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, p is 1. In some embodiments, p is 2.

[0315] In other implementations, (AA) p A dipeptide containing valine and alanine.

[0316] In some embodiments, the chain 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 13Amino acid analogs include compounds that are structurally and / or in overall shape similar 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 having the same stereochemistry as naturally occurring D-type amino acids, and L-type amino acid analogs. In some cases, amino acid analogs share the backbone and / or side chain structure with one or more natural amino acids, differing only in one or more modified groups in the molecule. Such modifications can include, but are not limited to, substituting an atom (e.g., S) with an atom (e.g., N), adding a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g., Cl or Br, etc.), deleting a group, substituting a covalent bond (single bond substituting for double bond, etc.), or combinations thereof. For example, amino acid analogs can include α-hydroxy acids and α-amino acids, etc. Examples of amino acid analogs include, but are not limited to, sulfonylalanine, etc.

[0317] In some embodiments, the chain 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 formula -(CR 13 OH) x - The part described, where x is 0 or x is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, R 13The group is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic. In some embodiments, R 13 It is hydrogen. In some implementations, R 13 It is an alkyl or substituted alkyl group, 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 groups. In some embodiments, R 13 It is an alkenyl or substituted alkenyl group, 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 groups. In some embodiments, R 13 It is an alkynyl or substituted alkynyl group. In some embodiments, R 13 It is an alkoxy or a substituted alkoxy group. In some embodiments, R 13 It is an amino or substituted amino group. In some embodiments, R 13 It is a carboxyl group or a carboxyl ester. In some embodiments, R 13 It is an acyl or acyloxy group. In some embodiments, R 13 It is an acylamino or aminoacyl group. In some embodiments, R 13 It is an alkylamide or a substituted alkylamide. In some embodiments, R 13 It is a sulfonyl group. In some embodiments, R 13 It is a thioalkoxy or a substituted thioalkoxy. In some embodiments, R 13 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups. In some embodiments, R 13 It is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R13 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 Substituted cycloalkyl groups. In some embodiments, R 13 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic group or C 3-8 Substituted heterocyclic groups, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0318] In some implementations, R 13 The group is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl groups are as described above for R. 13 As stated above.

[0319] In some embodiments, the chain 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 chain group includes an acetal, a disulfide, a hydrazine, or an ester. In some embodiments, the chain group includes an acetal. In some embodiments, the chain group includes a hydrazine. In some embodiments, the chain group includes a disulfide. In some embodiments, the chain group includes an ester.

[0320] In some embodiments, the chain 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 13This includes m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), or p-hydroxy-phenyl (PHP).

[0321] In some embodiments, the chain linking group includes a MABO group described by the following structure: .

[0322] In some embodiments, the chain linking group includes an MABC group described by the following structure: .

[0323] In some embodiments, the chain linking group includes a PABO group described by the following structure: .

[0324] In some embodiments, the chain linking group includes a PABC group described by the following structure: .

[0325] In some embodiments, the chain linking group includes a PAB group described by the following structure: .

[0326] In some embodiments, the chain linking group includes a PABA group described by the following structure: .

[0327] In some embodiments, the chain linking group includes a PAP group described by the following structure: .

[0328] In some embodiments, the chain linking group includes a PHP group described by the following structure: .

[0329] In some implementations, each R 14 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic.

[0330] In some implementations, R 14 It is hydrogen. In some implementations, each R 14 It is hydrogen. In some implementations, R 14 It is an alkyl or substituted alkyl group, 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 groups. In some embodiments, R 14 It is an alkenyl or substituted alkenyl group, 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 groups. In some embodiments, R 14 It is an alkynyl or substituted alkynyl group. In some embodiments, R 14 It is an alkoxy or a substituted alkoxy group. In some embodiments, R 14 It is an amino or substituted amino group. In some embodiments, R 14 It is a carboxyl group or a carboxyl ester. In some embodiments, R 14 It is an acyl or acyloxy group. In some embodiments, R 14 It is an acylamino or aminoacyl group. In some embodiments, R 14 It is an alkylamide or a substituted alkylamide. In some embodiments, R 14 It is a sulfonyl group. In some embodiments, R 14 It is a thioalkoxy or a substituted thioalkoxy. In some embodiments, R 14 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups. In some embodiments, R 14 It is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R 14 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C3-5 Substituted cycloalkyl groups. In some embodiments, R 14 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic group or C 3-8 Substituted heterocyclic groups, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0331] In some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP chain structures shown above, the benzene ring may be substituted with one or more additional groups selected from the following: halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, and substituted heterocyclic.

[0332] In some embodiments, the chain group 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 One or more of them may be optionally replaced by a glycoside or a glycoside derivative. For example, in some cases, T 1 T 2 T 3 T 4 T 5 and T 6 Each can be optionally replaced by a glycoside. In some cases, T 7 T 8 T 9 T 10 T 11 T 12 and T 13 Each may optionally be replaced by a glycoside. In some embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0333] In some embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP chain structures shown above can be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP chain structures shown above, the benzene ring can be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In some embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. In some embodiments, PABC is substituted with a glycoside, for example, the hydrogen atom of PABC is replaced by a glycoside such as glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0334] For example, in some embodiments, the glycoside or glycoside derivative is selected from the following structures: , , , , , and .

[0335] Regarding the connection of the functional group 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 group can be used for the subject connector. Linking functional groups of interest include, but are not limited to, amino, carbonyl, amide, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxygen, phosphoryl, phosphoramidyl, thiophosphoramidyl, etc. In some embodiments, 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 V13 Each is independently selected from covalent bonds, -CO-, -NR 15 -、-NR 15 (CH2) q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where q is an integer from 1 to 6. In some embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. In some embodiments, q is 6. In some embodiments, each R 15 Independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic.

[0336] In some implementations, R 15 It is hydrogen. In some implementations, each R 15 It is hydrogen. In some implementations, R 15 It is an alkyl or substituted alkyl group, 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 groups. In some embodiments, R 15 It is an alkenyl or substituted alkenyl group, 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 groups. In some embodiments, R 15 It is an alkynyl or substituted alkynyl group. In some embodiments, R 15 It is an alkoxy or a substituted alkoxy group. In some embodiments, R 15It is an amino or substituted amino group. In some embodiments, R 15 It is a carboxyl group or a carboxyl ester. In some embodiments, R 15 It is an acyl or acyloxy group. In some embodiments, R 15 It is an acylamino or aminoacyl group. In some embodiments, R 15 It is an alkylamide or a substituted alkylamide. In some embodiments, R 15 It is a sulfonyl group. In some embodiments, R 15 It is a thioalkoxy or a substituted thioalkoxy. In some embodiments, R 15 It is an aryl or substituted aryl group, such as C 5-8 Aryl or C 5-8 Substituted aryl groups, such as C5 aryl or C5-substituted aryl groups, or C6 aryl or C6-substituted aryl groups. In some embodiments, R 15 It is a heteroaryl or substituted heteroaryl, such as C 5-8 heteroaryl or C 5-8 Substituted heteroaryl groups, such as C5 heteroaryl or C5-substituted heteroaryl groups, or C6 heteroaryl or C6-substituted heteroaryl groups. In some embodiments, R 15 It is a cycloalkyl or substituted cycloalkyl, such as C 3-8 cycloalkyl or C 3-8 Substituted cycloalkyl groups, such as C 3-6 cycloalkyl or C 3-6 Substituted cycloalkyl, or C 3-5 cycloalkyl or C 3-5 Substituted cycloalkyl groups. In some embodiments, R 15 It is a heterocyclic group or a substituted heterocyclic group, such as C 3-8 Heterocyclic group or C 3-8 Substituted heterocyclic groups, such as C 3-6 Heterocyclic group or C 3-6 Substituted heterocyclic groups, or C 3-5 Heterocyclic group or C 3-5 Substituted heterocyclic groups.

