B7H3 antibody drug conjugates

Abstract

The antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof capable of specifically binding to human B7H3 and a cytotoxic agent such as an exetecan analog. Pharmaceutical compositions include the antibody-drug conjugate. A method of using the antibody-drug conjugate is to treat 4Ig-B7H3-positive cancer. Provided herein is an antibody-drug conjugate having the formula (I): Ab-(L-(D)m)n(I), or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof capable of specifically binding to human B7H3, L is a linker, D is a residue of a cytotoxic agent, m is an integer from 1 to 8, and n is 1 to 10.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to PCT Application No. PCT / CN2022 / 143248, filed December 29, 2022, entitled "Anti-B7H3 Antibodies and Methods of Use," and PCT / CN2022 / 143246, filed December 29, 2022, entitled "Anti-B7H3 Antibodies and Methods of Use," each of which is incorporated herein by reference in its entirety.

[0002] 2. Electronic Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on December 27, 2023, is named "01368-0060-00PCT.xml" and is 446,058 bytes in size. The Sequence Listing contained in this .XML file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates generally to anti-B7H3 antibody drug conjugates and their use in the treatment of cancer. [Background technology]

[0004] B7 homology 3 protein (B7H3) (also known as CD276, B7-H3, and B7RP-2, referred to herein as "B7H3") is a type I transmembrane glycoprotein of the immunoglobulin superfamily. Human B7H3 contains a putative signal peptide, V-like and C-like Ig domains, a transmembrane region, and a cytoplasmic domain. Exon duplication in humans results in the expression of two B7-H3 isoforms, each with a single IgV-IgC-like domain (the 2IgB7-H3 isoform) or an IgV-IgC-IgV-IgC-like domain (the 4IgB7-H3 isoform), both of which contain several conserved cysteine ​​residues. The predominant B7H3 isoform in human tissues and cell lines is the 4IgB7-H3 isoform (Steinberger et al., J. Immunol. 172(4):2352-9 (2004)).

[0005] B7H3 has been reported to have both costimulatory and coinhibitory signaling functions (see, e.g., Chapoval et al., Nat. Immunol. 2: 269-74 (2001); Suh et al., Nat. Immunol. 4: 899-906 (2003); Prasad et al., J. Immunol. 173: 2500-6 (2004); and Wang et al., Eur. J. Immunol. 35: 428-38 (2005)). As an example of the costimulatory function of B7H3, in vitro studies have shown that B7H3 can increase the proliferation of cytotoxic T lymphocytes (CTLs) and upregulate the production of interferon gamma (IFN-γ) in the presence of anti-CD3 antibodies, mimicking T cell receptor signaling (Chapoval et al., 2001). Furthermore, in vivo studies using cardiac allografts in B7H3- / - mice demonstrated decreased production of key cytokine, chemokine, and chemokine receptor mRNA transcripts (e.g., IL-2, IFN-γ, monocyte chemoattractant protein-1 (MCP-1), and IFN-inducible protein (IP)-10) compared with wild-type controls (Wang et al., 2005). In contrast, B7H3 co-inhibitory function has been observed in mice, where, for example, B7H3 protein inhibits T cell activation and effector cytokine production (Suh et al., 2003). While no ligand has been identified for human B7H3, mouse B7H3 has been found to bind to triggering receptor expressed on myeloid cells (TREM-)-like transcript 2 (TLT-2), a regulator of adaptive and innate immune cell responses. Binding of mouse B7H3 to TLT-2 on CD8+ T cells enhances T cell effector functions such as proliferation, cytotoxicity, and cytokine production (Hashiguchi et al., Proc. Nat'l. Acad. Sci. USA 105(30):10495-500(2008)).

[0006] B7H3 is not constitutively expressed in many immune cells (e.g., natural killer (NK) cells, T cells, and antigen-presenting cells (APCs)), but its expression can be inducible. Furthermore, B7H3 expression is not limited to immune cells. B7H3 transcripts are expressed in various human tissues, including the colon, heart, liver, placenta, prostate, small intestine, testis, and uterus, as well as osteoblasts, fibroblasts, epithelial cells, and other non-lymphoid cells, potentially exhibiting immunological and non-immunological functions (Nygren et al. Front Biosci. 3:989-93 (2011)). However, protein expression in normal tissues is typically maintained at low levels and is therefore likely subject to post-transcriptional regulation.

[0007] B7H3 is also expressed in a variety of human cancers, including prostate cancer, clear cell renal cell carcinoma, glioma, melanoma, lung cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, pancreatic cancer, gastric cancer, acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), ovarian cancer, colorectal cancer, colon cancer, renal cancer, hepatocellular carcinoma, kidney cancer, head and neck cancer, hypopharyngeal squamous cell carcinoma, glioblastoma, neuroblastoma, breast cancer, endometrial cancer, and urothelial cell carcinoma. The role of B7H3 in cancer cells is unclear, but its expression may regulate signaling events that can protect cancer cells from innate and adaptive immune responses. For example, B7H3 is overexpressed in high-grade prostatic intraepithelial tumors and prostatic adenomas, and high levels of B7H3 expression in these cancer cells are associated with an increased risk of cancer progression after surgery (Roth et al. Cancer Res. 67(16):7893-900(2007)). Furthermore, tumor B7H3 expression in NSCLC is inversely correlated with the number of tumor-infiltrating lymphocytes and significantly correlated with lymph node metastasis (Sun et al. Lung Cancer 53(2):143-51(2006)). The level of circulating soluble B7H3 in NSCLC patients is also associated with higher tumor stage, tumor size, lymph node metastasis, and distant metastasis (Yamato et al., Br. J. Cancer 101(10):1709-16(2009)).

[0008] B7H3 may also play an important role in T cell-mediated antitumor responses in a context-dependent manner. For example, gastric cancer tumor cell expression of B7H3 positively correlates with survival time, depth of invasion, and histological type (Wu et al., World J. Gastroenterol. 12(3):457-9(2006)). Furthermore, high expression of B7H3 on pancreatic tumor cells was associated with patient survival after surgical resection and significantly correlated with the number of tumor-infiltrating CD8+ T cells (Loos et al., BMC Cancer 9:463(2009)).

[0009] Antibody drug conjugates (ADCs) represent a relatively new class of therapeutic agents that comprise an antibody conjugated to a cytotoxic drug via a chemical linker. The therapeutic concept of ADCs is to combine the binding capacity of an antibody with a drug, where the antibody is used to deliver the drug to tumor cells by binding to target surface antigens, including target surface antigens that are overexpressed in tumor cells. There remains a need in the art for anti-B7H3 ADCs that can be used for therapeutic purposes, for example, in the treatment of 4Ig-B7H3-positive cancers. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Steinberger et al., J.Immunol.172(4):2352-9(2004) [Non-patent document 2] Chapoval et al., Nat. Immunol. 2: 269-74 (2001) [Non-patent document 3] Suh et al., Nat. Immunol. 4: 899-906 (2003) [Non-patent document 4] Prasad et al., J. Immunol. 173: 2500-6 (2004) [Non-patent document 5] Wang et al., Eur. J. Immunol. 35: 428-38 (2005) [Non-patent document 6] Hashiguchi et al.,Proc.Nat'l.Acad.Sci.USA105(30):10495-500(2008) [Non-Patent Document 7] Nygren et al.Front Biosci.3:989-93(2011) [Non-patent document 8] Roth et al.Cancer Res.67(16):7893-900(2007) [Non-Patent Document 9] Sun et al.Lung Cancer 53(2):143-51(2006) [Non-Patent Document 10] Yamato et al.,Br. J. Cancer 101(10):1709-16 (2009) [Non-Patent Document 11] Wu et al.,World J.Gastroenterol.12(3):457-9(2006) [Non-Patent Document 12] Loos et al.,BMC Cancer 9:463(2009) Summary of the Invention [Means for solving the problem]

[0011] Provided herein is an antibody drug conjugate having the formula (I): Ab-(L-(D)m)n(I), or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof capable of specifically binding to human B7H3; L is a linker, D is a residue of a cytotoxic agent; m is an integer from 1 to 8, and n is 1 to 10.

[0012] In one embodiment, m is 1.

[0013] In one embodiment, n is 3 to 10, for example, about 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, n is about 8.

[0014] In one embodiment, the antibody drug conjugate has formula (II): [ka] or Formula (IIo): [ka] or Formula (IIoo): [ka] or a pharmaceutically acceptable salt thereof, wherein Su is a hydrophilic residue.

[0015] In one embodiment, Su is [ka] is.

[0016] In one embodiment, the antibody drug conjugate has formula (III): [ka] or Formula (IIIo) [ka] or formula (IIIoo): [ka] or a pharmaceutically acceptable salt thereof, wherein Su is a hydrophilic residue.

[0017] In one embodiment, the antibody drug conjugate has formula (III): [ka] or Formula (IIIo) [ka] or a pharmaceutically acceptable salt thereof, wherein Su is a hydrophilic residue.

[0018] In one embodiment, Su is [ka] is.

[0019] In one embodiment, D is [ka] wherein: Y is -AB-C'-D'-*, where * indicates the bond connecting D to the antibody drug conjugate; A is bond, CR 1 R 2 , or NR 1 and B is a bond, —C(═O)—, or —C(═O)O—; C' is a bond or a divalent group, where the divalent group is an unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH, or O; R 1 and R 2 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 1 and R 2 taken together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 3 and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl.

[0020] In one embodiment, D is [ka] and In the formula, R 7 and R 8 are each independently hydrogen, halogen, or alkyl.

[0021] In one embodiment, D is [ka] is.

[0022] In one embodiment, D is [ka] is.

[0023] In one embodiment, the antibody drug conjugate has one of the following formulas, or a tautomer, stereoisomer, pharmaceutically acceptable salt, or solvate thereof: [ka] [ka] [ka] [ka] [ka] [ka]

[0024] In one embodiment, the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (viii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6, or (ix) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 300, (b) an HCDR2 of SEQ ID NO: 1700, and (c) an HCDR3 of SEQ ID NO: 500, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 600, (e) an LCDR2 of SEQ ID NO: 700, and (f) an LCDR3 of SEQ ID NO: 800.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 801). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 801).

[0026] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the IgV1 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the IgV2 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and binds to both the IgV1 domain and the IgV2 domain of human 4Ig-B7H3.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 145-238 of human 4Ig-B7H3 (SEQ ID NO: 801). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 363-456 of human 4Ig-B7H3 (SEQ ID NO: 801). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to the IgC1 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to the IgC2 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to the IgC1 or IgC2 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope that does not overlap with the epitope of the reference antibody DS-7300. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows ADC direct killing against the NCI-H1650 cell line. [Figure 2] 1 shows ADC direct killing against the Capan-1 cell line. [Figure 3] 1 shows ADC direct killing against MDA-MB-453 (B7H3 negative) cell line. [Figure 4]1 shows ADC direct killing against the NCI-H1650 cell line. [Figure 5] 1 shows ADC direct killing against the Capan-1 cell line. [Figure 6] 1 shows ADC direct killing against MDA-MB-453 (B7H3 negative) cell line. [Figure 7] 1 shows ADC direct killing against the NCI-H1650 cell line. [Figure 8] 1 shows ADC direct killing against the Capan-1 cell line. [Figure 9] 1 shows ADC direct killing against MDA-MB-453 (B7H3 negative) cell line. [Figure 10] 1 shows ADC direct killing against the NCI-H1650 cell line. [Figure 11] 1 shows ADC direct killing against the NCI-H1048 cell line. [Figure 12] 1 shows ADC direct killing against MDA-MB-453 (B7H3 negative) cell line. [Figure 13] 1 shows ADC direct killing against the NCI-H1650 cell line. [Figure 14] 1 shows ADC direct killing against the NCI-H1048 cell line. [Figure 15] 1 shows ADC direct killing against MDA-MB-453 (B7H3 negative) cell line. [Figure 16] 1 shows ADC bystander killing against NCI-H358 / MDA-MB-453(nano-Luc) in a co-culture assay. [Figure 17] We demonstrate ADC direct killing of MDA-MB-453 (nano-Luc) in a cell culture assay. In this system, the tested ADCs do not exhibit stronger nonspecific killing than the isotype ADC (isotype antibody-GGFG-DXd-DAR8 conjugate). [Figure 18] 1 shows ADC bystander killing against NCI-H358 / MDA-MB-453(nano-Luc) in a co-culture assay. [Figure 19]We demonstrate ADC direct killing of MDA-MB-453 (nano-Luc) in a cell culture assay. In this system, the tested ADCs do not exhibit stronger nonspecific killing than the isotype ADC (isotype antibody-GGFG-DXd-DAR8 conjugate). [Figure 20] 1 shows ADC bystander killing against NCI-H358 / MDA-MB-453(nano-Luc) in a co-culture assay. [Figure 21] This shows ADC direct killing of MDA-MB-453 (nano-Luc) in a co-culture assay. In this system, the tested ADCs do not exhibit stronger nonspecific killing than the isotype ADC (isotype antibody-GGFG-DXd-DAR8 conjugate). [Figure 22] 1 shows ADC bystander killing against NCI-H358 / MDA-MB-453(nano-Luc) in a co-culture assay. [Figure 23] This shows ADC direct killing of MDA-MB-453 (nano-Luc) in a co-culture assay. In this system, the tested ADCs do not exhibit stronger nonspecific killing than the isotype ADC (isotype antibody-GGFG-DXd-DAR8 conjugate). [Figure 24] 1 shows the mouse plasma stability of ADC3-1. [Figure 25] 1 shows the human plasma stability of ADC3-1. [Figure 26] 1 shows the mouse plasma stability of ADC3-2. [Figure 27] 1 shows the human plasma stability of ADC3-2. [Figure 28] 1 shows the plasma stability of ADC3-4 in mice. [Figure 29] 1 shows the human plasma stability of ADC3-4. [Figure 30] 1 shows the HIC-HPLC profile of ADC3-A (HIC method 2). [Figure 31A] 1 shows the mean results of the ADC efficacy studies of ADC3-1, ADC3-3, ADC3-14, and ADC3-15. [Figure 31B]Figure 31A shows the results of the vehicle group in the ADC efficacy study. [Figure 31C] Figure 31A shows the results of the ADC3-A (3mpk) group in the ADC efficacy study. [Figure 31D] Figure 31A shows the results of the ADC3-A (10 mpk) group in the ADC efficacy study. [Figure 31E] Figure 31A shows the results of the ADC3-1 (3mpk) group in the ADC efficacy study. [Figure 31F] Figure 31A shows the results of the ADC3-1 (10 mpk) group in the ADC efficacy study. [Figure 31G] Figure 31A shows the results of the ADC3-3 (3mpk) group in the ADC efficacy study. [Figure 31H] Figure 31A shows the results of the ADC3-3 (10 mpk) group in the ADC efficacy study. [Figure 31I] Figure 31A shows the results of the ADC3-14 (3mpk) group in the ADC efficacy study. [Figure 31J] Figure 31A shows the results of the ADC3-14 (10 mpk) group in the ADC efficacy study. [Figure 31K] Figure 31A shows the results of the ADC3-15 (3mpk) group in the ADC efficacy study. [Figure 31L] Figure 31A shows the results of the ADC3-15 (10 mpk) group in the ADC efficacy study. [Figure 32A] Average results of ADC efficacy studies for ADC3-2, ADC3-3, ADC3-4, ADC3-5, and ADC3-6 are shown. [Figure 32B] Figure 32A shows the results of the vehicle group in the ADC efficacy study. [Figure 32C] Figure 32A shows the results of the ADC3-A (10 mpk) group in the ADC efficacy study. [Figure 32D] Figure 32A shows the results of the ADC3-2 (3mpk) group in the ADC efficacy study. [Figure 32E] Figure 32A shows the results of the ADC3-4 (3mpk) group in the ADC efficacy study. [Figure 32F] Figure 32A shows the results of the ADC3-5 (3mpk) group in the ADC efficacy study. [Figure 32G] Figure 32A shows the results of the ADC3-6 (3mpk) group in the ADC efficacy study. [Figure 33A] Average results of the ADC efficacy studies of ADC3-8 and ADC3-9 are shown. [Figure 33B] Figure 33A shows the results of the vehicle group in the ADC efficacy study. [Figure 33C] Figure 33A shows the results of the ADC3-A (10 mpk) group in the ADC efficacy study. [Figure 33D] Figure 33A shows the results of the ADC3-8 (3mpk) group in the ADC efficacy study. [Figure 33E] Figure 33A shows the results of the ADC3-9 (3mpk) group in the ADC efficacy study. [Figure 34A] 1 shows the mean results of the ADC efficacy studies of ADC3-10, ADC3-11, ADC3-12, and ADC3-13. [Figure 34B] Figure 34A shows the results of the vehicle group in the ADC efficacy study. [Figure 34C] Figure 34A shows the results of the ADC3-A (10 mpk) group in the ADC efficacy study. [Figure 34D] Figure 34A shows the results of the ADC3-10 (3mpk) group in the ADC efficacy study. [Figure 34E] Figure 34A shows the results of the ADC3-11 (3mpk) group in the ADC efficacy study. [Figure 34F] Figure 34A shows the results of the ADC3-12 (3mpk) group in the ADC efficacy study. [Figure 34G] Figure 34A shows the results of the ADC3-13 (3mpk) group in the ADC efficacy study. [Figure 35A] 1 shows the mean results of the ADC efficacy studies of ADC3-1, ADC3-3, ADC3-14, and ADC3-15. [Figure 35B]Figure 35A shows the results of the vehicle group in the ADC efficacy study. [Figure 35C] Figure 35A shows the results of the ADC3-A (3mpk) group in the ADC efficacy study. [Figure 35D] Figure 35A shows the results of the ADC3-A (10 mpk) group in the ADC efficacy study. [Figure 35E] Figure 35A shows the results of the ADC3-1 (3mpk) group in the ADC efficacy study. [Figure 35F] Figure 35A shows the results of the ADC3-1 (10 mpk) group in the ADC efficacy study. [Figure 35G] Figure 35A shows the results of the ADC3-3 (3mpk) group in the ADC efficacy study. [Figure 35H] Figure 35A shows the results of the ADC3-3 (10 mpk) group in the ADC efficacy study. [Figure 35I] Figure 35A shows the results of the ADC3-14 (3mpk) group in the ADC efficacy study. [Figure 35J] Figure 35A shows the results of the ADC3-14 (10 mpk) group in the ADC efficacy study. [Figure 35K] Figure 35A shows the results of the ADC3-15 (3mpk) group in the ADC efficacy study. [Figure 35L] Figure 35A shows the results of the ADC3-15 (10 mpk) group in the ADC efficacy study. DETAILED DESCRIPTION OF THE INVENTION

[0029] Provided herein are compounds, compositions, ADCs, and methods useful for treating a wide variety of human cancers.