[0337] In some implementations, each R 15 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, heterocyclic, and substituted heterocyclic. 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, heterocyclic, and substituted heterocyclic are as described above for R. 15 As stated above.

[0338] As described above, in some implementations, L A It contains -(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 - The first connector, where a, b, c, d, e, and f are each independently 0 or 1, with the constraint that at least one of a, b, c, d, e, and f is 1.

[0339] In some implementations, in the first joint L A middle: T 1 Selected from (C1-C) 12 )alkyl and substituted (C1-C 12 )alkyl; T 2 T 3 T 4 T 5 and T 6 Each is independently selected from (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, disulfide, hydrazine and ester; and V 1 V 2 V 3 V 4 V 5 and V 6 Each is independently selected from covalent bonds, -CO-, -NR 15 -、-NR 15 (CH2)q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where q is an integer from 1 to 6; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , Where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ; AA represents an amino acid residue, where p is an integer from 1 to 20; and Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 It 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, heterocyclic and substituted heterocyclic.

[0340] In some implementations, L A Include: -(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 -V6 ) f -, in: a, b, c, d, e, and f are each independently 0 or 1, with the constraint that 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 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 1 V 2 V 3 V 4 V 5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 It 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, heterocyclic and substituted heterocyclic.

[0341] In L A Some implementation schemes: T 1 Selected from (C1-C) 12 )alkyl and substituted (C1-C 12 )alkyl; T 2 T 3 T 4 T 5 and T 6 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, hydrazine, and esters; and V 1 V 2 V 3 V 4 V 5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , Where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ; Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; a, b, c, and d are each 1; and e and f are 0.

[0342] In some implementations, T 1 T 2 T 3 T 4 T 5 and T 6 Each can be substituted with a glycoside at its own discretion.

[0343] In some implementations, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally replaced by glycosides.

[0344] In some embodiments, the glycoside is selected from glucuronide, galactoside, glucoside, mannoside, fucoidan, O-GlcNAc, and O-GalNAc.

[0345] In some implementations, 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 Selected from the following: in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CO-; T 2 Yes (AA) p And V 2 It does not exist (e.g., covalent bonds); T 3It is PABC and V 3 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and d, e, and f are each 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is (PEG) n And V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABC and V 4 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and e and f are both 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CO-; T 2 It is an amino acid analog and V 2 It is -NH-; T 3 It is (PEG) n And V 3 It is -CO-; T 4 Yes (AA) p And V 4 It does not exist (e.g., covalent bonds); T 5 It is PABC and V 5 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and f is 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is (PEG) n And V 2 It is -CO-; T 3 Yes (AA)p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABC and V 4 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and e and f are both 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABC and V 4 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and e and f are both 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is (PEG) n And V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABA and V 4 It is -CO-; T 5 It is (C1-C) 12 )alkyl and V 5 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and f is 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CO-; T2 It is 4AP and V 2 It is -CO-; T 3 It is (C1-C) 12 )alkyl and V 3 It is -CO-; T 4 Yes (AA) p And V 4 It does not exist (e.g., covalent bonds); T 5 It is PABC and V 5 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and f is 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CO-; T 2 It is 4AP and V 2 It is -CO-; T 3 It is (C1-C) 12 )alkyl and V 3 It is -O-; T 4 It is (C1-C) 12 )alkyl and V 4 It is -CO-; T 5 Yes (AA) p And V 5 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and T 6 It is PABC and V 6 It does not exist (e.g., covalent bonds); or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CO-; T 2 It is an amino acid analog and V 2 It does not exist (e.g., covalent bonds); T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABC and V 4It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and e and f are both 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is (PEG) n And V 2 It is -CONH-; T 3 It is a substitute (C1-C) 12 )alkyl and V 3 It is -CO-; T 4 Yes (AA) p And V 4 It does not exist (e.g., covalent bonds); T 5 It is PABC and V 5 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and f is 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CO-; T 2 Yes (AA) p And V 2 It is -NH-; T 3 It is (PEG) n And V 3 It is -CO-; T 4 Yes (AA) p And V 4 It does not exist (e.g., covalent bonds); T 5 It is PABC and V 5 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and f is 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is (PEG)n And V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PAP and V 4 It is -C(O)O-; p is an integer from 1 to 10; and e and f are both 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist (e.g., covalent bonds); T 4 It is PABC and V 4 It does not exist (e.g., covalent bonds); p is an integer from 1 to 10; and e and f are both 0; or in: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 It is PABC and V 3 There is no (e.g., covalent bond); and d, e, and f are each 0.

[0346] In some implementations, the first connector L A The left side of the aforementioned connector structure is connected to the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate portion, and the first connector L A The right side of the aforementioned connector structure is connected to the first drug or active agent.

[0347] As described above, in some implementations, L B It contains -(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 - The second connector, where g, h, i, j, k, l and m are each independently 0 or 1, with the constraint that at least one of g, h, i, j, k, l and m is 1.

[0348] In some implementations, at the second joint L B middle: T 7 Selected from (C1-C) 12 )alkyl and substituted (C1-C 12 )alkyl; T 8 T 9 T 10 T 11 T 12 and T 13 Each is independently selected from (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x -, 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, disulfide, hydrazine and ester; and V 7 V 8 V 9 V 10 V 11 V 12 and V 13 Each is independently selected from covalent bonds, -CO-, -NR15 -、-NR 15 (CH2) q -、-NR 15 (C6H4)-、-CONR 15 -、-NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where q is an integer from 1 to 6; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , Where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ; AA represents an amino acid residue, where p is an integer from 1 to 20; and Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 It 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, heterocyclic and substituted heterocyclic.

[0349] Any convenient chain group can be used for T 7 T 8 T 9 T 10 T 11 T 12 and T 13 For example, regarding T in the above text... 1 T 2 T 3 T 4 T 5 and T 6 Any chain group mentioned can be used for chain group T. 7 T8 T 9 T 10 T 11 T 12 and T 13 .

[0350] Any convenient connective functional group can be used for V 7 V 8 V 9 V 10 V 11 V 12 and V 13 For example, regarding V in the above text... 1 V 2 V 3 V 4 V 5 and V 6 Any of the connecting functional groups mentioned can be used to connect functional group V. 7 V 8 V 9 V 10 V 11 V 12 and V 13 .

[0351] In some implementations, each R 13 The groups are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, the alkyl, substituted alkyl, aryl, and substituted aryl groups are as described above for R. 13 As stated above.

[0352] In some implementations, each R 15 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, heterocyclic, and substituted heterocyclic. 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, heterocyclic, and substituted heterocyclic are as described above for R. 15 As described above, in these embodiments, various possible substituents are as described above for R. 15 As stated above.

[0353] Second connector L B In some embodiments, the chain group T 7 T 8 T 9 T 10 T 11 T12 and T 13 One or more of these components may be optionally replaced by a glycoside or a glycoside derivative. In some embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0354] Second connector L B In some embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP chain structures shown above may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. For example, in some embodiments of the MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP chain structures shown above, the benzene ring may be substituted with one or more additional groups selected from glycosides and glycoside derivatives. In some embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

[0355] In some implementations, T 7 T 8 T 9 T 10 T 11 T 12 and T 13 Each can be substituted with a glycoside at its own discretion.

[0356] In some implementations, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally replaced by glycosides.

[0357] In some embodiments, the glycoside is selected from glucuronide, galactoside, glucoside, mannoside, fucoidan, O-GlcNAc, and O-GalNAc.

[0358] In L B Some implementation schemes: g, h, i, j, and k are each 1; l and m are both 0; T 7 It is a covalent bond; T 8 T 9 T 10 T 11 and T 12 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, hydrazine, and esters; and V 7 V 8 V 9 V 10 V 11 and V 12 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , Where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ;and Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring.

[0359] In some implementations, T 1 T 2 T 3 T 4 T 5 T 6 T 7 T 8 T 9T 10 T 11 and T 12 Each can be substituted with a glycoside at its own discretion.

[0360] In some implementations, MABO, MABC, PABO, PABC, PAB, PABA, PAP, and PHP are each optionally replaced by glycosides.

[0361] In some embodiments, the glycoside is selected from glucuronide, galactoside, glucoside, mannoside, fucoidan, O-GlcNAc, and O-GalNAc.

[0362] In some implementations, 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 Selected from the following: in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 Yes (AA) p And V 9 It does not exist (e.g., covalent bonds); T 10 It is PABC and V 10 There is no (e.g., covalent bond); and k, l, and m are each 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is (PEG) n And V 9 It is -CO-; T 10 Yes (AA) p And V 10 There is no (e.g., covalent bond); and T 11 It is PABC and V 11 There is no (e.g., covalent bond); and l and m are both 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 It is an amino acid analog and V 9 It is -NH-; T 10 It is (PEG) n And V 10 It is -CO-; T 11 Yes (AA) p And V 11 It does not exist (e.g., covalent bonds); T 12 It is PABC and V 12 There is no (e.g., covalent bond); and m is 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is (PEG) n And V 9 It is -CO-; T 10 Yes (AA) p And V 10 It does not exist (e.g., covalent bonds); T 11 It is PABC and V 11 There is no (e.g., covalent bond); and l and m are both 0; or in: T7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is a substitute (C1-C) 12 )alkyl and V 9 It is -CO-; T 10 Yes (AA) p And V 10 It does not exist (e.g., covalent bonds); T 11 It is PABC and V 11 There is no (e.g., covalent bond); and l and m are both 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is (PEG) n And V 9 It is -CO-; T 10 Yes (AA) p And V 10 It does not exist (e.g., covalent bonds); T 11 It is PABA and V 11 It is -CO-; T 12 It is (C1-C) 12 )alkyl and V 12 There is no (e.g., covalent bond); and m is 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 It is 4AP and V 9 It is -CO-; T 10 It is (C1-C) 12 )alkyl and V 10 It is -CO-; T 11 Yes (AA) p And V 11 It does not exist (e.g., covalent bonds); T 12 It is PABC and V 12 There is no (e.g., covalent bond); and m is 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 It is 4AP and V 9 It is -CO-; T 10 It is (C1-C) 12 )alkyl and V 10 It is -O-; T 11 It is (C1-C) 12 )alkyl and V 11 It is -CO-; T 12 Yes (AA) p And V 12 There is no (e.g., covalent bond); and T 13 It is PABC and V 13 It does not exist (e.g., covalent bonds); or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 It is an amino acid analog and V 9 It does not exist (e.g., covalent bonds); T 10 Yes (AA) p And V 10 It does not exist (e.g., covalent bonds); T11 It is PABC and V 11 There is no (e.g., covalent bond); and l and m are both 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is (PEG) n And V 9 It is -CONH-; T 10 It is a substitute (C1-C) 12 )alkyl and V 10 It is -CO-; T 11 Yes (AA) p And V 11 It does not exist (e.g., covalent bonds); T 12 It is PABC and V 12 There is no (e.g., covalent bond); and m is 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 Yes (AA) p And V 9 It is -NH-; T 10 It is (PEG) n And V 10 It is -CO-; T 11 Yes (AA) p And V 11 It does not exist (e.g., covalent bonds); T 12 It is PABC and V 12 There is no (e.g., covalent bond); and m is 0; or in: T 7There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is (PEG) n And V 9 It is -CO-; T 10 Yes (AA) p And V 10 It does not exist (e.g., covalent bonds); T 11 It is PAP and V 11 It is -C(O)O-; and l and m are both 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CO-; T 9 Yes (AA) p And V 9 It does not exist (e.g., covalent bonds); T 10 It is PABC and V 10 It does not exist (e.g., covalent bonds); T 11 It is PAP and V 11 It is -C(O)O-; and l and m are both 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is a substitute (C1-C) 12 )alkyl and V 9 It is -CO-; T 10 It is PABC and V 10 There is no (e.g., covalent bond); and k, l, and m are each 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It does not exist (e.g., covalent bonds); T 9 It is a heteroaryl and V 9 It does not exist (e.g., covalent bonds); T 10 It is (C1-C) 12 )alkyl and V 10 It is -CONH-; T 11 It is (PEG) n And V 11 It is -CO-; and l and m are both 0; or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It does not exist (e.g., covalent bonds); T 9 It is a heteroaryl and V 9 It does not exist (e.g., covalent bonds); T 10 It is (C1-C) 12 )alkyl and V 10 It is -CONH-; T 11 It is a substitute (C1-C) 12 )alkyl and V 11 It is -CO-; T 12 Yes (AA) p And V 12 There is no (e.g., covalent bond); and T 13 It is PAB and V 13 It does not exist (e.g., covalent bonds); or in: T 7 There is no (e.g., covalent bond) and V 7 It is -NHCO-; T 8 It is (C1-C)12 )alkyl and V 8 It does not exist (e.g., covalent bonds); T 9 It is a heteroaryl and V 9 It does not exist (e.g., covalent bonds); T 10 It is (C1-C) 12 )alkyl and V 10 It is -CONH-; T 11 It is a substitute (C1-C) 12 )alkyl and V 11 It is -CO-; T 12 Yes (AA) p And V 12 There is no (e.g., covalent bond); and T 13 It is PABC and V 13 It does not exist (e.g., covalent bonds).

[0363] In some implementations, the second connector L B The left side of the aforementioned connector structure is connected to the hydrazyl-indolyl or hydrazyl-pyrrolo-pyridyl conjugate portion, and the second connector L B The right side of the aforementioned connector structure is connected to a second drug or active agent.

[0364] In some embodiments, the conjugate is an antibody-drug conjugate, wherein the ROR1 antibody and the drug are linked together via a connector as described above. In some cases, the connector m (e.g., L) A and / or L B A cleavable linker is a linker comprising one or more cleavable portions, wherein the cleavable portions include one or more bonds that can dissociate under certain conditions, thereby separating the cleavable linker into two or more separable portions. For example, the cleavable portions may include one or more covalent bonds that can dissociate or break under certain conditions, thereby separating the cleavable linker into two or more portions. Therefore, the linker included in an antibody-drug conjugate can be a cleavable linker, such that under appropriate conditions, the cleavable linker cleaves to separate or release the drug from the antibody at the desired target site of action of the drug.

[0365] In some cases, the cleavable linker includes two cleavable portions, such as a first cleavable portion and a second cleavable portion. The cleavable portions can be configured such that cleavage of both cleavable portions is required to separate or release the drug from the ROR1 antibody at the desired target site of action. For example, cleavage of the cleavable linker can be achieved by initially cleaving one of the two cleavable portions, and then cleaving the other of the two cleavable portions. In some embodiments, the cleavable linker includes a first cleavable portion and a second cleavable portion, the second cleavable portion hindering the cleavage of the first cleavable portion. "Hindering cleavage" means that the presence of the uncracked second cleavable portion reduces the likelihood of cleavage of the first cleavable portion or substantially inhibits its cleavage, thereby substantially reducing the amount of the cleavable linker or preventing its cleavage. For example, the presence of the uncracked second cleavable portion can hinder the cleavage of the first cleavable portion. The presence of the second cleavable portion hinders the cleavage of the first cleavable portion, which in turn substantially reduces the amount of drug released from the antibody or prevents the release of the drug from the antibody. For example, it can largely reduce or prevent the premature release of drugs from antibodies until the antibody-drug conjugate reaches or approaches the target site for drug action.

[0366] In some cases, because the second cleavable portion hinders the cleavage of the first cleavable portion, the cleavage of the cleavable linker can be achieved by initially cleaving the second cleavable portion, followed by cleavage of the first cleavable portion. Cleavage of the second cleavable portion can reduce or eliminate the hindrance to the cleavage of the first cleavable portion, thereby allowing the first cleavable portion to cleave. Cleavage of the first cleavable portion can lead to the dissociation or separation of the cleavable linker into two or more portions as described above, to release the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable portion does not generally occur in the presence of an uncleavable second cleavable portion. Generally speaking, this means that in the presence of an unfractured second fractured portion, the first fractured portion undergoes about 10% or less of fracture, for example, in the presence of an unfractured second fractured portion, the first fractured portion undergoes about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less of fracture.