[0030] It is understood that, within the scope of the present disclosure, the disclosure is not limited to the particular methods and / or experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0031] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0032] 1.Definition When referring to the compounds provided herein, the following terms have the following meanings unless otherwise specified. 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 disclosure pertains. In the event that there are multiple definitions for terms provided herein, these definitions prevail unless otherwise stated.

[0033] As used herein, and in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly indicates otherwise.

[0034] As used herein, unless otherwise indicated, the terms "about" and "approximately," when used in connection with an amount or weight percentage of a component of a composition, refer to an amount or weight percentage that would be recognized by one of ordinary skill in the art to provide an equivalent pharmacological effect to that obtained from the specified amount or weight percentage. In certain embodiments, the terms "about" and "approximately," when used in this context, contemplate an amount or weight percentage that is within 30%, 20%, 15%, 10%, or 5% of the specified amount or weight percentage.

[0035] As used herein, unless otherwise indicated, the terms "about" and "approximately," when used in connection with a numerical value or range of values ​​provided to characterize a particular solid form, e.g., a particular temperature or temperature range, e.g., one describing melting, dehydration, desolvation, or glass transition temperature, mass change, e.g., mass change as a function of temperature or humidity, solvent or water content (e.g., in terms of mass or percentage), or peak position, e.g., in analysis by IR or Raman spectroscopy or XRPD, indicate that the value or range of values ​​may deviate to an extent that would be considered reasonable by one of ordinary skill in the art and still describe the solid form. Techniques for characterizing crystalline forms and amorphous solids include, but are not limited to, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), single crystal X-ray diffraction, vibrational spectroscopy such as infrared (IR) and Raman spectroscopy, solid-state and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility studies, and dissolution studies. In certain embodiments, the terms "about" and "approximately" when used in this context indicate that a numerical value or range of values ​​may vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the recited value or range of values. For example, in some embodiments, the value of an XRPD peak position can vary by up to ±0.2 degrees 2θ (or ±0.2 degrees 2θ) and still describe a particular XRPD peak.

[0036] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0037] As used herein, the terms "administration" and "administering," when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid when the fluid is in contact with the cell.

[0038] The term "subject" or "patient" as used herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, primate), and most preferably a human (e.g., a patient having or at risk of having a disorder described herein).

[0039] In one aspect, "treating" any disease or disorder refers to ameliorating the disease or disorder (i.e., delaying, preventing, or reducing the onset of the disease or at least one of its clinical symptoms). In another aspect, "treat," "treating," or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another aspect, "treat," "treating," or "treatment" refers to modulating the disease or disorder either physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both.

[0040] The term "effective amount" in reference to a compound means an amount that can alleviate the symptoms, in whole or in part, or that can slow or stop the further progression or worsening of those symptoms. As will be apparent to one skilled in the art, it should be expected that the effective amount of the compounds disclosed herein may vary depending on the severity of the indication being treated.

[0041] The term "antibody," as used herein, refers to a polypeptide of the immunoglobulin family that can bind to a corresponding antigen in a reversible and specific manner other than by covalent bonds. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or Vκ) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0042] The locations of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, AbM, and IMGT (e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997); Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003).

[0043] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0044] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a large number of different antibodies with different amino acid sequences within their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be obtained by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; U.S. Patent No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any immunoglobulin class, such as IgG, IgM, IgD, IgE, IgA, etc., and any subclass thereof, e.g., IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies can be cultivated in vitro or in vivo. High-titer monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites fluid containing high concentrations of the desired antibody. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluid or from the culture supernatant using column chromatography techniques well known to those skilled in the art.

[0045] Unless otherwise specified, "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to the antigen bound by the full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, bispecific antibodies, linear antibodies, single-chain antibody molecules such as single-chain Fv (ScFv), nanobodies and antibodies formed from antibody fragments, and bicyclic peptides (Hurov, K. et al., 2021. Journal for ImmunoTherapy of Cancer, 9(11)).

[0046] As used herein, an antibody or antigen-binding antibody fragment "specifically binds" or "selectively binds" to an antigen (e.g., a protein) means that the antibody exhibits preferential binding to its target relative to other proteins, although this specificity does not require absolute binding specificity. A "specific" or "selective" binding reaction determines the presence of an antigen in a heterogeneous population of proteins and other biologics, for example, in a blood, serum, plasma, or tissue sample. Thus, under certain designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least two-fold stronger than background levels and does not specifically bind in significant amounts to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, an antibody or antigen-binding fragment thereof specifically binds to a particular antigen at least ten-fold stronger than background levels of binding and does not specifically bind in significant amounts to other antigens present in the sample.

[0047] The term "human antibody" herein refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse glycosylation if produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse immunoglobulin protein sequences or only rat immunoglobulin protein sequences, respectively.

[0048] The terms "humanized" or "humanized antibody" refer to forms of antibodies containing sequences derived from non-human (e.g., murine) and human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all FR regions being those of a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). When necessary to distinguish a humanized antibody from a rodent parent antibody, the prefix "hum," "hu," "Hu," or "h" is added to the name of the antibody clone. Humanized forms of rodent antibodies generally contain the same CDR sequences of the rodent parent antibody but can include certain amino acid substitutions to enhance affinity, increase the stability of the humanized antibody, remove post-translational modifications, or for other reasons.

[0049] As used herein, the term "knob-into-hole" technology refers to amino acids that together direct the pairing of two polypeptides, either in vitro or in vivo, by introducing a spatial protuberance (knob) in one polypeptide and a socket or cavity (hole) in the other polypeptide (at the interface where they interact). For example, knob-into-hole technology can be used to engineer the Fc:Fc binding interface of an antibody, C L :C H I interface, or V H / V LIn some embodiments, knob-into-hole amino acids have been introduced into the antibody's interface (see, e.g., US2011 / 0287009, US2007 / 0178552, WO96 / 027011, WO98 / 050431, and Zhu et al., 1997, Protein Science 6:781-788). In some embodiments, knob-into-hole amino acids ensure proper pairing of two different heavy chains together during antibody production. For example, antibodies with knob-into-hole amino acids in their Fc region may further comprise a single variable domain linked to each Fc region, or may further comprise a different heavy chain variable domain paired with a similar or different light chain variable domain. Knob-into-hole technology can also be used with VH or VL regions to ensure proper pairing.

[0050] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as starting points for searches to find longer HSPs containing them. Word hits are extended outward along each end of each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted if the cumulative alignment score falls by an amount X from the maximum achieved value, if the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or if either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLAST program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, M=5, N=−4, and a comparison of both strands.

[0051] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two sequences of nucleotides or amino acids will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0052] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17, (1988), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), which has been incorporated into the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0053] In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-B7H3 antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0054] The term "human 4Ig-B7H3" refers to the 4Ig isoform of the type I transmembrane protein B7H3 in humans. Human 4Ig-B7H3 (Uniprot Q5ZPR3) has the following sequence: >sp|Q5ZPR3|CD276_HUMAN CD276 antigen OS=Homo sapiens OX=9606 GN=CD276 PE=1 SV=1 MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA (SEQ ID NO: 802)

[0055] The term "toxin" or "payload" or "cytotoxic agent" is used herein to refer to a molecule that inhibits or reduces the expression of a molecule in a cell, inhibits or reduces the function of a cell, induces apoptosis of a cell, and / or causes cell death. This term includes toxins (including fragments and / or variants thereof), such as radioisotopes, chemotherapeutic agents, and small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin. Examples of cytotoxic agents include auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), auromycin, maytansinoids, pyrrolobenzodiazepines (PBDs), ricin, ricin A chain, comblastatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, and the like. Examples of suitable exotoxins include, but are not limited to, thiatoxin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogenin, letostrictocin, phenomycin, enomycin, curicin, crotin, and calicheamicin, as well as radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and Lu177.

[0056] As used herein, the term "residue" refers to the chemical moiety in a compound that remains after a chemical reaction. For example, the term "amino acid residue" or "N-alkylamino acid residue" refers to the product of amide or peptide coupling of an amino acid or an N-alkylamino acid with a suitable coupling partner, e.g., a water molecule is expelled after amide or peptide coupling of the amino acid or N-alkylamino acid, resulting in the incorporation of the amino acid residue or N-alkylamino acid residue into the product.

[0057] As used herein, a "sugar" or "sugar group" or "sugar residue" refers to a carbohydrate moiety that may contain a 3-carbon (triose) unit, a 4-carbon (tetrose) unit, a 5-carbon (pentose) unit, a 6-carbon (hexose) unit, a 7-carbon (heptose) unit, or a combination thereof, and may be a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, oligosaccharide, or any other polysaccharide. In some cases, a "sugar" or "sugar group" or "sugar residue" includes a furanose (e.g., ribofuranose, fructofuranose), or a pyranose (e.g., glucopyranose, galactopyranose), or a combination thereof. In some cases, a "sugar" or "sugar group" or "sugar residue" includes an aldose or ketose, or a combination thereof. Non-limiting examples of monosaccharides include ribose, deoxyribose, xylose, arabinose, glucose, mannose, galactose, and fructose. Non-limiting examples of disaccharides include sucrose, maltose, lactose, lactulose, and trehalose. Other "sugars" or "sugar groups" or "sugar residues" include polysaccharides and / or oligosaccharides, including, but not limited to, amylose, amylopectin, glycogen, inulin, and cellulose. In some cases, the "sugar" or "sugar group" or "sugar residue" is an amino sugar. In some cases, the "sugar" or "sugar group" or "sugar residue" is a glucamine residue (1-amino-1-deoxy-D-glucitol), which is linked to the rest of the molecule via its amino group to form an amide bond with the rest of the molecule (i.e., a glucamide).

[0058] As used herein, "binding agent" refers to any molecule, e.g., an antibody, that can specifically bind to a given binding partner, e.g., an antigen.

[0059] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, as well as a side chain (R group) specific to each amino acid. Amino acids can be proteinogenic or non-proteinogenic. "Proteinogenic" means that the amino acid is one of the 20 naturally occurring amino acids found in proteins. Proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. "Non-proteinogenic" means that the amino acid is either not naturally found in proteins or is not produced directly by cellular machinery (e.g., is a product of post-translational modification). Non-limiting examples of non-proteinogenic amino acids include gamma-aminobutyric acid (GABA), taurine (2-aminoethanesulfonic acid), theanine (L-γ-glutamylethylamide), hydroxyproline, beta-alanine, ornithine, and citrulline.

[0060] As used herein, "peptide," in its various grammatical forms, is defined in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The subunits may be linked by peptide bonds or by other bonds, e.g., esters, ethers, etc. As used herein, the term "amino acid" refers to natural and / or non-natural, proteinogenic, non-proteinogenic, or synthetic amino acids, e.g., glycine and both its D- and L-optical isomers, as well as amino acid analogs and peptidomimetics. Peptide chains, when short, e.g., two, three, or more amino acids, are commonly referred to as oligopeptides. When peptide chains are longer, peptides are typically referred to as polypeptides or proteins. Full-length proteins, analogs, variants, and fragments thereof are encompassed by definition. The term also includes post-expression modifications of polypeptides, e.g., glycosylation, acetylation, phosphorylation, etc. Furthermore, due to the presence of ionizable amino and carboxyl groups in the molecule, certain peptides may be obtained as acid or base salts or in neutral form. Peptides can be obtained directly from a source organism, or can be produced recombinantly or synthetically.

[0061] The amino acid sequence of an antibody can be numbered using any known numbering scheme, such as that described in Kabat et al., ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme). Unless otherwise specified, the numbering scheme used herein is the Kabat numbering scheme. However, the choice of numbering scheme is not intended to imply sequence differences where they do not exist, and one of skill in the art can readily ascertain sequence positions by examining the amino acid sequences of one or more antibodies. Unless otherwise specified, the "EU numbering scheme" is generally used when referring to residues within antibody heavy chain constant regions (e.g., as reported in Kabat et al., supra).

[0062] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, e.g., small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, e.g., gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0063] In some embodiments, the cancer is colorectal cancer, prostate cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, e.g., squamous non-small cell lung cancer or small cell lung cancer), or esophageal cancer (e.g., esophageal squamous cell carcinoma).

[0064] As used herein, the term "cytocidal activity" refers to the activity of decreasing or reducing the cell viability of the cell line tested.

[0065] As used herein, the term "bystander killing" refers to a situation in which a drug from an ADC is released from a target cell after internalization and degradation of the ADC, or the drug is released into the extracellular space. In either case, the drug is then taken up by and kills surrounding or bystander cells, which may or may not themselves express the ADC target antigen.

[0066] An "alkyl" group is a saturated straight or branched chain acyclic hydrocarbon having from 1 to 10 carbon atoms, typically from 1 to 8 carbon atoms, or in some embodiments, from 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and n-hexyl; saturated branched chain alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. Alkyl groups can be substituted or unsubstituted. In certain embodiments, when alkyl groups described herein are said to be "substituted," they can be substituted with any substituent or substituents (as found in the exemplary compounds and embodiments disclosed herein), as well as halogen (chloro, iodo, bromo, or fluoro), hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonate, phosphine, thiocarbonyl, sulfonyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, B(OH), or O(alkyl)aminocarbonyl.

[0067] An "alkenyl" group is a straight-chain or branched-chain acyclic hydrocarbon having 2 to 10 carbon atoms, typically 2 to 8 carbon atoms, and containing at least one carbon-carbon double bond. Representative straight-chain and branched (C2C8) alkenyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, 2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, and the like. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.

[0068] As used herein, "alkynyl" refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is optionally substituted and can be straight-chained, branched, or cyclic. Alkynyl includes radicals having 2 to 20 carbon atoms, i.e., C 2-20 Alkynyl radicals, radicals having 2 to 12 carbon atoms, i.e., C 2-12 Alkynyl radicals, radicals having 2 to 8 carbon atoms, i.e., C 2-8 Alkynyl radicals, radicals having 2 to 6 carbon atoms, i.e., C 2-6 Alkynyl radicals and radicals having 2 to 4 carbon atoms, i.e., C 2-4 Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, and butynyl.

[0069] A "cycloalkyl" group is a saturated or partially saturated cyclic alkyl group of 3 to 10 carbon atoms, having a single ring or multiple fused or bridged rings, which may be optionally substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members; in other embodiments, the number of ring carbon atoms ranges from 3 to 5, 3 to 6, or 3 to 7. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the like, or multiple ring or bridged ring structures such as adamantyl, and the like. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, among others. The cycloalkyl group can be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanone, and the like.

[0070] An "aryl" group is an aromatic carbocyclic group of 6 to 14 carbon atoms, having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). In some embodiments, aryl groups have 6 to 14 carbons, and in other embodiments, 6 to 12 or even 6 to 10 carbon atoms in the ring portion of the group. Specific aryl groups include phenyl, biphenyl, naphthyl, and the like. Aryl groups can be substituted or unsubstituted. The phrase "aryl group" also includes groups containing fused rings, for example, fused aromatic aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like).

[0071] As used herein, "aryloxy" refers to a monovalent moiety that is a radical of an aromatic compound, where the ring atoms are carbon atoms and the ring is substituted with an oxygen radical, i.e., the aromatic compound contains a single bond to the oxygen atom and the radical is localized at the oxygen atom (e.g., in the case of phenoxy, C6H5-O-). The aryloxy substituent is attached to the compound it substitutes through this oxygen atom. The aryloxy is optionally substituted. Aryloxy includes radicals having 6 to 20 ring carbon atoms, i.e., C 6-20 Aryloxy radicals, radicals having 6 to 15 ring carbon atoms, i.e., C 6-15 Aryloxy radicals and radicals having 6 to 10 ring carbon atoms, i.e., C 6-10 Examples of aryloxy moieties include, but are not limited to, phenoxy, naphthoxy, and anthroxy.

[0072] As used herein, "haloalkyl" refers to an alkyl, as defined above, containing at least one substituent selected from a halogen, e.g., fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). Examples of haloalkyl include, but are not limited to, -CF, -CHCF, -CClF, and -CCl.

[0073] As used herein, "haloalkoxy" refers to an alkoxy, as defined above, that contains at least one substituent selected from a halogen, for example, F, Cl, Br, or I.

[0074] A "heteroaryl" group is an aryl ring system having 1 to 4 heteroatoms as ring atoms in the heteroaromatic ring system, with the remainder of the atoms being carbon atoms. In some embodiments, heteroaryl groups contain 5 to 6 ring atoms, and in other embodiments, 6 to 9 or even 6 to 10 atoms in the ring portion of the group. Suitable heteroatoms include oxygen, sulfur, and nitrogen. In certain embodiments, heteroaryl ring systems are monocyclic or bicyclic. Non-limiting examples include pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl (e.g., isobenzofuran-1,3-diimine), indolyl, azaindolyl (e.g., pyrrolopyridyl or 1H-pyrrolo[2,3-b]pyridyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), imidazole, and the like. These include, but are not limited to, groups such as azabenzoimidazolyl (e.g., azabenzimidazolyl, 3H-imidazo[4,5-b]pyridyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.