[0367] In other words, the second cleavable portion can protect the first cleavable portion from cleavage. For example, the presence of an uncleavable second cleavable portion can protect the first cleavable portion from cleavage and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate reaches or approaches the desired target site of drug action. Therefore, cleavage of the second cleavable portion exposes the first cleavable portion (e.g., deprotects it), allowing the first cleavable portion to cleave, which leads to the cleavage of the cleavable linker, thereby separating or releasing the drug from the antibody at the desired target site of drug action, as described above. In some cases, cleavage of the second cleavable portion exposes the first cleavable portion for subsequent cleavage, but the cleavage of the second cleavable portion itself does not lead to the cleavage of the cleavable linker (e.g., cleavage of the first cleavable portion is still required to cleave the cleavable linker).

[0368] The cleavable portions included in the cleavable linker can each be an enzyme-cleavable portion. For example, the first cleavable portion can be a first enzyme-cleavable portion, and the second cleavable portion can be a second enzyme-cleavable portion. An enzyme-cleavable portion is a cleavable portion that can be separated into two or more portions as described above by the enzymatic action of an enzyme. An enzyme-cleavable portion can be any cleavable portion that can be cleaved by the enzymatic action of an enzyme, such as, but not limited to, esters, peptides, glycosides, etc. In some cases, the enzyme that cleaves the enzyme-cleavable portion is present at the desired target site, such as the target site for the drug to be released from the antibody-drug conjugate. In some cases, the enzyme that cleaves the enzyme-cleavable portion is not present in large quantities in other regions (such as whole blood, plasma, or serum). Therefore, the cleavage of the enzyme-cleavable portion can be controlled so that a large amount of cleavage occurs at the desired site, while cleavage does not occur significantly in other regions or before the antibody-drug conjugate reaches the desired site.

[0369] For example, as described herein, the antibody-drug conjugates of this disclosure can be used to treat cancer, such as for delivering cancer therapeutic drugs to desired sites of action where cancer cells are present. In some cases, the enzyme (such as an esterase that cleaves ester bonds or a glycosidase that cleaves glycosidic bonds) can be a biomarker of cancer that is overexpressed in cancer cells. The overexpression of certain enzymes in cancer and the resulting localization can be used in the context of an enzyme-cleavable moiety included in the cleavable linker of the antibody-drug conjugate of this disclosure to specifically release the drug at the desired site of action (e.g., the cancer site (and the site of the overexpressed enzyme)). Thus, in some embodiments, the enzyme-cleavable moiety is a cleavable moiety (e.g., an ester or glycoside) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme can be an esterase. Thus, in some cases, the enzyme-cleavable moiety is a cleavable moiety (e.g., an ester) that can be cleaved by an esterase. In some cases, the enzyme can be a glycosidase. Thus, in some cases, the enzyme-cleavable moiety is a cleavable moiety (e.g., a glycoside or a glycoside derivative) that can be cleaved by a glycosidase.

[0370] In some embodiments, the enzymatically cleavable moiety is an ester bond. For example, the aforementioned first cleavable moiety (e.g., a cleavable moiety protected from premature cleavage by a second cleavable moiety) may comprise an ester. The presence of the uncleavable second cleavable moiety can protect the first cleavable moiety (ester) from cleavage by the esterase, and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate reaches or approaches the desired target site of drug action. In some cases, a portion of the linker adjacent to the first cleavable moiety is attached to or includes a substituent, wherein the substituent comprises the second cleavable moiety. In some cases, the second cleavable moiety comprises a glycoside or a glycoside derivative.

[0371] In some embodiments, the enzyme-cleavable portion is a sugar moiety, such as a glycoside (or glycosyl group) or a glycoside derivative. In some cases, a glycoside or glycoside derivative can promote increased hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside or glycoside derivative. The glycoside or glycoside derivative can be any glycoside or glycoside derivative suitable for use in a cleavable linker and capable of cleavage by the enzymatic action of the enzyme. For example, a second cleavable portion (e.g., a cleavable portion that protects the first cleavable portion from premature cleavage) can be a glycoside or glycoside derivative. For example, in some embodiments, the first cleavable portion comprises an ester, and the second cleavable portion comprises a glycoside or glycoside derivative. In some embodiments, the second cleavable portion is a glycoside or glycoside derivative selected from: glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable portion is a glucuronide. In some cases, the second cleavable portion is a galactoside. In some cases, the second cleavable portion is a glucoside. In some cases, the second cleavable moiety is mannoside. In some cases, the second cleavable moiety is fucoside. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

[0372] Glycosides or glycoside derivatives can be covalently linked to a cleavable linker via a glycosidic bond. The glycosidic bond can connect the glycoside or glycoside derivative to the cleavable linker through various types of bonds, such as, but not limited to, O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyl groups). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside or glycoside derivative can be cleaved from the cleavable linker to which it is attached by an enzyme (e.g., through enzyme-mediated glycosidic bond hydrolysis). The glycoside or glycoside derivative can be removed or cleaved from the cleavable linker by any readily available enzyme capable of cleaving (hydrolyzing) the glycosidic bond connecting the glycoside or glycoside derivative to the cleavable linker. Examples of enzymes that can mediate the cleavage (hydrolysis) of the glycosidic bond connecting a glycoside or glycosidic derivative to a cleavable linker are glycosidases, such as glucuronidase, galactosidase, glucosidase, mannosidase, fucosidase, etc. Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond connecting a glycoside or glycosidic derivative to a cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond connecting a glycoside or glycosidic derivative to a cleavable linker is present at or near the desired site of action of the drug in the antibody-drug conjugate. For example, the enzyme may be a lysosomal enzyme, such as a lysosomal glycosidase, present in cells at or near the desired site of action of the drug in the antibody-drug conjugate. In some cases, the enzyme is present at or near the target site where the enzyme mediating the cleavage of the first cleavable moiety is located.

[0373] In some implementations, the ROR1-ADC is represented by equation (I): in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4 Each is selected from hydrogen and (C1-C) 12 )alkyl; L A It is the first connector, in which: T 1 It is (C1-C) 12 )alkyl and V1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 Yes (AA) p Where p is an integer from 1 to 20 and V 3 It is a covalent bond; T 4 It is PABC and V 4 It is a covalent bond; a, b, c, and d are each 1; e and f are both 0; and L B It is the second connector, in which: T 7 It is a covalent bond and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is a substitute (C1-C) 12 )alkyl and V 9 It is -CO-; T 10 Yes (AA) p Where p is an integer from 1 to 20 and V 10 It is a covalent bond; T 11 It is PABC and V 11 It is a covalent bond; and h, i, j, and k are each 1; and l and m are both 0; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

[0374] In some implementation schemes, W 1 and W 2 One or both of them are camptothecin analogues, such as belotecone.