[0075] A "heterocyclyl" is a non-aromatic cycloalkyl in which 1 to 4 of the ring carbon atoms are independently replaced with a heteroatom independently selected from the group consisting of O, S, and N. In some embodiments, a heterocyclyl group contains 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6, or 3 to 8 ring members. A heterocyclyl can also be attached to other groups at any ring atom (i.e., any carbon atom or heteroatom of the heterocyclic ring). Heterocyclyl groups can be substituted or unsubstituted. Heterocyclyl groups encompass unsaturated, partially saturated, and saturated ring systems, such as imidazolyl, imidazolinyl, and imidazolidinyl groups. The term "heterocyclyl" includes fused ring species, such as those containing fused aromatic and non-aromatic groups, such as benzotriazolyl, 2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase also includes, but is not limited to, bridged polycyclic ring systems containing heteroatoms, such as quinuclidyl.Representative examples of heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropi pyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithionyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxazolyl Azolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl, e.g., 1H-imidazo[4,5-b]pyridyl, or 1H-imidazo[4,5-b]pyridin-2(3H)-onyl), triazolopyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalyl Examples of aryl groups include, but are not limited to, phenyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthalenyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.Representative substituted heterocyclyl groups may be mono- or multiply substituted, for example, but not limited to, a pyridyl or morpholinyl group may be 2-, 3-, 4-, 5-, or 6-substituted or di-substituted with various substituents such as those described below.

[0076] A "cycloalkylalkyl" group is a radical of the formula: -alkyl-cycloalkyl, where alkyl and cycloalkyl are defined above. Substituted cycloalkylalkyl groups can be substituted on the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the group. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, and cyclohexylpropyl. Representative substituted cycloalkylalkyl groups can be mono-substituted or substituted more than once.

[0077] An "aralkyl" group is a radical of the formula: -alkyl-aryl, where alkyl and aryl are defined above. Substituted aralkyl groups can be substituted on the alkyl, aryl, or both the alkyl and aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl groups, and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.

[0078] A "heterocyclylalkyl" group is a radical of the formula: -alkyl-heterocyclyl, where alkyl and heterocyclyl are defined above. Substituted heterocyclylalkyl groups can be substituted on the alkyl, heterocyclyl, or both the alkyl and heterocyclyl portions of the group. Representative heterocyclylalkyl groups include, but are not limited to, 4-ethylmorpholinyl, 4-propylmorpholinyl, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, (tetrahydro-2H-pyran-4-yl)methyl, (tetrahydro-2H-pyran-4-yl)ethyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylethyl, and indol-2-ylpropyl.

[0079] "Halogen" is chloro, iodo, bromo, or fluoro.

[0080] An "alkoxy" or "alkoxyl" group is an --O(alkyl), where alkyl is as defined above.

[0081] An "alkoxyalkyl" group is -(alkyl)O(alkyl), where each alkyl is independently as defined above.

[0082] An "amine" group is a radical of the formula: --NH.sub.2.

[0083] A “hydroxylamine” group has the formula: N(R # )OH or NHOH radical, where R # is a substituted or unsubstituted alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0084] An “alkoxyamine” group is a group of the formula: —N(R # )O-alkyl or -NHO-alkyl radicals, where R # is as defined above.

[0085] An “aralkoxyamine” group has the formula: N(R # ) O-aryl or NHOaryl radicals, where R # is as defined above.

[0086] An "alkylamine" group is a radical of the formula: NH alkyl or N(alkyl) 2 , where each alkyl is independently as defined above.

[0087] An "aminocarbonyl" group is a group of the formula: -C(=O)N(R # )2, -C(=O)NH(R # ), or a radical of C(=O)NH2, where each R # is as defined above.

[0088] An "acylamino" group is a group of the formula: NHC(=O)(R # ) or N(alkyl)C(=O)(R # ) radical, where each alkyl and R # are independently as defined above.

[0089] An "O(alkyl)aminocarbonyl" group is a group of the formula: -O(alkyl)C(=O)N(R # )2, -O(alkyl)C(=O)NH(R # ), or a radical of —O(alkyl)C(═O)NH2, where each R # are independently as defined above.

[0090] An "N-oxide" group is a group of the formula: -N + -O - is a radical of

[0091] A "carboxy" group is a radical of the formula: C(=O)OH.

[0092] A "ketone" group is a group of the formula: C(=O)(R # ) radical, where R # is as defined above.

[0093] An "aldehyde" group is a radical of the formula: --CH(.dbd.O).

[0094] An "ester" group is an ester of the formula: C(=O)O(R # ) or OC(=O)(R # ) radical, where R # is as defined above.

[0095] A "urea" group is a group of the formula: -N(alkyl)C(=O)N(R # )2, -N(alkyl)C(=O)NH(R # ), -N(alkyl)C(=O)NH2, -NHC(=O)N(R # )2, -NHC(=O)NH(R # ), or NHC(=O)NH2 # where each alkyl and R # are independently as defined above.

[0096] An "imine" group is a group of the formula: -N=C(R # )2 or -C(R # )=N(R # ) radical, where each R # are independently as defined above.

[0097] An "imido" group is a group of the formula: -C(=O)N(R#)C(=O)(R # ) or N((C=O)(R # ))2 radical, wherein each R # are independently as defined above.

[0098] A "urethane" group has the formula: -OC(=O)N(R # )2, -OC(=O)NH(R # ), -N(R # )C(=O)O(R # ), or -NHC(=O)O(R # ) radical, where each R # are independently as defined above.

[0099] An "amidine" group is a group of the formula: -C(=N(R # ))N(R # )2, -C(=N(R # ))NH(R # ), -C(=N(R # ))NH2, -C(=NH)N(R # )2, -C(=NH)NH(R # ), -C(=NH)NH2, -N=C(R # )N(R # )2, -N=C(R # )NH(R # ), -N=C(R # )NH2, -N(R # )C(R # )=N(R # ), -NHC(R # )=N(R # ), -N(R # )C(R # )=NH, or -NHC(R # )=NH radical, and each R # are independently as defined above.

[0100] A "guanidine" group has the formula: -N(R # )C(=N(R # ))N(R # )2, -NHC(=N(R # ))N(R # )2, -N(R # )C(=NH)N(R # )2, -N(R # )C(=N(R # ))NH(R # ), -N(R # )C(=N(R # ))NH2, -NHC(=NH)N(R # )2, -NHC(=N(R # ))NH(R # ), -NHC(=N(R # ))NH2, -NHC(=NH)NH(R # ), -NHC(=NH)NH2, -N=C(N(R # )2)2, -N=C(NH(R # ))2, or -N=C(NH2)2 radical, where each R# are independently as defined above.

[0101] An "enamine" group is a group of the formula: -N(R # )C(R # )=C(R # )2, -NHC(R # )=C(R # )2, -C(N(R # )2)=C(R # )2, -C(NH(R # ))=C(R # )2, -C(NH2)=C(R # )2, -C(R # )=C(R # )(N(R # )2), C(R # )=C(R # )(NH(R # )), or -C(R # )=C(R # )(NH2), where each R # are independently as defined above.

[0102] An "oxime" group is a group of the formula: -C(=NO(R # ))(R # ), -C(=NOH)(R # ), -CH(=NO(R # )), or —CH(═NOH), where each R # are independently as defined above.

[0103] A "hydrazide" group has the formula: -C(=O)N(R # )N(R # )2, -C(=O)NHN(R # )2, -C(=O)N(R # )NH(R # ), -C(=O)N(R # )NH2, -C(=O)NHNH(R # )2, or —C(═O)NHNH2, where each R # are independently as defined above.

[0104] A “hydrazine” group has the formula: —N(R # )N(R # )2, -NHN(R # )2, -N(R # )NH(R # ) 、 -N(R # )NH2, -NHNH(R # )2, or -NHNH2 radicals, where each R # are independently as defined above.

[0105] A "hydrazone" group has the formula: -C(=NN(R # )2)(R # )2, -C(=NNH(R # ))(R # )2, -C(=N-NH2)(R # )2, -N(R # )(N=C(R # )2), or -NH(N=C(R # )2) is a radical of the formula # are independently as defined above.

[0106] An "azido" group is a radical of the formula: -N3.

[0107] An "isocyanate" group is a radical of the formula: N=C=O.

[0108] An "isothiocyanate" group is a radical of the formula: N=C=S.

[0109] A "cyanate" group is a radical of the formula: OCN.

[0110] A “thiocyanate” group is a radical of the formula: SCN.

[0111] A “thioether” group has the formula: —S(R # ) radical, where R # is as defined above.

[0112] A "thiocarbonyl" group is a group of the formula: -C(=S)(R# ) radical, where R # is as defined above.

[0113] A "sulfinyl" group is a group of the formula: -S(=O)(R # ) radical, where R # is defined above.

[0114] A "sulfone" group is a group of the formula: -S(=O)(R # ) radical, where R # is as defined above.

[0115] A "sulfonylamino" group is a group of the formula: -NHSO(R # ) or -N(alkyl)SO2(R # ) radical, where each alkyl and R # is defined above.

[0116] A "sulfonamide" group is a group of the formula: -S(=O)N(R # )2, -S(=O)2NH(R # ), or a radical of —S(═O)2NH2, where each R # are independently as defined above.

[0117] A "phosphonate" group has the formula: -P(=O)(O(R # ))2, -P(=O)(OH)2, -OP(=O)(O(R # ))(R # ), or -OP(=O)(OH)(R # ) radical, where each R # are independently as defined above.

[0118] A "phosphine" group has the formula: -P(R # )2 radical, wherein each R # are independently as defined above.

[0119] With the exception of alkyl groups, when groups described herein are said to be "substituted," they can be substituted with any suitable substituent(s). Illustrative examples of substituents include those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chloro, iodo, bromo, or fluoro), alkyl, hydroxyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, nitro, cyano, thiol, thioether, imine, imide, amidine, guanidine, enamine, aminocarbonyl, acylamino, phosphonate, phosphine, thiocarbonyl, sulfinyl, sulfone, sulfonamide, ketone, aldehyde, ester, urea, urethane, oxime, hydroxylamine, alkoxyamine, aralkoxyamine, N-oxide, hydrazine, hydrazide, hydrazone, azide, isocyanate, isothiocyanate, cyanate, thiocyanate, oxygen (=O), B(OH), O(alkyl)aminocarbonyl, cycloalkyl (which may be simply and heterocyclyl (which may be a single ring or a fused or non-fused multiple rings) (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), or heterocyclyl (which may be a single ring or a fused or non-fused multiple rings) (e.g., pyrrolidyl, piperidyl, piperazinyl, morpholinyl, or thiazinyl), single ring or a fused or non-fused multiple rings aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl), aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy.

[0120] As used herein, the term "pharmaceutically acceptable salt(s)" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and bases and organic acids and bases.

[0121] As used herein, unless otherwise indicated, the term "solvate" means a compound or salt thereof that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. In one embodiment, the solvate is a hydrate.

[0122] As used herein, unless otherwise indicated, the term "hydrate" refers to a compound or salt thereof that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

[0123] All pharmaceutically acceptable salts, solvates, and / or hydrates of the compounds depicted herein are within the scope of this disclosure.

[0124] As used herein, unless otherwise indicated, the terms "stereoisomer" or "stereoisomerically pure" refer to one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of that compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of that compound. A typical stereoisomerically pure compound will contain greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of that compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of that compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of that compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of that compound. Compounds may contain chiral centers and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomers, including mixtures thereof, are included in the embodiments disclosed herein. The use of stereomerically pure forms of such compounds, as well as mixtures of these forms, is encompassed by the embodiments disclosed herein. For example, mixtures containing equal or unequal amounts of the enantiomers of a particular compound can be used in the methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or resolved using standard techniques, such as chiral columns or chiral resolving agents.See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, SH, et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., University of Notre Dame Press, Notre Dame, IN, 1972).

[0125] It should also be noted that the compounds can include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In certain embodiments, the compounds are isolated as either cis or trans isomers. In other embodiments, the compounds are mixtures of cis and trans isomers.

[0126] "Tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of isomeric forms may vary depending on the environment in which the compound is found, for example, whether the compound is solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other: [ka]

[0127] As will be readily understood by one of ordinary skill in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of the compounds are within the scope of the present disclosure.

[0128] It should also be noted that the compounds may contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may contain unnatural proportions of atomic isotopes, such as tritium ( 3 H), iodine-125( 125 I), sulfur-35( 35 S), or carbon-14 ( 14 It may be radiolabeled with a radioisotope such as deuterium ( 2 H), carbon-13( 13 C), or nitrogen-15( 15 The compound may be isotopically enriched, such as with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 120, 121, 132, 143, 144, 150, 165, 170, 171, 182, 191, 192, 193, 194, 195, 196, 197, 198,

[0129] Certain groups, moieties, substituents, and atoms are represented by a wavy line crossing a bond or bonds to indicate the atom to which the group, moiety, substituent, or atom is attached. For example: [ka] The propyl-substituted phenyl group, depicted as: [ka] is.

[0130] As used herein, a diagram showing a substituent attached to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) via a bond between ring atoms is meant to indicate that the cyclic group can be substituted with that substituent at any ring position of a cyclic group, or on any ring of a fused ring group, in accordance with techniques described herein or known in the art to which this disclosure pertains, unless otherwise specified.

[0131] Diagrams showing substituents attached to an acyclic group via a bond between two atoms are meant to indicate that the substituent may be attached to either atom of the bond through which the substituent bond passes, unless otherwise specified, in accordance with techniques described herein or known in the art to which this disclosure pertains. Thus, for example, [ka] teeth, [ka] Includes.

[0132] It should be noted that if there is a discrepancy between a depicted structure and the name for that structure, the depicted structure should prevail.

[0133] In the appended claims and in the foregoing description, unless the context requires otherwise, either by express words or necessary implication, the term "comprise" or variations such as "comprises" or "comprising" are used in their inclusive sense, i.e., to clearly indicate the presence of stated features, but not to exclude the presence or addition of further features in various embodiments of the disclosure.

[0134] 2. Conjugates Provided herein are anti-B7H3 antibody-drug conjugates. In some embodiments, the conjugates comprise an antibody or antigen-binding fragment thereof (Ab) capable of specifically binding to human B7H3, and a cytotoxic agent (D). Optionally, the cytotoxic agent is covalently attached to the antibody or antigen-binding fragment thereof via a linker.

[0135] International Publication No. WO2023 / 125530, the entire contents of which are incorporated herein by reference, discloses antibody-drug conjugates, linker-payload moieties thereof that are suitable for use in the context of the present disclosure, and linker payloads that are suitable for use in the context of the present disclosure. In some embodiments, the linker payload is a linker payload disclosed in WO2023 / 125530.

[0136] In embodiments, the antibody drug conjugate has formula (XI): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, or isotopic substitution thereof, wherein BA is Ab, and the variables are as described for the antibody drug conjugates of the present disclosure (e.g., the antibody drug conjugates of Formula (I)), L is a covalent linker, PA is a payload residue (e.g., a residue of a cytotoxic agent (D)), and the variables are as described for the antibody drug conjugates of the present disclosure (e.g., the antibody drug conjugates of Formula (I)), and subscript x is 1 to 30.

[0137] In some cases, x is the variable n described with respect to the antibody drug conjugates of the present disclosure (e.g., antibody drug conjugates of Formula (I)). In some cases, x is 1 to 4. In some cases, x is about 1. In some cases, x is about 2. In some cases, x is about 3. In some cases, x is about 4.

[0138] In a further embodiment, the antibody drug conjugate has formula (XIa): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopically substituted variant, or prodrug thereof, wherein: RG 1 is a reactive group residue, RG 2 is an optional reactive group residue, SP 1 and SP 2 is, independently in each instance, the residue of an optional spacer group, HG is a hydrophilic residue, PAB is an optional self-immolative unit, the subscript p is 0 or 1, and the subscript x is 1 to 30. The values ​​of the remaining variables (e.g., AA 2 , A.A. 3 ) and alternative values ​​of variables (e.g., x, p, PAB, HG, RG 1 , R.G. 2 , SP 1 , SP 2 , BA) are as defined elsewhere herein.

[0139] In some embodiments, x is 1 to 15. In some embodiments, x is 2 to 10. In some embodiments, x is 3 to 9. In one embodiment, x is about 3. In one embodiment, x is about 4. In one embodiment, x is about 5. In one embodiment, x is about 6. In one embodiment, x is about 7. In one embodiment, x is about 8. In one embodiment, x is about 9.

[0140] In some embodiments of the compound of Formula (XIa), AA 2 is the formula (W): [ka] Including AA 3 is -valine-alanine-, -valine-citrulline-, or [ka] where R 6 is -CH3, or -(CH2)3-NHC(=O)NH2.

[0141] In some embodiments, [ka] teeth, [ka] is.

[0142] In some embodiments, AA 3 teeth, [ka] where R 6 is —CH, or —(CH)—NHC(═O)NH. In a further embodiment, R 6 is -CH3.

[0143] In some embodiments, AA 2 is glycine or [ka] are the amino acid residues of

[0144] In some embodiments, AA 2 is the formula (W): [ka] Including AA 3 is -glycine-glycine-phenylalanine-glycine- or [ka] is a tetrapeptide residue of

[0145] In some embodiments, PAB is —NH—CH—O—, a group of formula (Y1): [ka] or formula (Y2): [ka] wherein: [ka] indicates the bond that connects PAB to the adjacent group of the formula.

[0146] In some embodiments, PAB is —NH—CH 2 —O—.

[0147] In some embodiments, RG 1 teeth, [ka] -(succinimide-3-yl-N)-, [ka] In some embodiments, RG 1 teeth, [ka] is.

[0148] In some embodiments, RG 1 teeth, [ka] where EWG is an electron withdrawing group, e.g., -CN, -NO, halogen, -CF, -C(=O)OR 1 , or -C(=O)R 1 and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.

[0149] In some embodiments, RG 1 teeth, [ka] is.

[0150] In some embodiments, RG 1 teeth, [ka] where EWG is an electron withdrawing group, e.g., -CN, -NO, halogen, -CF, -C(=O)OR 1 , or -C(=O)R 1 and R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl.

[0151] In some embodiments, RG 1 teeth, [ka] The ring-open heterocycle is a product obtained from conjugation of the maleimide ring of RG with an antibody. In this regard, it will be understood that conjugation of the antibody to the maleimide ring can occur at either of the two carbons in the carbon-carbon double bond of the maleimide. Similarly, in the context of a ring-open heterocycle, conjugation can occur at either of the two carbon atoms of the double bond. In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] In some embodiments, RG 1 teeth, [ka] is.

[0152] In some embodiments, RG 2 is a bond, —C(═O)—NH—, or —NHC(═O)—. In some embodiments, RG 2 is -C(=O)-NH-.

[0153] In some embodiments, SP 1 is -(CH2) n1 -C(=O)-, -(CH2CH2O) n2 -CH2CH2-C(=O)-, -CH[-(CH2) n3 -COOH]-C(=O)-, -CH2-C(=O)-NH-(CH2) n4 -C(=O)-, -CH2-C(=O)-NH-(CH2) n3 -C(=O)-NH-(CH2) n4 -C(=O)-, or -C(=O)-(CH2) n5 -C(=O)-, wherein n1, n2, n3, n4, and n5 each independently represent an integer from 1 to 8. In some embodiments, SP 1 is *-CH2C(O)N(H)CH2CH2C(O)-, and the asterisk indicates RG 1 In some embodiments, SP 1 is *-(CH2)5C(O)-, and the asterisk indicates RG 1 In some embodiments, SP 1 is *-C(H)(CH2NH2)-(CH2)2OC(O)N(H)(CH2)2C(O)-, and the asterisk indicates RG1 indicates the bond that connects to

[0154] In some embodiments, SP 2 is -(CH2) n6 - and n6 represents an integer from 1 to 8. In some embodiments, n6 is 2.

[0155] In some embodiments, HG is [ka] [ka] wherein each n7 is independently 1 to 15, each n8 is independently 0 or 1, each n9 is independently 1 or 2, each n10 is independently an integer of 4 to 16, for example, 4, 8, or 12, each n11 is independently an integer of 0 to 5, n12 is an integer of 0 to 3, d is 0 to 3, and R 2 is H or Me, and R 3 -OH, -NH2, -NHCH2-CH2-(PEG) x -OH, or -NHCH2-CH2-(PEG) x -OMe and R 4 is OH or NH2, and each of X, Y, and Z is independently -CH2-, -NH-, -S-, or -O-.

[0156] In some embodiments, HG is [ka] [ka] wherein each n7 is independently 1 to 15, each n8 is independently 0 or 1, each n9 is independently 1 or 2, each n10 is independently an integer from 4 to 16, e.g., 4, 8, or 12, d is 0 to 3, and R 2 is H or Me, and R 3 -OH, -NH2, -NHCH2-CH2-(PEG)x -OH, or -NHCH2-CH2-(PEG) x -OMe and R 4 is OH or NH2.

[0157] In some embodiments, HG is [ka] wherein each n8 is independently 0 or 1; R 1 is H or Me.

[0158] In some embodiments, HG is [ka] wherein n8 is independently 0 or 1.

[0159] In some embodiments, HG is [ka] wherein each n11 is independently an integer from 0 to 5, n12 is an integer from 0 to 3, and each of X, Y, and Z is independently -CH2-, -NH-, -S-, or -O-.

[0160] In some embodiments, HG is —NHSO2NH2, —SO3H, —SO2NH2, —PO3H2, and RG 2 is a bond.

[0161] In some embodiments, each PA independently represents a chromophore functional group.

[0162] In some embodiments, each chromophore functional group is independently a functional group selected from the classes or subclasses of xanthophores, erythrophores, iridophores, leucophores, melanophores, and cyanophores, the classes or subclasses of fluorophore molecules, which are fluorescent compounds that emit light when exposed to excitation light, the classes or subclasses of visual phototransduction molecules, the classes or subclasses of photophore molecules, the classes or subclasses of luminescent molecules, and the class or subclass of luciferin compounds.

[0163] In some embodiments, each PA is a residue of a cytotoxic agent independently selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), mertansine (maytansinoid DM1 / DM4), paclitaxel, docetaxel, epothilone B, epothilone A, CYT997, auristatin tyramine phosphate, auristatin aminoquinoline, halocombstatin, calicheamicin theta, 7-ethyl-10-hydroxy-camptothecin (SN-38), pyrrolobenzodiazepine (PBD), pancratistatin, cyclophosphate, cribrostatin-6, kitastatin, turbostatin 1-4, halocombstatin, eribulin, hemiasterin, PNU, and cilstatin.

[0164] In some embodiments, each PA independently has the formula (D1): [ka] wherein R 4 , R 5a , and R 5b each independently represents hydrogen, a sugar residue, a substituted or unsubstituted inorganic or organic acid residue, a substituted or unsubstituted C 1-8 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted non-aromatic heterocyclyl, substituted or unsubstituted cycloalkylalkyl, or substituted or unsubstituted heterocyclylalkyl; R 5a and R 5btogether with the atom to which they are attached form a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted non-aromatic heterocyclyl.

[0165] In some embodiments, R 4 is hydrogen, [ka] where R 5a and R 5b each is independently H, CH, or CF, or R 5a and R 5b together with the atom to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted non-aromatic heterocyclyl.

[0166] In some embodiments, R 4 is hydrogen, [ka] where R 5a and R 5b each is independently H, CH, or CF, or R 5a and R 5b together with the atom to which they are attached form a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted non-aromatic heterocyclyl.

[0167] In some embodiments, each PA independently has the following structure: [ka] It has one of the following.

[0168] In some embodiments, each PA independently has the formula (D2): [ka] wherein ring B is a substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heteroaryl.

[0169] In some embodiments, each PA independently has the following structure: [ka] It has one of the following.

[0170] In some embodiments, each PA independently has the formula (D3): [ka] wherein S 2 is an enzymatically hydrolyzable hydrophilic group.

[0171] In some embodiments, S 2 The group is hydrogen or has the formula: [ka] represents one of the following:

[0172] In some embodiments, each PA independently has the formula (E1): [ka] wherein R 7 and R 8 Each of is independently hydrogen, halogen, or alkyl.

[0173] In some embodiments, R 7 and R 8 is hydrogen.

[0174] In some embodiments, R 7 and R 8 is methyl.

[0175] In some embodiments, R 7is methyl and R 8 is F.

[0176] In some embodiments, R 7 and R 8 The carbon to which is attached is in the S configuration.

[0177] In some embodiments, R 7 and R 8 The carbon to which is attached is in the R configuration.

[0178] In some embodiments, each PA independently has the following formula: [ka] It has.

[0179] In some embodiments, each PA is independently Dxd or independently has the formula: [ka] It has.

[0180] In some embodiments, each PA independently has the following formula: [ka] It has.

[0181] In a further embodiment, the antibody drug conjugate has formula (XIb): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein: AA 2 is the formula (W): [ka] Including AA 1 is -valine-alanine-, -valine-citrulline-, or [ka] where R 6 is -CH3, or -(CH2)3-NHC(=O)NH2. The values ​​of the remaining variables (e.g., x, p, BA, HG, RG 1 , R.G. 2 , SP 1 , SP 2 , PAB, PA) and alternative values ​​of the variables (e.g., AA 1 , A.A. 2 ) are as described elsewhere herein, for example, with respect to compounds of formula XIa.

[0182] In some embodiments, AA 2 is the formula (W): [ka] Including AA 1 is -glycine-glycine-phenylalanine-glycine- or [ka] is a tetrapeptide residue of

[0183] In a further embodiment, the antibody drug conjugate has formula (XIc): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopically substituted derivative thereof, or a prodrug thereof, wherein AA 3 is -valine-alanine-, -valine-citrulline-, or [ka] where R 6 is -CH3, or -(CH2)3-NHC(=O)NH2. The values ​​of the remaining variables (e.g., BA, RG 1 , SP1 ,PAB,p,PA,x) and alternative values ​​of variables (AA 3 , R 6 ) are as described elsewhere herein, for example, with respect to compounds of formula XIa.

[0184] In some embodiments (e.g., compounds of Formula (XIc)), AA 3 is -glycine-glycine-phenylalanine-glycine- or [ka] is a tetrapeptide residue of

[0185] In some embodiments, the antibody drug conjugate is selected from one of the following compounds, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, or isotopic substitution thereof: [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Table A-8] [Table A-9] [Table A-10] [Table A-11] [Table A-12] [Table A-13] [Table A-14] [Table A-15] wherein Ab is any of the anti-B7H3 antibodies disclosed herein.

[0186] PCT Application No. PCT / CN2022 / 123665, the entire contents of which are incorporated herein by reference, discloses antibody-drug conjugates, linker-payload moieties thereof that are suitable for use in the context of the present disclosure, and linker payloads that are suitable for use in the context of the present disclosure. In some embodiments, the linker payload is a linker payload disclosed in PCT / CN2022 / 123665.

[0187] In some embodiments (e.g., of a compound of Formula XI), PA is: [ka] where Y is -AB-C'-D'-H, A is a bond, CR 1 R 2 , or NR 1 B is a bond, —C(═O)—, or —C(═O)O—; C′ is a bond or a divalent group, where the divalent group is an unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH, or O; R 1 , and R2 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 1 , and R 2 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl, and R 3 , and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 , and R 4 taken together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl. In some embodiments, R 3 is methyl and R 4 is F, then Y is not -NH-C(=O)-CDH.

[0188] In embodiments, in the residue of the payload, Y is -AB-C'-D'-, resulting from removal of -H from -AB-C'-D'-H. It will be understood that the payload residue may result from removal of a hydrogen atom from a payload depicted herein, but may also result from removal of a hydroxy group, such as the hydroxy group formed when D' is O in the payload depicted herein (or the corresponding hydroxy group in any of the other payload structures depicted herein).

[0189] In some embodiments, the PA is: [ka] where A is a residue of CR 1 R 2 , NH, or NR 1 and B is a bond, —C(═O)—, or —C(═O)O—; R1 , and R 2 each independently represents H, or C 1-4 is alkyl, R 3 , and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 5 , and R 6 is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl; and n is 1, 2, 3, 4, or 5.

[0190] In some embodiments, A is -CH2- and B is a bond.

[0191] In some embodiments, R 5 and R 6 is hydrogen and n is 1, 2, or 3.

[0192] In some embodiments, R 3 is methyl and R 4 is F.

[0193] In some embodiments, the PA is [ka] is a residue of

[0194] In some embodiments, R 3 and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted dioxole ring.

[0195] In some embodiments, the PA is [ka] is a residue of

[0196] In some embodiments, A is —N(CH 3 )— and B is a bond.

[0197] In some embodiments, R 5 and R 6 is hydrogen and n is 2.

[0198] In some embodiments, the PA is [ka] is a residue of

[0199] In some embodiments, A is -NH- and B is -C(=O)O-. In further embodiments, R 5 and R 6 is hydrogen and n is 2.

[0200] In some embodiments, the PA is [ka] is a residue of

[0201] In some embodiments, A is -NH- and B is -C(=O)-. In further embodiments, R 5 and R 6 is hydrogen and n is 2.

[0202] In some embodiments, the PA is [ka] is a residue of

[0203] In some embodiments, R 3 is Cl and R 4 is F and B is -C(=O)-.

[0204] In some embodiments, the PA is [ka] is a residue of

[0205] In some embodiments, R 3 is methyl and R 4 is Cl and B is -C(=O)-.

[0206] In some embodiments, the PA is [ka] is a residue of

[0207] In some embodiments, R 3 and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted heterocyclyl.

[0208] In some embodiments, R 3 , and R 4 together with the atoms to which they are attached form an unsubstituted or substituted dioxole ring, and B is -C(=O)-.

[0209] In some embodiments, the PA is [ka] is a residue of

[0210] In some embodiments, the PA is [ka] where the variables (e.g., R 3 , R 4 ) values ​​and alternative values ​​are as described elsewhere herein.

[0211] In some embodiments, R 3is methyl and R 4 is Cl.

[0212] In some embodiments, the PA is [ka] is a residue of

[0213] In some embodiments, R 3 is Cl and R 4 is F.

[0214] In some embodiments, the PA is [ka] is a residue of

[0215] In some embodiments, R 3 is F and R 4 is F.

[0216] In some embodiments, the PA is [ka] is a residue of

[0217] In some embodiments, R 3 is H and R 4 is F.

[0218] In some embodiments, the PA is [ka] is a residue of

[0219] In some embodiments, R 3 is H and R 4 is OH.

[0220] In some embodiments, the PA is [ka] is a residue of

[0221] In some embodiments, R 3 is methyl and R 4 is methyl.

[0222] In some embodiments, the PA is [ka] is a residue of

[0223] In some embodiments, R 3 is methoxyl and R 4 is F.

[0224] In some embodiments, the PA is [ka] is a residue of

[0225] In some embodiments, R 3 is H and R 4 is methoxyl.

[0226] In some embodiments, the PA is [ka] is a residue of

[0227] In some embodiments, R 3 is H and R 4 is Cl.

[0228] In some embodiments, the PA is [ka] is a residue of

[0229] In some embodiments, R3 and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted heterocyclyl.

[0230] In some embodiments, R 3 and R 4 taken together with the atoms to which they are attached form an unsubstituted or substituted dioxole ring.

[0231] In some embodiments, the PA is [ka] is a residue of

[0232] In some embodiments, the PA is [ka] is a residue of

[0233] In some embodiments, the PA is [ka] is a residue of

[0234] In some embodiments, the PA is [ka] is a residue of

[0235] In some embodiments, the PA is [Table B-1] [Table B-2] [Table B-3] [Table B-4] [Table B-5] is a residue of

[0236] In some embodiments, the PA is [Table C-1] [Table C-2] [Table C-3] [Table C-4] [Table C-5] [Table C-6] [Table C-7] [Table C-8] [Table C-9] [Table C-10] [Table C-11] is a residue of

[0237] In some embodiments, the PA is [ka] where R 7’ and R 8’ each independently is hydrogen, or substituted or unsubstituted alkyl, or R7’ and R 8’ taken together with the nitrogen atom to which they are attached form an unsubstituted or substituted heterocyclyl, or an unsubstituted or substituted heteroaryl.

[0238] In some embodiments, the PA is [ka] is a residue of

[0239] In some embodiments, the antibody drug conjugate has formula (XV): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein the values ​​and alternative value variables (e.g., A, B, C', D', L, R 3 , R 4 , and x) are as described elsewhere herein.

[0240] In some embodiments, the antibody drug conjugate has the structure of Formula (XVIIIa), (XVIIIb), or (XVIIIc): [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, or isotopic substitution thereof, wherein the values ​​and alternative value variables (e.g., L, R 7 , R 8 , and x) are as described elsewhere herein.

[0241] In some embodiments, the antibody drug conjugate has the structure of any one of the following formulas: [ka] or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, isotopic substitution, or prodrug thereof, wherein the values ​​and alternative values ​​of the variables (e.g., L and x) are as defined elsewhere herein.

[0242] In some embodiments, L is [ka] where the bond indicated by an asterisk is attached to BA.

[0243] In some embodiments, L is [ka] where the bond indicated by an asterisk is attached to BA.

[0244] In some embodiments, L is: [ka] where the values ​​of the variables and substitution values ​​(e.g., RG 1 , SP 1 , A.A. 2 , A.A. 3 ,PAB,p,SP 2 RG 2 , and HG) are as described elsewhere herein.

[0245] In some embodiments, L is: [ka] where the values ​​of the variables and substitution values ​​(e.g., RG 1 , SP 1 , A.A. 1 , A.A. 2 ,PAB,p,SP 2 RG 2 , and HG) are as described elsewhere herein.

[0246] In some embodiments, L is: [ka] where the values ​​of the variables and substitution values ​​(e.g., RG 1 , SP 1 , A.A. 3 , PAB, and p) are as described elsewhere herein.

[0247] In some embodiments, -AA 2 (SP 2 -RG 2 -HG)-AA 3 -(PAB) p -teeth, [ka] where * is SP 1 indicates the bond that connects to

[0248] In some embodiments, the antibody drug conjugate is selected from the following, or a pharmaceutically acceptable salt, tautomer, solvate, stereoisomer, enantiomer, or isotopic substitution thereof, wherein Ab is any of the anti-B7H3 antibodies disclosed herein: [Table D-1] [Table D-2] [Table D-3] [Table D-4] [Table D-5] [Table D-6]

[0249] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (viii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6, or (ix) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 300, (b) an HCDR2 of SEQ ID NO: 1700, and (c) an HCDR3 of SEQ ID NO: 500, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 600, (e) an LCDR2 of SEQ ID NO: 700, and (f) an LCDR3 of SEQ ID NO: 800.

[0250] In some embodiments, the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (ii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8; (iii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (iv) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (v) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (vi) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21; (vii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (viii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (ix) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1800, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1400; (viii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 900, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1000; or (ix) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1300, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1400.

[0251] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within each of SEQ ID NOs: 26 and 24, each of SEQ ID NOs: 7 and 8, each of SEQ ID NOs: 12 and 8, each of SEQ ID NOs: 15 and 8, each of SEQ ID NOs: 18 and 8, each of SEQ ID NOs: 7 and 21, each of SEQ ID NOs: 7 and 24, each of SEQ ID NOs: 29 and 24, each of SEQ ID NOs: 1800 and 1400, each of SEQ ID NOs: 900 and 1000, or each of SEQ ID NOs: 1300 and 1400 are inserted, deleted, or substituted in the antibody or antigen-binding fragment.