[0375] In some implementations, the ROR1-ADC is represented by equation (I): in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4 Each is selected from hydrogen and (C1-C) 12 )alkyl; L A It is a connector, in which: T 1 It is a (C1-C6) alkyl group and V 1 It is -CONH-; T 2 It is -NHCO(PEG) k Substituted (C1-C6) alkylene groups, where k is an integer from 2 to 10, and V 2 It is -CO-; T 3 It is (AA)2 and V 3 It is a covalent bond; T 4 It is PABC substituted with glycosides and V 4 It is a covalent bond; a, b, c, and d are each 1; and e and f are both 0; and L B It is a connector, in which: T 7 It is a covalent bond and V 7 It is -NHCO-; T 8 It is a (C1-C6) alkyl group and V 8 It is -CONH-; T 9 It is -NHCO(PEG) k Substituted (C1-C6) alkylene groups, where k is an integer from 2 to 10, and V 9 It is -CO-; T 10 It is (AA)2 and V 10 It is a covalent bond; T 11 It is PABC substituted with glycosides and V 11 It is a covalent bond; h, i, j, and k are each 1; and l and m are both 0; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

[0376] In some implementations, the ROR1-ADC is represented by equation (I): in: Ab indicates an antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4 Each is selected from hydrogen and (C1-C) 12 )alkyl; L A It is a connector, in which: T 1 It is a (C1-C6) alkyl group and V 1 It is -CONH-; T 2 It is -NHCO(PEG) t Substituted (C1-C6) alkylene groups, wherein (PEG) t yes And t is an integer from 2 to 10, arbitrarily 8, and V 2 It is -CO-; T 3 It is (AA)2 and V 3 It is a covalent bond; T 4 It is PABC substituted with glycosides and V 4 It is a covalent bond; a, b, c, and d are each 1; and e and f are both 0; and L B It is a connector, in which: T 7 It is a covalent bond and V 7 It is -NHCO-; T 8 It is a (C1-C6) alkyl group and V 8 It is -CONH-; T 9 It is -NHCO(PEG) t Substituted (C1-C6) alkylene groups, wherein (PEG) t yes And t is an integer from 2 to 10, arbitrarily 8, and V 9 It is -CO-; T 10 It is (AA)2 and V 10 It is a covalent bond; T 11 It is PABC substituted with glycosides and V 11 It is a covalent bond; h, i, j, and k are each 1; and l and m are both 0; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

[0377] In some implementations, T 4 and T 11 One or both of the PABCs are replaced with glucuronide. In some embodiments, T 1 and T 8 One or both of them are ethyl. In some embodiments, T 2 and T 9 One or both of them are C5 alkylene groups substituted with -NHCO(PEG)k, where k is an integer from 5 to 10. In some embodiments, W 1 and W 2 One or both of them are camptothecin analogues, such as belotecone.

[0378] In some implementations, T 4 and T 11 One or both of the PABCs are replaced with glucuronide. In some embodiments, T 1 and T 8 One or both of them are ethyl. In some embodiments, T 2 and T 9 One or both of them are -NHCO(PEG) t Substituted C5 alkylene groups, of which (PEG) t yes And t is an integer from 5 to 10. In some implementations, W 1 and W 2 One or both of them are camptothecin analogues, such as belotecone.

[0379] In some implementations, s is an integer from 1 to 4. In other implementations, s is 4.

[0380] In some implementations, ROR1-ADC is represented by equation (II): in: Ab indicates an antibody that binds to ROR1; and s is an integer from 1 to 10.

[0381] In some implementations, s is an integer from 1 to 4.

[0382] Any chemical entity, connector, and conjugate described in the above structures can be adapted to the subject compound and conjugate.

[0383] Further disclosures regarding hydrazyl-indolyl and hydrazyl-pyrrolo-pyridyl compounds and methods for generating conjugates can be found in U.S. Patent Nos. 9,310,374, 9,493,413, 11,564,989 and International Publication No. WO 2022 / 187370, the disclosures of each of which are incorporated herein by reference.

[0384] 7.3 Tyrosine protein kinase receptor 1 (ROR1) antibody As described above, the subject conjugate comprises an antibody (Ab) that binds to ROR1. The amino acid sequence of the antibody may be modified to include 2-formylglycine (fGly) residues. As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and / or their standard single-letter abbreviations, 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.

[0385] In some embodiments, this disclosure provides antibodies against tyrosine protein kinase receptor 1 (ROR1), which can be used herein as therapeutic agents for treating cancer. Such agents include antibodies that bind to ROR1 (e.g., monospecific or multispecific, including bispecific). Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, and heteroconjugated antibodies, as well as variants thereof having increased or decreased affinity or other properties.

[0386] In some embodiments, this document describes ROR1 antibodies that bind to ROR1, wherein ROR1 comprises a ROR1 polypeptide, a ROR1 polypeptide fragment, a ROR1 peptide, or a ROR1 epitope. In some embodiments, the ROR1 antibody is a human or humanized antibody (e.g., containing a human constant region) that binds to ROR1, wherein ROR1 comprises a ROR1 polypeptide, a ROR1 polypeptide fragment, a ROR1 peptide, or a ROR1 epitope. In some embodiments, the ROR1 antibody (such as a human ROR1 antibody) may bind to ROR1 expressed on the surface of mammalian (e.g., human) cells (including cancer cells expressing ROR1). In some embodiments, the ROR1 antibody (such as a human ROR1 antibody) may bind to ROR1 expressed on the surface of mammalian (e.g., human) cells (including cancer cells overexpressing ROR1). In some embodiments, the ROR1 antibody binds to ROR1 extracellular epitopes exposed on cells (such as cancer cells). In some embodiments, this document describes a ROR1 antibody that binds to ROR1 (such as human ROR1 or a portion thereof). In some embodiments, ROR1 is human ROR1. In some embodiments, the ROR1 antibody is a human ROR1 antibody (e.g., an antibody that binds to human ROR1). In some embodiments, the ROR1 antibody binds to both human and cyno ROR1. In other embodiments, the ROR1 antibody binds to human ROR1 but not to cyno ROR1.

[0387] In some embodiments, the ROR1 antibody provided herein is available in concentrations of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) DThe antibody binds to ROR1 (e.g., human ROR1, cyno ROR1, mouse ROR1, and / or rat ROR1). Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure, including by RIA, for example with the Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. MolBiol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR), and by Octet... ® Use, for example, Octet ® This can be done using the Red96 system, or via Biacore. ® Use, for example, Biacore ® TM-2000 or Biacore ® The TM-3000 is used for this purpose. The "on-rate of association rate" or "k-association" can also be measured using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, employing, for example, Octet. ® Red96, Biacore ® TM-2000, Biacore ® TM-3000 system, Biacore ® TM-8K or Biacore ® The TM-8K+ system is used to determine this.

[0388] 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 has a higher affinity for ROR1 than for 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 twice that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least five times that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 10 times that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 100 times that to ROR2. In some embodiments, the binding affinity of the ROR1 antibody provided herein to ROR1 is at least 1000 times that to ROR2.

[0389] In some embodiments, the ROR1 antibody described herein comprises the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 of any antibody described herein, such as the amino acid sequences of the VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and / or VL CDR3 depicted in Table 1. Therefore, in some embodiments, the 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, the 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, the CDR of the ROR1 antibody used herein is disclosed in U.S. Patent Application Publication No. US20210155692A1, which is incorporated herein by reference in its entirety.

[0390] In some embodiments, the ROR1 antibody comprises a VH region containing VH CDR1, VH CDR2, and / or VHCDR3, and / or a VL region containing VL CDR1, VL CDR2, and / or VL CDR3, said regions being derived from any of the antibodies described herein (see, for example, any of those in Tables 1-2). Therefore, in some embodiments, the 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.

[0391] In some embodiments, the ROR1 antibody provided herein comprises (i) VH CDR1, VH CDR2 and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:25, and / or (ii) VL CDR1, VL CDR2 and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:26.

[0392] In some embodiments, the ROR1 antibody provided herein comprises VH CDR1, VH CDR2, and / or VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:25; and / or VL CDR1, VL CDR2, and / or VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein comprises VH CDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:25; and VL CDR1, VL CDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:26. The CDR sequences can be determined according to well-known numbering systems or combinations thereof. In some embodiments, the CDR is based on exemplary numbering. In some embodiments, the CDR is based on IMGT numbering. In some embodiments, the CDR is based on Kabat numbering. In some embodiments, the CDR is based on AbM numbering. In other embodiments, the CDR is based on Chothia numbering. In other embodiments, the CDR is based on a Contact number. In some embodiments, the CDR sequence is determined based on any two or more combinations of the numbering systems described above (e.g., a combination of Kabat and Chothia). Various exemplary CDR numbering systems are described and illustrated in Section 7.1 above.