[0252] In some embodiments, the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 24; (ii) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8; (iii) a heavy chain variable region comprising SEQ ID NO: 12 and a light chain variable region comprising SEQ ID NO: 8; (iv) a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 8; (v) a heavy chain variable region comprising SEQ ID NO: 18 and a light chain variable region comprising SEQ ID NO: 8; (vi) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 21; (vii) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 24; (viii) a heavy chain variable region comprising SEQ ID NO: 29 and a light chain variable region comprising SEQ ID NO: 24; (ix) a heavy chain variable region comprising SEQ ID NO: 900 and a light chain variable region comprising SEQ ID NO: 100; (x) a heavy chain variable region comprising SEQ ID NO: 1300 and a light chain variable region comprising SEQ ID NO: 1400; or (xi) a heavy chain variable region comprising SEQ ID NO: 1800, and a light chain variable region comprising SEQ ID NO: 1400.

[0253] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues 29-139 of human 4Ig-B7H3 (SEQ ID NO: 801). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising, consisting essentially of, or consisting of amino acid residues 243-357 of human 4Ig-B7H3 (SEQ ID NO: 801).

[0254] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the IgV1 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the IgV2 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and binds to both the IgV1 domain and the IgV2 domain of human 4Ig-B7H3.

[0255] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 145-238 of human 4Ig-B7H3 (SEQ ID NO: 801). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 363-456 of human 4Ig-B7H3 (SEQ ID NO: 801). In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to the IgC1 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human 4Ig-B7H3 and does not bind to the IgC2 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to the IgC1 or IgC2 domain of human 4Ig-B7H3. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope that does not overlap with the epitope of the reference antibody DS-7300.

[0256] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0257] In some embodiments, the antibody or antigen-binding fragment comprises an scFv comprising a VH having the amino acid sequence of SEQ ID NO: 26 and a VL having the amino acid sequence of SEQ ID NO: 24, optionally wherein the VH and VL are connected via an amino acid linker, and optionally wherein the amino acid linker is any of the sequences of SEQ ID NO: 35 to SEQ ID NO: 77.

[0258] In some embodiments, the antibody or antigen-binding fragment comprises an scFv having the amino acid sequence of SEQ ID NO:32.

[0259] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

[0260] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1 subclass and a light chain constant region of the kappa type.

[0261] All possible combinations of anti-B7H3 antibodies, linkers, and payloads are contemplated herein.

[0262] The antibody-drug conjugates disclosed herein can be produced by any method known in the art.In one example, the host cell transformed with the isolated nucleic acid comprising the sequence encoding anti-B7H3 antibody or its antigen-binding fragment is cultured under suitable culture conditions.The antibody or its antigen-binding fragment is thereby expressed and can be recovered from the cell culture.

[0263] The cytotoxic agent is conjugated to the antibody or antigen-binding fragment thereof using a linker disclosed herein, e.g., using a conjugator disclosed herein, to generate an antibody drug conjugate.

[0264] 2.1. Aspect 1 Provided herein is an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof and a cytotoxic agent.

[0265] 2.2. Aspect 2 Provided herein is an antibody drug conjugate having the formula (I): Ab-(L-(D)m)n(I), or a pharmaceutically acceptable salt thereof, wherein: Ab is an antibody or antigen-binding fragment thereof that binds to B7H3, e.g., an antibody or antigen-binding fragment thereof that can specifically bind to human B7H3; L is a linker, D is the residue of a cytotoxic agent; m is an integer from 1 to 8, and n is 1 to 10.

[0266] In one embodiment, m is 1.

[0267] In one embodiment, n is 3 to 10, e.g., 4 to 10, 5 to 10, 6 to 10, or 7 to 9. In certain embodiments, n is about 8. In embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10.

[0268] In one embodiment, the antibody drug conjugate has formula (II): [ka] or Formula (IIo): [ka] or Formula (Iioo): [ka] where Su is a hydrophilic residue.

[0269] In one embodiment, the antibody drug conjugate has formula (II): [ka] or Formula (IIo): [ka] where Su is a hydrophilic residue.

[0270] In one embodiment, Su is [ka] is.

[0271] In one embodiment, the antibody drug conjugate has formula (III): [ka] or Formula (IIIo): [ka] or Formula (IIIoo): [ka] where Su is a hydrophilic residue.

[0272] In one embodiment, the antibody drug conjugate has formula (III): [ka] or Formula (IIIo): [ka] where Su is a hydrophilic residue.

[0273] In one embodiment, Su is [ka] is.

[0274] In one embodiment, D is [ka] where: Y is -AB-C'-D'-*, where * indicates the bond connecting D to the antibody drug conjugate; A is bond, CR 1 R 2 , or NR 1 and B is a bond, —C(═O)—, or —C(═O)O—; C' is a bond or a divalent group, where the divalent group is an unsubstituted or substituted C 1-8 alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH, or O; R 1 and R 2 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 1 and R 2 together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R 3 and R 4 each is independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R 3 and R 4 together with the atoms to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl.

[0275] In one embodiment, D is [ka] and In the formula, R 7 and R 8are each independently hydrogen, halogen, or alkyl.

[0276] In one embodiment, D is [ka] is.

[0277] In one embodiment, D is [ka] is.

[0278] In one embodiment, the antibody drug conjugate has any one of the following structures: [ka] [ka] [ka] [ka] [ka] where Ab and n are as described herein.

[0279] In one embodiment, the anti-B7H3 antibody drug conjugate has any one of the following structures: [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] wherein n is as described herein.

[0280] In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one embodiment, n is 8.

[0281] In one embodiment, provided herein is a pharmaceutical composition comprising an antibody drug conjugate provided herein and a pharmaceutically acceptable carrier.

[0282] 2.3. Aspect 3 Provided herein are antibodies or antigen-binding fragments (Abs) that bind to B7H3, including antibodies or antigen-binding fragments thereof: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (viii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6, or (IX) A heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 300, (b) an HCDR2 of SEQ ID NO: 1700, and (c) an HCDR3 of SEQ ID NO: 500, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 600, (e) an LCDR2 of SEQ ID NO: 700, and (f) an LCDR3 of SEQ ID NO: 800.

[0283] In one embodiment, the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (ii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8; (iii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (iv) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (v) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (vi) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21; (vii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (viii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; or (ix) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1800, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1400.

[0284] In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within each of SEQ ID NOs: 26 and 24, each of SEQ ID NOs: 7 and 8, each of SEQ ID NOs: 12 and 8, each of SEQ ID NOs: 15 and 8, each of SEQ ID NOs: 18 and 8, each of SEQ ID NOs: 7 and 21, each of SEQ ID NOs: 7 and 24, each of SEQ ID NOs: 29 and 24, or each of SEQ ID NOs: 1800 and 1400 are inserted, deleted, or substituted in the antibody or antigen-binding fragment.

[0285] In one embodiment, the antibody or antigen-binding fragment comprises: (i) a heavy chain variable region comprising SEQ ID NO: 26 and a light chain variable region comprising SEQ ID NO: 24; (ii) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8; (iii) a heavy chain variable region comprising SEQ ID NO: 12 and a light chain variable region comprising SEQ ID NO: 8; (iv) a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 8; (v) a heavy chain variable region comprising SEQ ID NO: 18 and a light chain variable region comprising SEQ ID NO: 8; (vi) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 21; (vii) a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 24; (viii) a heavy chain variable region comprising SEQ ID NO: 29 and a light chain variable region comprising SEQ ID NO: 24, or (ix) a heavy chain variable region comprising SEQ ID NO: 1800, and a light chain variable region comprising SEQ ID NO: 1400.

[0286] In one embodiment, the antibody or antigen-binding fragment is a monoclonal antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

[0287] In one embodiment, the antibody or antigen-binding fragment comprises an scFv comprising a VH having the amino acid sequence of SEQ ID NO: 26 and a VL having the amino acid sequence of SEQ ID NO: 24, optionally wherein the VH and VL are connected via an amino acid linker, and optionally wherein the amino acid linker is any of the sequences of SEQ ID NO: 35 to SEQ ID NO: 77.

[0288] In one embodiment, the antibody or antigen-binding fragment comprises an scFv having the amino acid sequence of SEQ ID NO:32.

[0289] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, and / or a light chain constant region of the kappa or lambda type.

[0290] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1 subclass and a light chain constant region of the kappa type.

[0291] 2.4. Aspect 4 Provided herein is an antibody drug conjugate, the antibody drug conjugate comprising: [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] or a pharmaceutically acceptable salt thereof; n is 4, 5, 6, 7, 8, 9, or 10; Ab, an antibody or antigen-binding fragment thereof that binds to B7H3, the antibody or antigen-binding fragment including: (i) a heavy chain variable region (VH) comprising (a) an HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (viii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6, or (IX) A heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 300, (b) an HCDR2 of SEQ ID NO: 1700, and (c) an HCDR3 of SEQ ID NO: 500, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 600, (e) an LCDR2 of SEQ ID NO: 700, and (f) an LCDR3 of SEQ ID NO: 800.

[0292] In one embodiment, n is 8.

[0293] Provided herein are pharmaceutical compositions comprising an antibody drug conjugate provided herein and a pharmaceutically acceptable carrier.

[0294] 3.Treatment method The antibody-drug conjugates of the present disclosure are useful in a variety of applications, including, but not limited to, methods for treating B7H3-associated disorders or diseases. In one aspect, the B7H3-associated disorder or disease is cancer. In some embodiments, the cells are 4Ig-B7H3 positive.

[0295] Thus, provided herein are methods for treating cancer, comprising administering to a patient in need of cancer treatment an effective amount of an antibody-drug conjugate provided herein or a pharmaceutical composition provided herein. In some embodiments, the cancer is 4Ig-B7H3 positive. In some embodiments, the cancer is colorectal cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, renal cancer, lung cancer, or esophageal cancer. In some embodiments, the cancer is lung cancer, e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). In some embodiments, the cancer is squamous non-small cell lung cancer. In some embodiments, the cancer is esophageal cancer, e.g., esophageal squamous cell carcinoma.

[0296] The antibody-drug conjugates disclosed herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, including localized treatment and intralesional administration, if desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0297] The antibody-drug conjugates of the present disclosure can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical professionals. The antibody-drug conjugates are optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disorder or treatment, and the other factors discussed above. These will generally be used at the same dosages and via the same routes of administration as those described herein, or at about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0298] 4. Combination therapy The antibody drug conjugates described herein are, in some embodiments, administered in combination with another therapeutic agent. Other therapeutic agents that can be used with the antibody drug conjugates of the present disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel formulations, (e.g., Abraxane®), docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20 targeted agents (e.g., rituximab, oncolytics, steroid ... fatumumab, RO5072759, LFB-R603), CD52-targeted agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19-targeted agents (e.g., MEDI-551, MOR208 ), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), EGEN-001, or polo-like kinase 1 inhibitors (e.g., BI 672).

[0299] In some embodiments, the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine.

[0300] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor, hi some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0301] Anti-PD-1 antibodies include, but are not limited to, tislelizumab, pembrolizumab, and nivolumab. Tislelizumab is disclosed in U.S. Pat. No. 8,735,553. Pembrolizumab (formerly known as MK-3475), disclosed by Merck in U.S. Pat. Nos. 8,354,509 and 8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the treatment of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC) and is currently undergoing clinical investigation for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. US8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin's lymphoma. In some embodiments, the anti-PD-1 antibody is tislelizumab.

[0302] 5. Pharmaceutical Compositions and Formulations Also provided are compositions, e.g., pharmaceutical compositions, comprising the antibody drug conjugates described herein. These compositions can further comprise suitable carriers, e.g., pharmaceutically acceptable excipients such as buffers, which are well known in the art.

[0303] Pharmaceutical compositions of the antibody drug conjugates described herein may be prepared in the form of a lyophilized formulation or aqueous solution by mixing an antibody drug conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and may include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins; Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. US 7,871,607 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0304] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0305] 6. Linker Payload As used herein, the compound of formula (IIa): [ka] or a pharmaceutically acceptable salt thereof, wherein Su is a hydrophilic residue and D is a residue of a cytotoxic agent. In some embodiments, Su is HG-N(H)-, where HG is as described herein. In some embodiments, Su is [ka] is.

[0306] As used herein, the compound of formula (IIIa): [ka] or a pharmaceutically acceptable salt thereof, wherein Su is a hydrophilic residue and D is a residue of a cytotoxic agent. In some embodiments, Su is HG-N(H)-, where HG is as described herein. In some embodiments, Su is [ka] is.

[0307] Any value of D described herein is suitable for use in connection with formulas (IIa) and (IIIa). In some embodiments, D is [ka] is.

[0308] In some embodiments, the linker payload is one of the following structures: [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof is provided. [Example]

[0309] The following examples are intended to be merely illustrative and should not be construed as limiting in any way. Unless otherwise specified, experimental methods in the examples described below are conventional. Unless otherwise specified, all reagents and materials are commercially available. All solvents and chemicals used are of analytical grade or chemical purity. All solvents are redistilled before use. All anhydrous solvents are prepared according to standard or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin-layer chromatography (TLC) are commercially available from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd., China. Unless otherwise specified, all elutions were performed with petroleum ether (60-90°C) / ethyl acetate (v / v) and visualized with iodine or molybdophosphate in ethanol. Unless otherwise specified, all extraction solvents were dried over anhydrous Na2SO4. 1H NMR spectra were recorded on a Bruck-400, Varian 400MR nuclear magnetic resonance spectrometer using TMS (tetramethylsilane) as the internal standard. Coupling constants are given in Hertz. Peaks are reported as singlets (s), doublets (d), triplets (t), quartets (q), quintets (p), sextets (h), septets (hept), multiplets (m), or combinations thereof. br means broad. LC / MS data were recorded using an Agilent 1100 or 1200 high-performance liquid chromatography-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) and an ion trap (ESI source) detecting at 214 nm and 254 nm. All compound names, except for reagents, were generated by ChemDraw® 18.0.

[0310] For the purposes of brevity, certain abbreviations are used herein. One example is the one-letter abbreviations that represent amino acid residues. The amino acids and their corresponding three-letter and one-letter abbreviations are as follows: [Table 7]

[0311] In the examples below, the following abbreviations are used: [Table 8]

[0312] UPLC analysis method:

[0313] Method A: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.2 min, 10% to 95% B, 5.8 min, 95% B hold for 0.5 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0314] Method B: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.5 min, 10% to 90% B for 2.5 min, 90% B hold for 0.2 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0315] Method C: Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold for 0.2 min, 10% to 90% B for 1.3 min, 90% B hold for 0.3 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm

[0316] Example 1 Example 1-1 [ka]

[0317] Step 1 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropanamide (1-1)

[0318] To a mixture of 1-1a (5 mg, 0.042 mmol) and HATU (16 mg, 0.042 mmol) in DMF (1 mL) was added DIPEA (21 μL, 16 mg, 0.13 mmol) and exatecan mesylate (purchased from ShangHai HaoYuanMedChemExpress CO. LTD, 23 mg, 0.043 mmol). The resulting brown mixture was stirred at room temperature for 2 h. Upon completion of the reaction, the mixture was purified by preparative HPLC (TFA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A - water (0.1% TFA): B - acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give 1-1 (15 mg, 65.5% yield) as a white powder.

[0319] 1 H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.59-5.54 (m, 1H), 5.42 (s, 2H), 5.18 (q, J = 19.2 Hz, 2H), 4.87 (t, J = 5.2 Hz, 1H), 3.45 (dd, J = 10.2, 4.8 Hz, 1H), 3.41-3.28 (m, 1H), 3.15 (t, J = 5.6 Hz, 2H), 2.40 (s, 3H), 2.24-2.07 (m, 2H), 1.92-1.80 (m, 2H), 1.11 (d, J = 7.6 Hz, 6H), 0.87 (t, J = 7.2 Hz, 3H). MS (ESI) m / z: 536.4 [M+H] + .

[0320] Example 1-2: [ka]

[0321] Step 1 Diethyl 2-fluoro-2-methylmalonate (1-2b)

[0322] A solution of compound 1-2a (10.0 g, 57.4 mmol) in THF (200 mL) was cooled to 0 °C. 60% NaH in oil (3.21 g, 80.37 mmol) was added portionwise to the mixture and stirred at 0 °C for 30 minutes. N-fluoro-N-(phenylsulfonyl)benzenesulfonamide (NSFI, 19.91 g, 63.2 mmol) was then added portionwise to the mixture at 0 °C, followed by warming to room temperature and stirring for 16 hours. After the reaction was complete, the suspension was filtered and the filtrate was concentrated. PE (100 mL) was added to the residue, the precipitate was filtered, and the filtrate was concentrated to give compound 1-2b (12.5 g, crude) as a pale yellow oil.

[0323] 1 H NMR (400 MHz, CDCl3) δ 4.30 (q, J = 7.2 Hz, 4H), 1.79 (d, J = 22.0 Hz, 3H), 1.31 (t, J = 7.2 Hz, 6H); 19 F NMR (376 MHz, CDCl3) δ -157.50.

[0324] Step 2 3-Ethoxy-2-fluoro-2-methyl-3-oxopropanoic acid (1-2c)

[0325] To a solution of compound 1-2b (1.0 g, 5.2 mmol) in EtOH (5 mL) was added H 2 A solution of KOH (321 mg) in 50 mL of HCl and 2 mL of EtOH was added dropwise at 0° C. The mixture was stirred at room temperature for 2 hours. The mixture was diluted with 20 mL and washed with DCM (20 mL*3). The aqueous solution was adjusted to pH=3 with 1N HCl and then extracted with EtOAc (50 mL*3). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated to give compound 1-2c (470 mg, 55.0% yield) as a colorless oil.

[0326] 1 H NMR (400 MHz, CDCl3) δ 8.31 (br s, 1H), 4.32 (q, J = 7.2 Hz, 2H), 1.83 (d, J = 22.0 Hz, 3H), 1.33 (t, J = 7.2 Hz, 3H); 19 F NMR (376 MHz, CDCl3) δ -157.59.

[0327] Step 3 2-Fluoro-3-hydroxy-2-methylpropanoic acid (1-2d)

[0328] To a solution of compound 1-2c (200 mg, 1.22 mmol) in isopropanol (4 mL) was added 2 M LiBH (1.22 mL, 2.44 mmol) at 0 °C. The mixture was stirred at room temperature for 2 h. The mixture was quenched by dropwise addition of 2 N HCl (1.22 mL) at 0 °C, diluted with HO (10 mL), and extracted with EtOAc (50 mL * 3). The organic layers were combined, dried over anhydrous NaSO, filtered, and concentrated to give compound 1-2d (92 mg, 61.7% yield) as a colorless oil.

[0329] 1 H NMR (400 MHz, CDCl3) δ 4.01-3.81 (m, 2H), 1.58 (d, J = 21.2 Hz, 3H); 19 F NMR (376 MHz, CDCl3) δ -163.98.

[0330] Step 4 N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-fluoro-3-hydroxy-2-methylpropanamide (1-2)

[0331] To a solution of compound 1-2d (23 mg, 0.19 mmol) in DMF (2 mL), exatecan mesylate (50 mg, 0.094 mmol), HATU (54 mg, 141 mmol), and DIEA (36 mg, 0.28 mmol) were added. The mixture was stirred at room temperature for 1 hour. The mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give the following two isomers:

[0332] 1-2-1: White solid (11 mg, 21.9% yield). PLC-MS, RT=3.52 min.

[0333] 1 H NMR (400 MHz, DMSO-d6) δ 9.06 (dd, J = 9.0, 2.8 Hz, 1H), 8.00 (d, J = 10.9 Hz, 1H), 7.54 (s, 1H), 6.75 (s, 1H), 5.82 (d, J = 8.0 Hz, 1H), 5.65 (s, 2H), 5.43 (dt, J = 77.8, 12.4 Hz, 3H), 4.17-3.91 (m, 1H), 3.83 (ddd, J = 18.0, 12.4, 5.6 Hz, 1H), 3.40-3.27 (m, 1H), 2.62 (s, 3H), 2.50-2.34 (m, 2H), 2.22-1.98 (m, 2H), 1.81 (d, J = 21.4 Hz, 3H), 1.11 (t, J = 7.2 Hz, 3H); MS (ESI) m / z: 540.3 [M+H] + .

[0334] Heterogene 2: white solid, 1-2-2 (8.4 mg, yield 16.6%). UPLC-MS, RT=3.86 points.

[0335] 1 H NMR (400 MHz, DMSO-d6) δ 8.72 (dd, J = 8.4, 2.4 Hz, 1H), 7.78 (d, J = 11.2 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58 (d, J = 8.0 Hz, 1H), 5.42 (s, 2H), 5.32 - 5.05 (m, 3H), 3.83 (dd, J = 26.8, 12.0 Hz, 1H), 3.61 (dd, J = 21.6, 12.0 Hz, 1H), 3.22-3.07 (m, 2H), 2.46-2.30 (m, 3H), 2.28-2.05 (m, 2H), 2.02-1.74 (m, 2H), 1.45 (d, J = 21.4 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H); MS (ESI) m / z: 540.3 [M+H] + .

[0336] Examples 1-3 [ka]

[0337] Step 1 2-((tert-butyldiphenylsilyl)oxy)ethyl(4-nitrophenyl)carbonate (1-3b)

[0338] To a solution of 1-3a (100 mg, 0.33 mmol) and bis(4-nitrophenyl)carbonate (123 mg, 0.40 mmol) in dry CHCl (2 mL), DIEA (176 μL, 1.0 mmol) and DMAP (4.1 mg, 0.033 mmol) were added and stirred at room temperature overnight. The solution was poured into 1 N HCl (2 mL) and extracted with CHCl (2 mL × 3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 5:1) to give compound 1-3b (148 mg, 95.5% yield) as a colorless oil.

[0339] 1 H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 9.1 Hz, 2H), 7.69 (d, J = 6.5 Hz, 4H), 7.50 - 7.31 (m, 8H), 4.47 - 4.36 (m, 2H), 3.99 - 3.89 (m, 2H), 1.07 (s, 9H).

[0340] Step 2 2-((tert-Butyldiphenylsilyl)oxy)ethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (1-3c)

[0341] To a solution of exatecan mesylate (50 mg, 0.094 mmol), 1-3b (53 mg, 0.11 mmol), and HOBt (1.3 mg, 0.009 mmol) in dry DMF (1 mL) was added DIEA (50 μL, 0.28 mmol) and stirred overnight at room temperature. The solution was poured into saturated NH4Cl (5 mL) and extracted with EtOAc (5 mL * 3). The organic phase was concentrated and purified by flash column chromatography (silica gel, petroleum ether / ethyl acetate = 1:3) to give compound 1-3c (68 mg, 94.9% yield) as a yellow solid.

[0342] MS (ESI) m / z: 762.4 [M+H] + .

[0343] 1 H NMR (400 MHz, CDCl3) δ 7.70 - 7.60 (m, 5H), 7.59 - 7.52 (m, 1H), 7.36 - 7.27 (m, 7H), 5.68 (d, J = 16.2 Hz, 1H), 5.33 (d, J = 16.3 Hz, 1H), 5.25 - 5.15 (m, 1H), 5.09 - 4.96 (m, 2H), 4.56 - 4.44 (m, 1H), 4.33 - 4.21 (m, 1H), 4.04 - 3.89 (m, 2H), 3.70 (s, 1H), 3.17 - 2.99 (m, 2H), 2.47 - 2.34 (m, 4H), 2.19 - 2.07 (m, 1H), 2.05 - 1.88 (m, 2H), 1.08 (t, J = 7.3 Hz, 3H), 1.04 (s, 9H).

[0344] Step 3 2-Hydroxyethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (1-3)

[0345] To a solution of 1-3c (65 mg, 0.085 mmol) in dry THF (2 mL) was added 1 M TBAF in THF (102 μL, 0.102 mmol) at 0 °C and stirred at room temperature for 40 min. The solution was poured into saturated NH4Cl (5 mL) and extracted with CHCl2 / MeOH (5 / 1, 6 mL*3). The organic phase was concentrated and purified by flash column chromatography (silica gel, CHCl2 / MeOH = 10:1) to give compound 1-3 (39 mg, 87.3% yield) as a pale yellow solid.

[0346] MS (ESI) m / z: 524.4 [M+H] + .

[0347] 1 H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 10.9 Hz, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.33 - 5.17 (m, 3H), 4.78 (t, J = 5.4 Hz, 1H), 4.19 - 4.01 (m, 2H), 3.68 - 3.57 (m, 2H), 3.30 - 3.20 (m, 1H), 3.17 - 3.06 (m, 1H), 2.38 (s, 3H), 2.26 - 2.07 (m, 2H), 1.95 - 1.78 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H).

[0348] Examples 1-4 [ka]

[0349] Step 1 N-(3-fluoro-7-(3-methoxypropyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-4c)

[0350] To a solution of LDA (1.40 mL, 2.81 mmol, 2 M in THF) in THF (19 mL) was added 1-4a (300.00 mg, 1.28 mmol) at −78 °C. The mixture was stirred at the same temperature for 2 h, and then a solution of 1-4b (0.38 g, 1.91 mmol) in THF (1 mL) was added dropwise. The reaction mixture was slowly warmed to 0 °C and continuously stirred for 4 h. The reaction was then quenched with saturated aqueous NH4Cl (50 mL), and the organic material was extracted with EtOAc (30 mL). The combined organic layers were washed with brine (50 mL) and dried over MgSO4, after which the combined extracts were concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (silica gel, Hex:EtOAc = 97:3) to give 1-4c (80.00 mg, 20.41% yield) as a colorless oil.

[0351] MS (ESI) m / z: 308.2 [M+H] + .

[0352] Step 2 8-Amino-6-fluoro-2-(3-methoxypropyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-4d)

[0353] 2N HCl (20 mL) was added to a solution of 1-14f (836 mg, 2.72 mmol) in MeOH (20 mL). The reaction mixture was purged with N three times and reacted under N at 60 °C for 5 h. The mixture was concentrated and purified by silica gel column chromatography (CHCl:MeOH) to give 1-4d (652 mg, 90.3% yield) as a gray solid.

[0354] MS (ESI) m / z: 266.2[M+H] + .

[0355] Step 3 (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-methoxypropyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-4f)

[0356] A mixture of 1-4d (650 mg, 2.45 mmol), 1-4e (787 mg, 2.99 mmol), and a catalytic amount of PPTS (200 mg, 0.80 mmol) in toluene (40 mL) was heated to reflux (135–140 °C) for 48 h using a Dean-Stark trap. The reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (CHCl:MeOH) to give 1-4f (857 mg, 71.0% yield) as a gray solid. UPLC analysis: 1-4f, Peak 1, retention time = 2.66 min, Peak 2, retention time = 2.77 min (Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 10% B hold 1 min, 10% to 95% B, 5 min, 95% B hold 1 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm).

[0357] MS (ESI) m / z: 493.1 [M+H] + .

[0358] Step 4 (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(3-hydroxypropyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-4)

[0359] To a solution of 1-4f (70.00 mg, 0.14 mmol) in CHCl (20 mL) was added BBr (71.26 mg, 0.28 mmol) at 0 °C, and the mixture was stirred at the same temperature for 1 h. The reaction mixture was slowly warmed to 25 °C and continuously stirred for 3 h. Then, the reaction was quenched with saturated aqueous NaHCO, and the organic material was extracted three times with EtOAc (30 mL * 3). The combined organic layers were washed with brine (50 mL) and dried over MgSO, and the combined extracts were concentrated in vacuo. The resulting crude residue was purified by flash column chromatography (silica gel, CHCl:MeOH = 90:10) to give 1-4 (4.20 mg, 6.18% yield) as a gray solid.

[0360] MS (ESI) m / z: 479.4 [M+H] + .

[0361] UPLC analysis: 1-4-1, peak 1, retention time = 4.49 min, 1-4-2, peak 2, retention time = 4.65 min (Mobile phase A: 0.1% FA in water, B: MeCN, Gradient: 15% B hold 1 min, 15% to 95% B 9 min, 95% B hold 2 min, Flow rate: 0.6 mL / min, Column: ACQUITY UPLC® BEH C18 1.7 μm).

[0362] 1-4-1: 1 H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J = 11.1 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.43 (s, 2H), 5.36 (d, J = 18.7 Hz, 1H), 5.22 (d, J = 18.7 Hz, 1H), 3.52 - 3.39 (m, 2H), 3.18 - 2.97 (m, 2H), 2.38 (s, 3H), 2.34 - 2.25 (m, 2H), 2.01 - 1.78 (m, 3H), 1.78 - 1.51 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0363] 1-4-2: 1H NMR (400 MHz, DMSO-d6) δ 7.74 (d, J = 11.1 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.43 (s, 2H), 5.37 (d, J = 18.8 Hz, 1H), 5.23 (d, J = 18.7 Hz, 1H), 3.51 - 3.40 (m, 2H), 3.16 - 2.98 (m, 2H), 2.38 (s, 3H), 2.35 - 2.24 (m, 2H), 1.99 - 1.81 (m, 3H), 1.78 - 1.53 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0364] Examples 1-5 [ka]

[0365] (1S,9S)-4-Chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-aminium 2,2,2-trifluoroacetate (1-5a)

[0366] 1-5a was synthesized according to a reported procedure (US Pat. No. 1,318,212 B2).

[0367] N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropanamide (1-5)

[0368] 1-5 (5.8 mg, purity 99.82%, yield 67.9%) was synthesized according to the same procedure as in Step 1 of Example 1-1.

[0369] MS (ESI) m / z: 556.3 [M+H] + .

[0370] Examples 1-6 [ka]

[0371] Step 1 N-(benzo[d][1,3]dioxol-5-yl)acetamide (1-6b)

[0372] To a solution of 1-6a (25.00 g, 182 mmol) in CH2Cl2 (200 mL) was added Ac2O (27.85 g, 273 mmol) and Et3N (36.76 g, 364 mmol) at 0 °C. The reaction mixture was reacted at 20 °C for 3 h. LCMS showed that the reaction was complete. The reaction mixture was concentrated to 50 mL. To the residue was added EtOAc (200 mL) and washed with saturated NaHCO3 (200 mL * 3). The organic layer was dried over Na2SO4 and filtered. The filtrate was concentrated in vacuo, and the residue was used in the next step without further workup and purification (28.25 g, crude).

[0373] MS (ESI) m / z: 180.1 [M+H] + .

[0374] Step 2 N-(6-bromobenzo[d][1,3]dioxol-5-yl)acetamide (1-6c)

[0375] A solution of 1-6b (28.25 g, crude) and sodium acetate (15.4 g, 188.3 mmol) in acetic acid (100 mL) was heated to 60 °C, and then a mixture of bromine (30.1 g, 188.3 mmol) and acetic acid (60 mL) was added dropwise to the reaction solution. The reaction temperature was increased to 80 °C and stirred at 80 °C for 2 h. LCMS showed that the reaction was complete. The reaction solution was poured into ice water, and a yellow solid was formed. The solid was filtered and washed three times with water to give the crude product, which was recrystallized with EtOH to give 1-6c (17.1 g, 42% yield).

[0376] MS (ESI) m / z: 258.1 / 260.1 [M+H] + .

[0377] 1H NMR (400 MHz, DMSO) δ 9.36 (s, 1H), 7.22 (s, 1H), 7.08 (d, J = 3.7 Hz, 1H), 6.07 (s, 2H), 2.02 (s, 3H).

[0378] Step 3 N-(6-(1-hydroxycyclobutyl)benzo[d][1,3]dioxol-5-yl)acetamide (1-6d)

[0379] A solution of compound 1-6c (8.5 g, 33.01 mmol) in THF (200 mL) was cooled to -90 °C, and n-BuLi (15.84 mL, 39.60 mmol) was added to the mixture over 2 h under N protection. The internal temperature was kept below -85 °C. The mixture was stirred at -85 °C for 20 min. A solution of cyclobutanone (2.7 g, 39.60 mmol) in THF (50 mL) was added dropwise to the mixture. The mixture was stirred at -85 °C for 30 min and warmed to room temperature for 1 h. The mixture was quenched with saturated NH Cl solution (200 mL) and extracted with EtOAc (150 mL * 3). The combined organic layers were dried over Na SO . After filtration and evaporation, the residue was washed with MTBE (5 mL * 3) and recrystallized with EtOH to give 1-6d (3.1 g, 37.5% yield).

[0380] MS (ESI) m / z: 250.1 [M+H] + .

[0381] Step 4 N-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-6e)

[0382] To a solution of compound 1-6d (3.1 g, 12.44 mmol) in CHCl (20 mL) and HO (20 mL) was added AgNO (12.4 mL, 6.22 mmol) and KSO (8.4 g, 31.1 mmol). The mixture was stirred at 20 °C for 16 h. The mixture was filtered through Celite, and the filter residue was washed with CHCl:MeOH (1:1) (50 mL). The filtrate was poured into water and extracted with CHCl (300 mL). The combined organic layers were dried over NaSO. After filtration and evaporation, the residue was purified by silica gel column chromatography (eluent: petroleum ether / CHCl 100 / 0 to 0 / 100) to give 1-6e (1.9 g, 61.8% yield) as a pale yellow solid.

[0383] MS (ESI) m / z: 248.1 [M+H] + .

[0384] Step 5 (Z)-N-(7-(hydroxyimino)-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-6f)

[0385] To a solution of compound 1-6e (1.9 g, 7.69 mmol) in THF (20 mL) and t-BuOH (5 mL) was added t-BuOK (1 M in THF, 9.28 mL, 9.28 mmol) dropwise at 0 °C. The atmosphere was purged with N2, and the mixture was cooled to 0 °C using an ice-water bath. After 5 min, isopentyl nitrite (1.09 g, 9.28 mmol) was added to the stirred mixture. LCMS showed the reaction was complete, and the reaction mixture was allowed to warm to room temperature. 1 N HCl was added to adjust the pH to 1. The aqueous layer was extracted with CHCl:THF (2:1, v / v, 50 mL*3). The combined organic layers were washed with brine (100 mL) and dried over NaSO4. After filtration and evaporation, the residue was triturated with MTBE (20 mL x 2) to give 1-6f (1.5 g, 70.6% yield).

[0386] MS (ESI) m / z: 277.1 [M+H] + .

[0387] Step 6 N,N'-(6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxole-5,7-diyl)diacetamide (1-6g)

[0388] To a solution of compound 1-6f (1.5 g, 5.43 mmol) in AcO (5 mL) was added PtO (150 mg). The mixture was stirred under H (15 Psi) for 16 h at 20 °C. LCMS showed the reaction was complete. The mixture was filtered through a pad of Celite. The filtrate was diluted with EtOAc (100 mL) and washed with saturated NaHCO (100 mL * 3). The organic layer was dried over NaSO. After filtration and evaporation, the residue 1-6g (1.3 g, crude) was used in the next step without further workup and purification.

[0389] MS (ESI) m / z: 305.2 [M+H] + .

[0390] Step 7 N-(5-amino-6-oxo-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-7-yl)acetamide (1-6h)

[0391] To a solution of compound 1-6g (1.3 g, crude) in MeOH (10 mL) was added HCl (2N, 10 mL). The mixture was stirred at 60 °C for 3 h. The mixture was cooled to room temperature, and the pH was adjusted to 8 using saturated NaHCO 3 . The mixture was extracted with EtOAc (50 mL*3). The organic layer was dried and concentrated. The residue was purified by silica gel column chromatography (eluent: CHClMeOH = 100 / 0 to 90 / 10) to give compound 1-6h (670 mg, 59.8% yield) as an off-white solid.