[0393] In some embodiments, the ROR1 antibody provided herein comprises (a) a VH region comprising VHCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, and 36; VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, and 10; and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, and 37; and / or (b) a VL region comprising VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, and 18; VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 20, and 21; and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 22, 23, and 24.

[0394] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:1; VH CDR2 containing the amino acid sequence of SEQ ID NO:6; and VHCDR3 containing the amino acid sequence of SEQ ID NO:11; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:15; VL CDR2 containing the amino acid sequence of SEQ ID NO:19; and VL CDR3 containing the amino acid sequence of SEQ ID NO:22.

[0395] In some embodiments, the ROR1 binder (e.g., antibody) provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:36; VH CDR2 containing the amino acid sequence of SEQ ID NO:10; and VH CDR3 containing the amino acid sequence of SEQ ID NO:37; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:15; VL CDR2 containing the amino acid sequence of SEQ ID NO:19; and VLCDR3 containing the amino acid sequence of SEQ ID NO:23.

[0396] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:2; VH CDR2 containing the amino acid sequence of SEQ ID NO:7; and VHCDR3 containing the amino acid sequence of SEQ ID NO:12; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:16; VL CDR2 containing the amino acid sequence of SEQ ID NO:20; and VL CDR3 containing the amino acid sequence of SEQ ID NO:22.

[0397] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:3; VH CDR2 containing the amino acid sequence of SEQ ID NO:6; and VHCDR3 containing the amino acid sequence of SEQ ID NO:11; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:15; VL CDR2 containing the amino acid sequence of SEQ ID NO:19; and VL CDR3 containing the amino acid sequence of SEQ ID NO:22.

[0398] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:4; VH CDR2 containing the amino acid sequence of SEQ ID NO:8; and VHCDR3 containing the amino acid sequence of SEQ ID NO:13; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:17; VL CDR2 containing the amino acid sequence of SEQ ID NO:20; and VL CDR3 containing the amino acid sequence of SEQ ID NO:23.

[0399] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:5; VH CDR2 containing the amino acid sequence of SEQ ID NO:9; and VHCDR3 containing the amino acid sequence of SEQ ID NO:14; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:18; VL CDR2 containing the amino acid sequence of SEQ ID NO:21; and VL CDR3 containing the amino acid sequence of SEQ ID NO:24.

[0400] In some embodiments, the ROR1 antibody provided herein comprises a VH region comprising VH CDR1 containing the amino acid sequence of SEQ ID NO:1; VH CDR2 containing the amino acid sequence of SEQ ID NO:10; and VHCDR3 containing the amino acid sequence of SEQ ID NO:11; and a VL region comprising VL CDR1 containing the amino acid sequence of SEQ ID NO:15; VL CDR2 containing the amino acid sequence of SEQ ID NO:19; and VL CDR3 containing the amino acid sequence of SEQ ID NO:22.

[0401] In some embodiments, the antibody further comprises one or more frame regions of SEQ ID NO: 25 and / or 26. In some embodiments, the antibody or a fragment thereof further comprises frame 1 (FR1), frame 2 (FR2), frame 3 (FR3), and / or frame 4 (FR4) sequences, as shown in any of SEQ ID NO: 25 and 26. In some embodiments, the antibody provided herein is a humanized antibody. The frame regions described herein are determined based on the boundaries of the CDR numbering system. In other words, if the CDR is determined by, for example, Kabat, IMGT, or Chothia, the frame region is the amino acid residues surrounding the CDR in the variable region, in the form of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. For example, FR1 is defined as the N-terminal amino acid residue of CDR1, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR2 is defined as the amino acid residue between CDR1 and CDR2, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; FR3 is defined as the amino acid residue between CDR2 and CDR3, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system; and FR4 is defined as the C-terminal amino acid residue of CDR3, as defined by, for example, the Kabat numbering system, the IMGT numbering system, or the Chothia numbering system.

[0402] In some embodiments, including human ROR1 antibodies, the ROR1 antibodies described herein (e.g., antibodies such as monospecific or bispecific antibodies) comprise a VH region or VH domain. Alternatively, in some embodiments, including human ROR1 antibodies, the ROR1 antibodies described herein (e.g., antibodies such as monospecific or bispecific antibodies) comprise a VL region or VL domain. In some embodiments, including human ROR1 antibodies, the ROR1 antibodies described herein (e.g., antibodies such as monospecific or bispecific antibodies) have a combination of: (i) a VH domain or VH region; and (ii) a VL domain or VL region.

[0403] In some embodiments, the ROR1 antibody provided herein comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, the ROR1 antibody provided herein comprises VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein comprises VH, wherein the VH comprises the amino acid sequence of SEQ ID NO:25; and VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:26.

[0404] In some embodiments, the ROR1 provided herein comprises an amino acid sequence having a certain percentage of 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 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 (e.g., CDR, VH, or VL in Table 1) or any full-length antibody chain disclosed herein. In some embodiments, the ROR1 antibody provided herein comprises the CDR of any antibody or fragment thereof provided herein (e.g., in Table 1). In other embodiments, the ROR1 antibody provided herein comprises an amino acid sequence having a certain percentage of 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 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 (e.g., VH or VL in Table 1) or any full-length antibody chain as disclosed herein.

[0405] The determination of the percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. Non-limiting examples of mathematical algorithms used to compare two sequences are those in the following literature: Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs (Altschul et al., J. Mol. Biol. 215:403 (1990)). BLAST nucleotide searches can be performed using NBLAST nucleotide program parameter settings (e.g., score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using XBLAST program parameter settings (e.g., score 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, vacancy BLAST can be used, as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). In some embodiments, the percentage of identity between two sequences is calculated by dividing the number of residues that vary between the two sequences in the alignment (excluding or including conserved amino acid substitutions or degenerate nucleotide substitutions) by the number of residues of either: (i) the full length of the shorter sequence, (ii) the full length of the longer sequence, (iii) the average length of the two sequences, (iv) the total length of the non-vacancy portion in the alignment, (v) the alignment length excluding protrusions, or (vi) the alignment length including protrusions. As used herein with respect to sequence alignment, a protrusion refers to either end or both ends of the alignment, where residues in one sequence are considered not aligned with residues in the other sequence (e.g., vacancy). Alternatively, PSIBLAST can be used for iterative searching to detect distant relationships between molecules (as above). When using the BLAST, vacancy BLAST, and PSIBlast procedures, the default parameters of their respective procedures (e.g., XBLAST and NBLAST) can be used (see, for example, National Center for Biotechnology Information (NCBI), ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the one in Myers and Miller, CABIOS 4:11-17 (1998).Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, regardless of whether vacancies are allowed. When calculating the percentage of identity, only perfect matches are typically counted.

[0406] In some embodiments, the antibodies provided herein contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the antibodies containing said sequence retain their ability to bind to ROR1. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the reference amino acid sequence. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDR (e.g., in FR and / or constant regions).

[0407] 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) regions of the ROR1 antibody (including human ROR1 antibody) described herein may vary by one, two, three, four, five, or six amino acid positions, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., 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 of a CDR defined in Table 1 may be varied by shifting the N-terminal and / or C-terminal boundary of the CDR relative to the current CDR position by one, two, three, four, five, or six amino acids, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%). 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) regions of the ROR1 antibody (including human ROR1 antibody) described herein may vary (e.g., shorten or lengthen) by one, two, three, four, five, or more amino acids, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, such as 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 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 CDRs described by SEQ ID NO: 1-24, 36 and 37, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). In other embodiments, the 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 CDRs described by SEQ ID NO: 1-24, 36 and 37, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., 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 the 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 CDRs described by SEQ ID NO: 1-24, 36 and 37, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%). Alternatively, in some embodiments, the carboxyl terminus of the 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 CDRs described by SEQ ID NO: 1-24, 36 and 37, provided that binding to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., 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 determine whether binding to ROR1 (e.g., human ROR1) is maintained, such as the binding assays and conditions described in the "Examples" section herein.