[0392] MS (ESI) m / z: 263.1 [M+H] + .

[0393] Step 8 N-((10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-6i)

[0394] To a solution of compound 1-6h (400 mg, 1.52 mmol) in toluene (40 mL) and o-cresol (0.5 mL) was added 1-4e (479 mg, 1.82 mmol) and PPTS (38 mg, 0.15 mmol). The mixture was refluxed at 140 °C for 24 h. LCMS showed the reaction was complete, and a black solid precipitated. After filtration, the filter cake was washed with acetone (10 mL*3) to give a dark brown solid 1-6i (580 mg, crude), which was used in the next step without further workup and purification.

[0395] MS (ESI) m / z: 490.3 [M+H] + .

[0396] Step 9 (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (1-6j) (1R,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (1-6k)

[0397] A solution of compound 1-6i (580 mg, crude) in MsOH (10 mL) and HO (10 mL) was refluxed at 110° C. for 5 hours. LCMS showed the reaction was complete. The mixture was filtered and purified by preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% TFA): B-acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give the following two isomers:

[0398] Compound 1-6j (110 mg, yield 20.8%) was obtained as a yellow solid.

[0399] MS (ESI) m / z: 448.2 [M+H] + . Retention time (0.82 minutes).

[0400] Compound 1-6k (120 mg, yield 22.7%) was obtained as a yellow solid.

[0401] MS (ESI) m / z: 448.2 [M+H] + . Retention time (1.87 minutes).

[0402] N-((1S,10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxy-2,2-dimethylpropanamide (1-6)

[0403] Compound 1-6 (1.8 mg, 17.9% yield) was synthesized according to the synthetic procedure of Step 1 in Example 1-1.

[0404] MS (ESI) m / z: 548.4 [M+H] + .

[0405] Examples 1-7 [ka]

[0406] Step 1 tert-Butyl(2-iodoethoxy)dimethylsilane (1-7b)

[0407] A solution of compound 1-7a (3000 mg, 17.44 mmol) in CHCl (40 mL) was cooled to 0 °C, and then imidazole (1779 mg, 26.16 mmol) and TBSCl (3139 mg, 20.93 mmol) were added portionwise at 0 °C. The mixture was stirred at 20 °C for 16 h. TLC (SiO, petroleum ether) showed that the reaction was complete. The reaction was poured into water (40 mL) and extracted with CHCl (50 mL * 3). The combined organic layers were dried over NaSO and concentrated to give a residue, which was purified by silica gel column chromatography (eluent: petroleum ether = 100) to give 1-7b (3.5 g, 70.2% yield) as a colorless oil.

[0408] Step 2 N-(7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-7d)

[0409] To a solution of compound 1-7c (2350 mg, 10 mmol) in THF (40 mL) was added tBuOK (1 M in THF) (22 mL, 22 mmol) dropwise at 0 °C, maintaining the internal temperature at 0 °C. The mixture was stirred at 0 °C for 30 min, and then 1-7b (3432 mg, 12 mmol) was added slowly. LCMS showed the reaction was complete. 1N HCl was added dropwise to the mixture to adjust the pH to 2. The mixture was poured into saturated aqueous NaHCO3 solution (50 mL) and extracted with EtOAc (60 mL * 3). The combined organic layers were dried over anhydrous Na2SO4 and concentrated to give the crude product, which was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 83 / 17) to give 1-7d (700 mg, 17.8% yield) as a yellow solid.

[0410] MS (ESI) m / z: 394.2 [M+H] + .

[0411] 1 H NMR (400 MHz, DMSO-d6) δ = 7.40 (s, 2H), 6.34 (d, J=12.6, 1H), 6.34 (d, J=12.6, 1H), 4.45 (t, J=5.2, 1H), 4.45 (t, J=5.2, 1H), 3.65 - 3.35 (m, 2H), 3.55 - 3.45 (m, 2H), 2.83 (s, 1H), 2.83 (s, 1H), 2.72 - 2.61 (m, 1H), 2.46 (d, J=5.0, 1H), 2.11 - 1.99 (m, 3H), 1.97 (d, J=1.0, 3H), 1.72 - 1.60 (m, 1H), 1.43 (d, J=6.2, 1H).

[0412] Step 3 8-Amino-6-fluoro-2-(2-hydroxyethyl)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (1-7e)

[0413] To a solution of compound 1-7d (700 mg, 1.78 mmol) in MeOH (3 mL) was added 2N HCl (3 mL). The mixture was stirred at 60 °C for 16 h. LCMS showed the reaction was complete. The mixture was poured into saturated aqueous NaHCO (20 mL) and extracted with EtOAc (30 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 76 / 24) to give 1-7e (200 mg, 47% yield) as an off-white solid.

[0414] MS (ESI) m / z: 238.2 [M+H] + .

[0415] Step 4 (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-7)

[0416] To a solution of compound 1-7e (100 mg, 0.42 mmol) in toluene (6 mL) and o-cresol (0.35 mL) was added 1-4e (110 mg, 0.42 mmol) and PPTS (16 mg, 0.06 mmol). The mixture was refluxed at 140 °C for 5 h. LCMS showed that the reaction was complete. The solvent was evaporated, and the residue was purified by silica gel column chromatography (eluent: CHCl / MeOH = 100 / 0 to 92 / 8) to give 1-7 (80 mg, 41% yield) as an orange solid.

[0417] MS (ESI) m / z: 465.2 [M+H] + .

[0418] 1 H NMR (400 MHz, DMSO-d6) δ = 7.74 (d, J=11.0, 1H), 7.31 (s, 1H), 6.53 (d, J=1.9, 1H), 5.44 (s, 2H), 5.30 (s, 2H), 4.77 (t, J=4.8, 1H), 3.69 - 3.56 (m, 4H), 3.10 (t, J=18.6, 2H), 2.38 (s, 3H), 2.29 (d, J=9.7, 1H), 1.99 - 1.81 (m, 3H), 1.73 (d, J=5.4, 2H), 0.93 - 0.81 (m, 3H).

[0419] Examples 1-8 [ka]

[0420] Step 1 2-(3-iodopropoxy)tetrahydro-2H-pyran (1-8b)

[0421] To a solution of compound 1-8a (2.0 g, 9.01 mmol) in acetone (20 mL) was added NaI (4.0 g, 27 mmol). The mixture was stirred at 60 °C for 3 h. The mixture was diluted with hexane (40 mL) and washed with water (40 mL) and brine (40 mL). The combined organic layers were dried over NaSO and concentrated to give 1-8b (1.3 g, 54% yield) as a colorless oil.

[0422] 1H NMR (400 MHz, CDCl3) δ 4.61 (dd, J = 4.4, 2.8 Hz, 1H), 3.91 - 3.83 (m, 1H), 3.83 - 3.77 (m, 1H), 3.56 - 3.49 (m, 1H), 3.45 (dt, J = 10.0, 5.9 Hz, 1H), 3.30 (td, J = 6.8, 1.0 Hz, 2H), 2.10 (ddd, J = 12.7, 6.8, 5.9 Hz, 2H), 1.90 - 1.63 (m, 3H), 1.61 - 1.48 (m, 5H).

[0423] Step 2 N-(6-oxo-7-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)-6,7,8,9-tetrahydronaphtho[1,2-d][1,3]dioxol-5-yl)acetamide (1-8c)

[0424] To a solution of compound 1-6e (100 mg, 3.52 mmol) in THF (5 mL) was added t-BuOK (1.2 mL, 1.21 mmol, 1 M in THF) dropwise at −40° C. under N. The mixture was stirred at −40° C. for 30 min under N. Then, compound 1-8b (164 mg, 0.606 mmol, dissolved in 0.2 mL of THF) was added dropwise to the mixture. The mixture was stirred for 16 h with gradual heating to room temperature under N. The mixture was quenched with saturated NH.sub.4Cl (3 mL) and extracted with EA (3 mL*3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to give a residue, which was purified by column chromatography (petroleum ether / ethyl acetate = 1:0 to 4:1) to give compound 1-8c (20 mg, 12.7% yield) as a yellow solid.

[0425] MS (ESI) m / z: 248.1 [M+H] + .

[0426] 1H NMR (400 MHz, CDCl3) δ 12.44 (s, 1H), 8.26 (s, 1H), 6.01 - 5.98 (m, 2H), 4.59 - 4.53 (m, 1H), 3.90 - 3.72 (m, 2H), 3.53 - 3.36 (m, 2H), 3.00 - 2.88 (m, 1H), 2.79 - 2.67 (m, 1H), 2.55 - 2.44 (m, 1H), 2.19 (s, 3H), 2.17 - 2.09 (m, 1H), 2.06 - 1.86 (m, 2H), 1.86 - 1.65 (m, 6H), 1.60 - 1.54 (m, 2H).

[0427] Step 3 5-Amino-7-(3-hydroxypropyl)-8,9-dihydronaphtho[1,2-d][1,3]dioxol-6(7H)-one (1-8d)

[0428] To a solution of compound 1-8c (20 mg, 0.05 mmol) in MeOH (5 mL) was added 2N HCl (5 mL). The mixture was stirred at 60 °C for 3 h. The mixture was concentrated in vacuo to remove MeOH. The mixture was then adjusted to pH 8 using saturated Na2CO3 and extracted with CHCl2 (5 mL * 3). The combined organic phase was concentrated in vacuo to give a residue, which was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 100 / 0 to 0 / 100) to give 1-8d (10 mg, 74% yield) as a yellow oil.

[0429] MS (ESI) m / z: 264.2 [M+H] + .

[0430] Step 4 (1R,10S)-10-Ethyl-10-hydroxy-1-(3-hydroxypropyl)-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (1-8-1) and (1S,10S)-10-Ethyl-10-hydroxy-1-(3-hydroxypropyl)-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (1-8-2)

[0431] To a solution of compound 1-8d (80 mg, 0.304 mmol) in toluene (14 mL) and o-cresol (2 mL) was added 1-4e (88 mg, 0.334 mmol) and PPTS (23 mg, 0.091 mmol). The mixture was refluxed at 140 °C for 16 h. The mixture was concentrated in vacuo to remove toluene and purified by silica gel column chromatography (eluent: CHClMeOH = 100 / 0 to 100 / 10) to give the crude product, which was further purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A - water (0.1% formic acid): B - acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give 1-8-1 (15 mg, 71% yield) as a white solid and 1-8-2 (20 mg, 13% yield) as a white solid.

[0432] Compound 1-8-1 (retention time: 2.54 minutes):

[0433] MS (ESI) m / z: 491.5 [M+H] + .

[0434] 1H NMR (400 MHz, DMSO-d6) δ 7.37 (s, 1H), 7.23 (s, 1H), 6.48 (s, 1H), 6.27 (d, J = 2.9 Hz, 2H), 5.42 (s, 2H), 5.31 (d, J = 18.9 Hz, 1H), 5.19 (d, J = 18.8 Hz, 1H), 4.42 (t, J = 5.1 Hz, 1H), 3.48 - 3.42 (m, 2H), 3.01 - 2.91 (m, 2H), 2.22 (d, J = 13.4 Hz, 1H), 1.93 - 1.75 (m, 4H), 1.73 - 1.63 (m, 2H), 1.61 - 1.54 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0435] Compound 1-8-2 (retention time: 2.66 min):

[0436] MS (ESI) m / z: 491.5 [M+H] + .

[0437] 1 H NMR (400 MHz, DMSO-d6) δ 7.37 (s, 1H), 7.23 (s, 1H), 6.49 (s, 1H), 6.27 (s, 2H), 5.42 (s, 2H), 5.31 (d, J = 18.9 Hz, 1H), 5.20 (d, J = 18.9 Hz, 1H), 4.43 (t, J = 5.2 Hz, 1H), 3.47 - 3.43 (m, 2H), 3.02 - 2.86 (m, 2H), 2.22 (d, J = 13.3 Hz, 1H), 1.94 - 1.78 (m, 3H), 1.73 - 1.63 (m, 2H), 1.63 - 1.53 (m, 3H), 0.87 (t, J = 7.3 Hz, 3H).

[0438] Example 1-9

change

[0439] Step 1 N-(3-fluoro-4-methyl-7-methylene-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-9a)

[0440] To a solution of 1-4a (470.5 mg, 2 mmol) in CHCN (5 mL) was added paraformaldehyde (150 mg, 5 mmol) and ZnCl (136.3 mg, 1 mmol). The mixture was stirred at room temperature for 10 min. Then, pyrrolidine (287 mg, 4 mmol) was added dropwise. Upon completion of the addition, the mixture was stirred at 70 °C for 2 h. The reaction solution was then cooled to room temperature, the solvent was removed by evaporation, and the residue was directly purified by silica gel column chromatography (eluent: hexane / EtOAc = 30 / 1 to 5 / 1) to give 1-9a as a white solid (210 mg, 42.5% yield).

[0441] 1 H NMR (400 MHz, CDCl3) δ 12.42 (s, 1H), 8.46 (d, J = 12.9 Hz, 1H), 6.19 (d, J = 1.3 Hz, 1H), 5.51 (d, J = 1.6 Hz, 1H), 2.93 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 6.4 Hz, 2H), 2.25 (s, 3H), 2.17 (d, J = 1.9 Hz, 3H).

[0442] MS (ESI) m / z: 248.2 [M+H] + .

[0443] Step 2 N-(3-fluoro-7-(((2-hydroxyethyl)amino)methyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-9b)

[0444] To a solution of 1-9a (200 mg, 0.8 mmol) in THF (10 mL) was added 2-aminoethan-1-ol (108.8 mg, 1.78 mmol). The mixture was stirred at room temperature for 40 min. The solvent was then removed by evaporation. The crude product, which was not stable under silica gel purification, was used directly in the next step without further purification (290 mg, crude).

[0445] MS (ESI) m / z: 309.3 [M+H] + .

[0446] Step 3 N-(3-fluoro-7-(((2-hydroxyethyl)amino)methyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-9c)

[0447] To a solution of 1-9b (80 mg, 0.26 mmol) in CHCl (2 mL) was added EtN (40 mg, 0.4 mmol). The solution was then cooled to 0 °C, and benzyl chloroformate (48.6 mg, 0.28 mmol) was added dropwise. The mixture was then warmed to room temperature and stirred at room temperature for 2 h. The solvent was removed by evaporation, and the residue was directly purified by silica gel column chromatography (eluent: CHCl / MeOH = 32 / 1) to give 1-9c as a colorless oil (110 mg, 95.6% yield).

[0448] 1H NMR (400 MHz, CDCl3) δ 12.09 (s, 1H), 8.39 (d, J = 13.0 Hz, 1H), 7.42 - 7.27 (m, 4H), 7.21 (s, 1H), 5.30 (s, 1H), 5.13 (d, J = 13.1 Hz, 2H), 4.94 (d, J = 12.0 Hz, 1H), 3.99 - 3.71 (m, 3H), 3.65 - 3.53 (m, 1H), 3.52 - 3.35 (m, 2H), 2.85 (t, J = 62.3 Hz, 3H), 2.21 (s, 3H), 2.10 (d, J = 27.8 Hz, 3H), 1.79 (s, 2H).

[0449] MS (ESI) m / z: 465.4 [M+Na] + .

[0450] Step 4 Benzyl ((8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methyl) (2-hydroxyethyl)carbamate (1-9d)

[0451] To a solution of compound 1-9c (100 mg, 0.23 mmol) in MeOH (3 mL) was added HCl (2N, 1.5 mL). The mixture was stirred at 60 °C for 3 h. The mixture was cooled to room temperature, and the pH was adjusted to 8 using saturated NaHCO3. The mixture was extracted with EtOAc (10 mL * 3). The organic layer was dried and concentrated. The crude product 1-9d (red oil) was used directly in the next step without further purification (crude: 81 mg, 89.6% yield).

[0452] MS (ESI) m / z: 401.4 [M+H] + .

[0453] Step 5 N-Benzyl (((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)methyl) (2-hydroxyethyl)carbamate (1-9e)

[0454] To a solution of 1-9d (80 mg, 0.2 mmol) in toluene (10 mL) and o-cresol (0.5 mL) was added 1-4e (60.5 mg, 0.23 mmol) and PPTS (30.2 mg, 0.12 mmol). The mixture was refluxed at 140 °C for 12 h. The solvent was removed by evaporation, and the crude product 1-9e was used directly in the next step without further purification (crude: 120 mg, 95.6% yield).

[0455] MS (ESI) m / z: 628.5 [M+H] + .

[0456] Step 6 N-Benzyl(9S)-9-ethyl-5-fluoro-9-hydroxy-1-(((2-hydroxyethyl)amino)methyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-9f)

[0457] To a solution of 1-9e (crude 120 mg, 0.2 mmol) in MeOH (2 mL) and THF (2 mL), wet Pd / C (20%, 24 mg) was added and stirred under a H atmosphere at room temperature for 8 h. The solution was filtered through Celite and concentrated in vacuo. The residue was purified by preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% TFA): B-acetonitrile, Flow rate: 20 mL / min). The fraction was lyophilized to give 1-9f (33.2 mg, 33.6% yield) as a white solid.

[0458] 1H NMR (400 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.74 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.44 (s, 2H), 5.41 - 5.34 (m, 2H), 5.31 (s, 1H), 3.55 (s, 1H), 3.49 (dd, J = 5.6, 3.8 Hz, 2H), 3.05 (dd, J = 27.9, 9.3 Hz, 3H), 2.89 - 2.81 (m, 2H), 2.74 - 2.67 (m, 2H), 2.37 (s, 3H), 2.03 - 1.81 (m, 4H), 0.87 (dd, J = 11.7, 4.3 Hz, 3H).