[0408] In other embodiments, the ROR1 antibodies (including human ROR1 antibodies) that bind to ROR1 provided herein also contain conserved sequence modifications. Conserved sequence modifications, relating to peptides that are ROR1 antibodies (such as human ROR1 antibodies), include conserved amino acid substitutions, including those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. Thus, in some embodiments, a predicted non-essential amino acid residue in ROR1 is replaced by another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions that do not eliminate antigen binding and their encoding nucleotides are well known in the art (see, for example, 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 conserved sequence modifications described herein modify the amino acid sequence of the ROR1 antibody (including the human ROR1 antibody) 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 modification refers to a maximum of 1, 2, 3, 4, 5, or 6 amino acid substitutions on the CDR, such as those described in any of Table 1. Thus, for example, each such CDR may contain up to 5 conserved amino acid substitutions, for example up to (but not exceeding) 4 conserved amino acid substitutions, for example up to (but not exceeding) 3 conserved amino acid substitutions, for example up to (but not exceeding) 2 conserved amino acid substitutions, or no more than 1 conserved amino acid substitution. In some embodiments, the ROR1 antibody (including the human ROR1 antibody) contains one or more (including six) CDRs that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the CDRs of A27 (see, for example, Tables 1-2).

[0409] In some embodiments, the ROR1 antibody (including the human ROR1 antibody) contains VH and VL, which contain the same CDRs as those in A27 (see, for example, Tables 1-2). In some embodiments, the amino acid sequence modifications do not include any modifications within the SDR. In some embodiments, the amino acid sequence modifications do not include any modifications within the CDRs (such as CDR1, CDR2, CDR3, or any combination thereof). Alternatively or additionally, the amino acid sequence modifications are in the frame, constant region, and / or fragment crystallizable region (Fc).

[0410] In some embodiments, the antibodies or fragments provided herein comprise 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 with 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 with the amino acid sequence of SEQ ID NO:26, and the binding of the antibody or fragment to ROR1 (e.g., human ROR1) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%).

[0411] In some embodiments, functional epitopes can be localized, for example, by combining alanine scans, to identify amino acids in the ROR1 protein essential for interaction with the ROR1 antibody provided herein. In some embodiments, epitopes can be identified using the conformation and crystal structure of a ROR1 antibody bound to ROR1. In some embodiments, this disclosure provides an antibody that specifically binds to the same epitope as any ROR1 antibody provided herein.

[0412] For example, in some embodiments, the ROR1 antibody (e.g., an antibody) provided herein binds to the same epitopes as an anti-ROR1 antibody, which comprises VHCDR1, VH CDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:25; and VL CDR1, VLCDR2, and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein binds to the same epitopes as an anti-ROR1 antibody, which comprises VH containing the amino acid sequence of SEQ ID NO:25; and VL containing the amino acid sequence of SEQ ID NO:26.

[0413] In some embodiments, the ROR1 antibody provided herein further comprises an Fc or a variant thereof. In some embodiments, the Fc comprises the amino acid sequence shown in SEQ ID NO: 38. In other embodiments, the Fc variant is a silent Fc (sFc). In other 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, 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 the amino acid sequence shown in SEQ ID NO: 39. Alternatively, the variant Fc region has reduced potential immunogenicity. In other embodiments, the 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 at position Leu358 (L358) according to the EU numbering system (also referred to herein as “EEM” or “D356E / E357E / L358M”). Other suitable Fcs can be found, for example, in US20230071196 and US20220389055, each of which is incorporated herein by reference in its entirety.

[0414] In some implementations, the ROR1 antibody provided herein competitively and specifically binds to ROR1 with any of the anti-ROR1 antibodies or fragments thereof described herein.

[0415] In some embodiments, the ROR1 antibody provided herein competitively and specifically binds to ROR1 with an anti-ROR1 antibody comprising VH CDR1, VHCDR2, and VH CDR3 as shown in VH containing the amino acid sequence of SEQ ID NO:25; and VL CDR1, VL CDR2, and VLCDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the ROR1 antibody provided herein competitively and specifically binds to ROR1 with an anti-ROR1 antibody comprising VH containing the amino acid sequence of SEQ ID NO:25; and VL containing the amino acid sequence of SEQ ID NO:26.

[0416] In some embodiments, the ROR1 antibody comprises six CDRs of an antibody designated A27. In other embodiments, the ROR1 antibody comprises six CDRs as listed in a column of Table 1. In some embodiments, the ROR1 antibody comprises three CDRs of the heavy chain variable region as shown in SEQ ID NO:25 and three CDRs of the light chain variable region as shown in SEQ ID NO:26. In some embodiments, the...

Claims

1. An antibody-drug conjugate (ADC) of formula (I), said antibody-drug conjugate comprising: a. Antibodies that bind to tyrosine protein kinase receptor 1 (ROR1); and b. Two or more drugs conjugated to pyridazine-pyrrole via a linker. in: Ab represents the antibody that binds to ROR1; Z 1 Z 2 and Z 4 Each is CR independently 4 ; Z 3 It is CL B -W 2 ; R 1 R 2 R 3 and R 4 Each is selected from hydrogen and alkyl groups; L A It is the first connector, which includes: -(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 -, in: a, b, c, d, e, and f are each independently 0 or 1, with the constraint that 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 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 1 V 2 V 3 V 4 V 5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 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, heterocyclic and substituted heterocyclic; L B It is the second connector, which includes: -(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 -, in: g, h, i, j, k, l, and m are each independently 0 or 1, with the constraint that 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 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonyl (PABC), p-amino-benzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), acetal, hydrazine, disulfide and ester, wherein EDA is the ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each x is an integer from 1 to 12; V 7 V 8 V 9 V 10 V 11 V 12 and V 13 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-, where each q is an integer from 1 to 6; Each R 13 Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and Each R 15 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, heterocyclic and substituted heterocyclic; s is an integer from 1 to 10; W 1 It is the first medicine; and W 2 It is the second drug.

2. The ADC as described in claim 1, wherein: T 1 Selected from (C1-C) 12 )alkyl and substituted (C1-C 12 )alkyl; T 2 T 3 T 4 T 5 and T 6 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, hydrazine, and esters; and V 1 V 2 V 3 V 4 V 5 and V 6 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; (PEG) n yes ; EDA is an ethylenediamine moiety having the following structure: ； 4-Amino-piperidine (4AP) is ; Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; q is an integer from 1 to 6; r is 0 or 1; and y is an integer from 1 to 6.

3. The ADC as described in claim 1 or 2, wherein: T 1 It is (C1-C) 12 )alkyl and V 1 It is -CONH-; T 2 It is a substitute (C1-C) 12 )alkyl and V 2 It is -CO-; T 3 Yes (AA) p And V 3 It does not exist; T 4 It is PABC and V 4 It does not exist; p is an integer from 1 to 10; and a, b, c, and d are each 1; and e and f are both 0.

4. The ADC as described in any one of claims 1-3, wherein: T 7 It is a covalent bond; T 8 T 9 T 10 T 11 and T 12 Each is independently selected from covalent bonds, (C1-C) 12 )alkyl, substituted (C1-C 12 Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic, (EDA) w (PEG) n (AA) p 、-(CR 13 OH) x - 4-amino-piperidine (4AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal, hydrazine, and esters; and V 7 V 8 V 9 V 10 V 11 and V 12 Each can be independently selected from the following groups: 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)-, -SO2-, -SO2NR 15 -、-NR 15 SO2- and -P(O)OH-; in: (PEG) n yes , where n is an integer from 1 to 30; EDA is an ethylenediamine moiety having the following structure: , where y is an integer from 1 to 6 and r is 0 or 1; 4-Amino-piperidine (4AP) is ;and Each R 12 Independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, wherein any two adjacent R 12 All groups can be linked in a ring to form a piperazine ring; g, h, i, j, and k are each 1; and l and m are both 0.

5. The ADC as described in any one of claims 1-4, wherein: T 7 It does not exist and V 7 It is -NHCO-; T 8 It is (C1-C) 12 )alkyl and V 8 It is -CONH-; T 9 It is a substitute (C1-C) 12 )alkyl and V 9 It is -CO-; T 10 Yes (AA) p And V 10 It does not exist; T 11 It is PABC and V 11 It does not exist; p is an integer from 1 to 10; and g, h, i, j, and k are each 1; and l and m are both 0.

6. The ADC as claimed in any one of claims 1-5, wherein T 2 and T 9 One or both of them are -NHCO(PEG) t Substituted (C1-C6) alkylene groups, wherein (PEG) t yes And t is an integer from 2 to 10, arbitrarily 8.

7. The ADC as claimed in any one of claims 1-6, wherein T 3 and T 10 The p of one or both of them is 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, wherein the glycoside is optionally selected from glucuronide, galactoside, glucoside, mannoside, fucoidan, O-GlcNAc and O-GalNAc.

10. The ADC of any one of claims 1-9, wherein W 1 and W 2 One or both of them are camptothecin analogs, optionally said camptothecin analog is belotecone.

11. The ADC as claimed in any one of claims 1-10, wherein W 1 and W 2 Each of them is belotecone.

12. An ADC represented by equation (II): in: 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: heavy chain variable region (VH) complementarity-determining region 1 (CDR1), VH complementarity-determining region 2 (CDR2) and VH complementarity-determining region 3 (CDR3) as shown in VH containing the amino acid sequence of SEQ ID NO:25; and light chain variable region (VL) CDR1, VL CDR2 and VL CDR3 as shown in VL containing the amino acid sequence of SEQ ID NO:

26.

17. The ADC of any one of claims 1-16, wherein the antibody Ab comprises: (a) VH, wherein VH comprises: (1) VH CDR1, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5 and 36; (2) VH CDR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 7, 8, 9 and 10; and (3) VH CDR3, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14 and 37; and (b) VL, wherein the VL comprises: (1) VL CDR1, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 16, 17 and 18; (2) VL CDR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, 20 and 21; and (3) VL CDR3, which contains an amino acid sequence selected from the group consisting of SEQ ID NO: 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) VH, which comprises VH CDR1 containing the amino acid sequence of SEQ ID NO:1; VH CDR2 containing the amino acid sequence of SEQ ID NO:6; VH CDR3 containing the amino acid sequence of SEQ ID NO:11; and VL, which comprises VLCDR1 containing the amino acid sequence of SEQ ID NO:15; VL CDR2 containing the amino acid sequence of SEQ ID NO:19; and VL CDR3 containing the amino acid sequence of SEQ ID NO:22; (ii) VH, which comprises VH CDR1 containing the amino acid sequence of SEQ ID NO:2; VH CDR2 containing the amino acid sequence of SEQ ID NO:7; VH CDR3 containing the amino acid sequence of SEQ ID NO:12; and VL, which comprises VLCDR1 containing the amino acid sequence of SEQ ID NO:16; VL CDR2 containing the amino acid sequence of SEQ ID NO:20; and VL CDR3 containing the amino acid sequence of SEQ ID NO:22; (iii) VH, wherein the VH comprises VH CDR1, which comprises the amino acid sequence of SEQ ID NO:3; VH CDR2, which comprises the amino acid sequence of SEQ ID NO:6; VH CDR3, which comprises the amino acid sequence of SEQ ID NO:11; and VL, wherein the VL comprises VL CDR1, which comprises the amino acid sequence of SEQ ID NO:15; VL CDR2, which comprises the amino acid sequence of SEQ ID NO:19; and VL CDR3, which comprises the amino acid sequence of SEQ ID NO:22; (iv) VH, wherein VH comprises VH CDR1, which comprises the amino acid sequence of SEQ ID NO:4; VH CDR2, which comprises the amino acid sequence of SEQ ID NO:8; VH CDR3, which comprises the amino acid sequence of SEQ ID NO:13; and VL, wherein VL comprises VLCDR1, which comprises the amino acid sequence of SEQ ID NO:17; VL CDR2, which comprises the amino acid sequence of SEQ ID NO:20; and VL CDR3, which comprises the amino acid sequence of SEQ ID NO:23; (v) VH, which comprises VH CDR1 containing the amino acid sequence of SEQ ID NO:5; VH CDR2 containing the amino acid sequence of SEQ ID NO:9; VH CDR3 containing the amino acid sequence of SEQ ID NO:14; and VL, which comprises VLCDR1 containing the amino acid sequence of SEQ ID NO:18; VL CDR2 containing the amino acid sequence of SEQ ID NO:21; and VL CDR3 containing the amino acid sequence of SEQ ID NO:24; (vi) VH, wherein VH comprises VH CDR1, which contains the amino acid sequence of SEQ ID NO:1; VH CDR2, which contains the amino acid sequence of SEQ ID NO:10; VH CDR3, which contains the amino acid sequence of SEQ ID NO:11; and VL, wherein VL comprises VLCDR1, which contains the amino acid sequence of SEQ ID NO:15; VL CDR2, which contains the amino acid sequence of SEQ ID NO:19; and VL CDR3, which contains the amino acid sequence of SEQ ID NO:22; or (vii) VH, which comprises VH CDR1 containing the amino acid sequence of SEQ ID NO:36; VH CDR2 containing the amino acid sequence of SEQ ID NO:10; VH CDR3 containing the amino acid sequence of SEQ ID NO:37; and VL, which comprises VL CDR1 containing the amino acid sequence of SEQ ID NO:15; VL CDR2 containing the amino acid sequence of SEQ ID NO:19; and VL CDR3 containing 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 frame 1 (FR1), a frame 2 (FR2), a frame 3 (FR3), and / or a frame 4 (FR4) sequence.

20. The ADC of any one of claims 1-19, wherein the antibody Ab further comprises a human frame sequence, optionally FR1, FR2, FR3 and / or FR4 as shown in any one of SEQ ID NO: 25 and 26.

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

26.

22. The ADC of any one of claims 1-21, wherein the antibody Ab comprises the sequence of formula (VIII). in: fGly' is an amino acid residue that is coupled to the drug via a linker; Z 20 It is a proline (P) or alanine (A) residue; Z 30 It 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). X 1 It may or may not be present, and when present it can be any amino acid residue, the limitation being that when the sequence of formula (VIII) is located at the N-terminus of the antibody Ab, X 1 Existence; and X 2 and X 3 It can be any amino acid residue independently. Optionally, 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')TPSR (SEQ ID NO:159), L(fGly')TPSK (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')APSK (SEQ ID NO:159), L(fGly')TPSK (SEQ ID NO:150), L(fGly')TPSR (SEQ ID NO:151), L(fGly')TPSR (SEQ ID NO:152), L(fGly' 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); In addition, the sequence of said formula (VIII) optionally includes 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 IgG1κ 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, the variant 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 Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a dual variable region antibody, a single variable region antibody, a linear antibody, a V region antibody, 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 with a diagnostic agent, a detectable agent, or a therapeutic agent, optionally wherein the therapeutic agent is a chemotherapeutic agent, a cytotoxic agent, or a drug.

30. The ADC of any one of claims 1-27, wherein the antibody Ab is a multispecific antibody, optionally a bispecific antibody.

31. A pharmaceutical composition comprising an ADC as described in any one of claims 1-30 and a pharmaceutically acceptable excipient.

32. The pharmaceutical composition according to claim 31, characterized in that... The antibody-drug ratio (DAR) for ADC drugs is approximately 1 to approximately 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 suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of an ADC as described in any one of claims 1-30 or a pharmaceutical composition as described in any one of claims 31-36.

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

39. The method of any one 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's sarcoma, chronic lymphocytic leukemia, mantle cell lymphoma, B-ALL, hematologic malignancies, prostate cancer, colon cancer, kidney 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.

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