[0459] MS (ESI) m / z: 494.4 [M+H] + .

[0460] Step 7 (9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(((2-hydroxyethyl)(methyl)amino)methyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-9)

[0461] To a solution of 1-9f (10 mg, 0.02 mmol) in MeOH (1 mL) was added 37% formaldehyde solution (2.8 μL, 0.04 mmol). The solution was then cooled to 0 °C, and NaBHCN (2 mg, 0.03 mmol) was added in one portion. The solution was then warmed to room temperature and stirred for an additional 2 h. The mixture was then quenched with HO and purified by preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% TFA): B-acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give 1-9 (5.5 mg, 54.2% yield) as a white solid.

[0462] MS (ESI) m / z: 508.5 [M+H] + .

[0463] General Procedure for Preparation of Linker-Payload

[0464] General Procedure A: Preparation of Example 2-1 [ka]

[0465] Step 1 Benzyl (S)-11-benzyl-1-(9H-fluoren-9-yl)-20,20-dimethyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazahenicosan-21-oate (2-1c)

[0466] To a solution of 2-1a (250 mg, 0.40 mmol) and 2-1b (83 mg, 0.40 mmol) in THF (5 mL) was added 4 Å molecular sieves. The mixture was stirred at room temperature for 10 min, then Sc(OTf) (195 mg, 0.40 mmol) was added and the mixture was allowed to react at room temperature for another 16 h. The suspension mixture was filtered through a Celite pad, and the cake was washed with THF (10 mL). The filtrate was then quenched with saturated NaHCO (10 mL) and extracted with EtOAc (30 mL * 2). After separation, the combined organic layer was washed with brine (50 mL), dried over NaSO, filtered, and the filtrate was concentrated in vacuo to give a residue. Purification by silica gel column chromatography (A - DCM; B - MeOH, MeOH / DCM = 0 / 100 to 95 / 5) gave 2-1c (90 mg, 29.2% yield).

[0467] MS (ESI) m / z: 800.5 [M+Na] + .

[0468] Step 2 (S)-11-Benzyl-1-(9H-fluoren-9-yl)-20,20-dimethyl-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16-pentaazahenicosan-21-oic acid (2-1d)

[0469] To a solution of 2-1c (80 mg, 0.10 mmol) in MeOH (3 mL) was added wet Pd / C (20 mg). The black suspension was purged with a H balloon three times and then reacted under a H balloon at room temperature for 2 h. After the reaction was complete, the black suspension was filtered through a pad of Celite, and the cake was washed with MeOH. The combined organic layers were concentrated in vacuo to give 2-1d (61 mg, 84.8% yield).

[0470] MS (ESI) m / z: 710.4 [M+Na] + .

[0471] Step 3 (9H-Fluoren-9-yl)methyl ((S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)carbamate (2-1f)

[0472] To a mixture of 2-1d (60 mg, 0.087 mmol) and HATU (33 mg, 0.087 mmol) in DMF (2 mL) was added DIPEA (43 μL, 34 mg, 0.26 mmol). The mixture was reacted at room temperature for 10 minutes. 2-1e (46 mg, 0.087 mmol) was added and reacted at the same temperature for another hour. After the reaction was complete, the mixture was filtered, and the filtrate was purified using preparative HPLC (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20 mL / min) to give 2-1f (85 mg, Yield: 88.2%).

[0473] MS (ESI) m / z: 1105.5 [M+H] + .

[0474] Step 4 3-(((S)-13-amino-7-benzyl-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,2-dimethylpropanamide (2-1g)

[0475] To a solution of 2-1f (85 mg, 0.062 mmol) in DMF (2 mL) was added EtNH (64 μL, 46 mg, 0.62 mmol). The mixture was stirred at room temperature for 0.5 h. Upon completion of the reaction, the mixture was concentrated in vacuo to give 2-1g (86 mg, crude) as a yellow solid.

[0476] MS (ESI) m / z: 883.5 [M+H] + .

[0477] Step 6 (9H-Fluoren-9-yl)methyl((6S,15S)-15-benzyl-25-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) Amino)-24,24-dimethyl-3,7,10,13,16,19,25-heptaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (2-1i)

[0478] To a solution of 2-1g (86 mg, crude) and 2-1h (43 mg, 0.079 mmol) in DMF (1.5 mL) was added DIPEA (26 μL, 21 mg, 0.16 mmol). The mixture was stirred at room temperature for 1.5 h. Upon completion of the reaction, the mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give 2-1i (70 mg, 62.6% yield) as a white powder.

[0479] MS (ESI) m / z: 1410.7 [M+H] + .

[0480] Step 7 (S)-2-Amino-N1-((S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-N5-(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide (2-1j)

[0481] To a solution of 2-1i (70 mg, 0.050 mmol) in DMF (1 mL) was added EtNH (51 μL, 36 mg, 0.50 mmol). The mixture was stirred at room temperature for 0.5 h. Upon completion of the reaction, the mixture was concentrated in vacuo to give 2-1j (71 mg, crude) as a yellow solid.

[0482] MS (ESI) m / z: 1188.2 [M+H] + .

[0483] Step 8 (S)-N 1 -((S)-7-benzyl-17-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-16,16-dimethyl-2,5,8,11,17-pentaoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-N 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide (2-1)

[0484] To a solution of 2-1k (19 mg) in DMF (3 mL) was added HATU (34 mg, 0.088 mmol) and DIPEA (10 μL, 7.6 mg, 0.059 mmol). The resulting yellow solution was stirred at room temperature for 5 min, and then 2-1j (71 mg, crude) was added. The mixture was stirred at room temperature for 60 min. Upon completion of the reaction, the mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give 2-1 (32 mg, 26.3% yield) as a white powder.

[0485] MS (ESI) m / z: 1381.1 [M+H] + .

[0486] General Procedure B: Preparation of Examples 2-4 [ka]

[0487] Step 1 Benzyl (5S,8S,14R)-1-(9H-fluoren-9-yl)-14-fluoro-5-isopropyl-8,14-dimethyl-3,6,9-trioxo-2,12-dioxa-4,7,10-triazapentadecan-15-oate (2-4c)

[0488] To a mixture of 2-4a (3.0 g, 6.23 mmol), 2-4b (1.98 g, 9.34 mmol), and freshly dried 4 Å molecular sieves (6 g) in anhydrous THF (30 mL) was added scandium trifluoromethanesulfonate (3.99 g, 8.10 mmol) and stirred overnight at room temperature under a N atmosphere. The solution was filtered through Celite, diluted with EtOAc (300 mL), and washed with saturated NaHCO (50 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: petroleum ether / EtOAc = 100 / 0 to 50 / 50) to give compound 2-4c (3.5 mg, 84% yield).

[0489] MS (ESI) m / z: 656.5 [M+Na] + .

[0490] Step 2 Benzyl (R)-3-(((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)methoxy)-2-fluoro-2-methylpropanoate (2-4d)

[0491] To a solution of compound 2-4c (3.5 g, 5.52 mmol) in DMF (30 mL) was added EtNH (4.04 g, 55.2 mmol). The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under high vacuum to give compound 2-4d (2.2 g, crude) as an off-white solid, which was used directly without further purification.

[0492] MS (ESI) m / z: 412.4 [M+H] + .

[0493] Step 3 Benzyl (5S,8S,11S,17R)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-17-fluoro-8-isopropyl-11,17-dimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadecane-18-oate (2-4f)

[0494] To a solution of compound 2-4d (2.2 g, 5.35 mmol) in DMF (20 mL) was added compound 2-4e (3.04 g, 5.61 mmol), HATU (3.05 g, 8.02 mmol), and DIEA (1.38 g, 10.69 mmol). The mixture was stirred at room temperature for 30 min. The mixture was poured into CHCN (50 mL) and stirred for 30 min. The solid was filtered and further purified by trituration with CHCN:HO (50 mL, 10:1 v:v) to give compound 2-4f (4.1 g, 82% yield) as an off-white solid.

[0495] MS (ESI) m / z: 957.8 [M+Na] + .

[0496] Step 4 (5S,8S,11S,17R)-5-(3-((((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)amino)-3-oxopropyl)-1-(9H-fluoren-9-yl)-17-fluoro-8-isopropyl-11,17-dimethyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaoctadecanoic acid (2-4g)

[0497] To a solution of compound 2-4f (3.0 g, 3.21 mmol) in the co-solvent DMF-MeOH (40 mL, 1:1, v:v) was added Pd / C (10%, 300 mg). The mixture was stirred under H atmosphere (15 psi) for 7 hours. The mixture was filtered through a pad of Celite and concentrated to give compound 2-4g (2.5 mg, crude) as a white solid.

[0498] MS (ESI) m / z: 867.7 [M+Na] + .

[0499] Step 5 (9H-Fluoren-9-yl)methyl((6S,9S,12S,18R)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-19-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13 ,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-18-fluoro-9-isopropyl-12,18-dimethyl-3,7,10,13,19-pentaoxo-16-oxa-2,8,11,14-tetraazanonadecane-6-yl)carbamate (2-4h)

[0500] To a solution of compound exatecan mesylate (1000 mg, 1.18 mmol) in DMF (20 mL), compound 2-4g (692 mg, 1.3 mmol), HATU (675 mg, 1.78 mmol), and DIEA (459 mg, 3.55 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH = 100 / 0 to 20 / 80) to obtain the title compound 2-4h (1320 mg, 88.6% yield) as an off-white solid.

[0501] MS (ESI) m / z: 1285.0 [M+Na] + .

[0502] Step 6 (S)-2-amino-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-1-(((S)-1-((((R)-3-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-4i)

[0503] To a solution of compound 2-4h (1000 mg, 0.793 mmol) in DMF (20 mL) was added EtNH (580 mg, 7.93 mmol). The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under high vacuum to give compound 2-4i (824.6 mg, crude) as an off-white solid, which was used directly without further purification.

[0504] MS (ESI) m / z: 1040.9 [M+H] + .

[0505] Step 7 (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamide)-N1-((S)-1-(((S)-1-((((R)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydro Oxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-fluoro-2-methyl-3-oxopropoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-4)

[0506] To a solution of compound 2-4i (824 mg, 0.792 mmol) in DMF (15 mL), compound 2-4j (233.0 mg, 1.03 mmol), HATU (391.6 mg, 1.03 mmol), and DIEA (204.8 mg, 1.58 mmol) were added. The mixture was stirred at room temperature for 30 minutes. The mixture was purified by preparative HPLC (FA) (Method: Column: XBridge Prep C18 OBD 5 μm 19*150 mm, Mobile phase: A-water (0.1% formic acid): B-acetonitrile, Flow rate: 20 mL / min). The fractions were lyophilized to give compound 2-4 (375 mg, 37.9% yield) as a white solid.

[0507] MS (ESI) m / z: 1271.0 [M+Na] + .

[0508] General Procedure C: Preparation of Examples 2-7 [ka]

[0509] Step 1 (9H-Fluoren-9-yl)methyl ((7S)-7-benzyl-17-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)carbamate (2-7a)

[0510] Following the procedure described in Step 1 of Example 2-1, compound 2-7a (103 mg, purity 97%) was obtained.

[0511] MS (ESI) m / z: 1048.6 [M+H] + .

[0512] Step 2 (2S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((3-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (2-7b)

[0513] Following the procedure described in Step 4 of Example 2-1, compound 2-7b (105 mg, crude) was obtained.

[0514] MS (ESI) m / z: 826.5 [M+H] + .

[0515] Step 3 (9H-Fluoren-9-yl)methyl ((6S,15S)-15-benzyl-25-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,7,10,13,16,19-hexaoxo-1-((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-22-oxa-2,8,11,14,17,20-hexaazapentacosan-6-yl)carbamate (2-7c)

[0516] Following the procedure described in Step 5 of Example 2-1, compound 2-7c (50 mg, purity 98%) was obtained.

[0517] MS (ESI) m / z: 1352.8 [M+H] + .

[0518] Step 4 (2S)-2-amino-N 1 -((7S)-7-benzyl-17-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-N 5 -((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide (2-7d)

[0519] Following the procedure described in Step 6 of Example 2-1, compound 2-7d (52 mg, crude) was obtained.

[0520] MS (ESI) m / z: 1130.7 [M+H] + .

[0521] Step 5 (2S)-N 1 -((7S)-7-Benzyl-17-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2,5,8,11-tetraoxo-14-oxa-3,6,9,12-tetraazaheptadecyl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-N 5 -(((2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl)pentanediamide (2-7)

[0522] Following the procedure described in Step 7 of Example 2-1, compound 2-7 (33 mg, 98% purity) was obtained.

[0523] MS (ESI) m / z: 1323.8 [M+H] + .

[0524] General Procedure D: Preparation of Examples 2-8 [ka]

[0525] Step 1 (9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-(((3-((S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-113-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (2-8a)

[0526] Following the procedure described in Step 1 of Example 2-4, compound 2-8a (81 mg, 91% purity) was obtained.

[0527] MS (ESI) m / z: 900.8 [M+H] + .

[0528] Step 2 (S)-2-Amino-N-((S)-1-(((3-((S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-113-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (2-8b)

[0529] Following the procedure described in Step 4 of Example 2-4, compound 2-8b (83 mg, crude) was obtained.

[0530] MS (ESI) m / z: 678.7 [M+H] + .

[0531] Step 3 (9H-Fluoren-9-yl)methyl ((6S,9S,12S)-1-((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)-19-((S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-113-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-9-isopropyl-12-methyl-3,7,10,13-tetraoxo-16-oxa-2,8,11,14-tetraazanonadecane-6-yl)carbamate (2-8c)

[0532] Following the procedure described in Step 5 of Example 2-4, compound 2-8c (65 mg, 98% purity) was obtained.

[0533] MS (ESI) m / z: 1201.9 [M+H] + .

[0534] Step 4 (S)-2-amino-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-N1-((S)-1-(((S)-1-(((3-((S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-113-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-8d)

[0535] Following the procedure described in Step 6 of Example 2-4, compound 2-8d (68 mg, crude) was obtained.

[0536] MS (ESI) m / z: 979.8 [M+H] + .

[0537] Step 5 (S)-N5-(((2R,3S,4R,5S)-5-(2-amino-2-oxoethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)propanamide)-N1-((S)-1-(((S)-1-(((3-((S)-9-ethyl-5-fluoro- 9-Hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-113-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propoxy)methyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)pentanediamide (2-8)

[0538] Following the procedure described in Step 7 of Example 2-4, compound 2-8 (25 mg, 95% purity) was obtained.

[0539] MS (ESI) m / z: 1187.9 [M+H] + .

[0540] Example 2-2 [ka]

[0541] 2-2 (38 mg, 96% purity) was synthesized according to general procedure B.

[0542] MS (ESI) m / z: 1266.7 [M+Na] + .

[0543] Example 2-3 [ka]

[0544] 2-3 (30 mg, 98% purity) was synthesized according to general procedure A.

[0545] MS (ESI) m / z: 1408.1 [M+Na] + .

[0546] Examples 2-5 [ka]

[0547] 2-5 (51 mg, 97% purity) was synthesized according to general procedure C.

[0548] MS (ESI) m / z: 1292.0 [M+Na] + .

[0549] Examples 2-6 [ka]

[0550] 2-6 (34 mg, 95% purity) was synthesized according to general procedure B.

[0551] MS (ESI) m / z: 1255.0 [M+Na] + .

[0552] Examples 2-9 [ka]

[0553] 2-9 (9.7 mg, 99% purity) was synthesized according to general procedure B.

[0554] MS (ESI) m / z: 1287.0 [M+Na] + .

[0555] Example 2-10 [ka]

[0556] 2-10 (15 mg, 95% purity) was synthesized according to general procedure B.

[0557] MS (ESI) m / z: 1179.0 [M+Na] + .

[0558] Example 2-11 [ka]

[0559] 2-11 (23 mg, 98% purity) was synthesized according to general procedure D.

[0560] MS (ESI) m / z: 1173.9 [M+H] + .

[0561] Example 2-12 [ka]

[0562] 2-12 (4.1 mg, 95% purity) was synthesized according to general procedure C.

[0563] MS (ESI) m / z: 1337.0 [M+H] + .

[0564] Example 2-13 [ka]

[0565] 2-13 (13 mg, 98% purity) was synthesized according to general procedure C.

[0566] MS (ESI) m / z: 1375.2 [M+Na] + . [Table 1-1] [Table 1-2] [Table 1-3] [Table 2-1] [Table 2-2] [Table 2-3]

[0567] Example 3 Preparation and characterization of ADCs Preparation of antibody drug conjugates

[0568] Preparation of antibody-drug conjugates with a drug-to-antibody ratio (DAR) of 8 Antibody in conjugation buffer (concentration 0.5-25 mg / mL, PBS buffer (pH 6.0-8.5)) was incubated at reducing temperature (0-40 °C) for 10 min and 8-15 equivalents. TCEP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to proceed at reducing temperature for 1-8 h. After the reduction mixture was cooled to 0-25 °C, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker-payload stock (10-25 equivalents, 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0-25 °C for 1-3 h, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was subjected to buffer exchange (spin desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween® 20, 60, 80, etc.).

[0569] Preparation of DAR4 antibody drug conjugates Antibody in conjugation buffer (concentration 0.5-25 mg / mL, PBS buffer (pH 6.0-7.4)) was incubated at reduced temperature (0-37 °C) for 10 min and 1-6 equiv. TCEP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to proceed at reduced temperature for 1-18 h. After reduction, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker-payload stocks (4-15 equiv., 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0-25 °C for 1-3 h, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was subjected to buffer exchange (spin desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween® 20, 60, 80, etc.).

[0570] Preparation of DAR6 antibody-drug conjugate Antibody in conjugation buffer (concentration 0.5-25 mg / mL, PBS buffer (pH 6.0-7.4)) was incubated at reduced temperature (0-37 °C) for 10 min and 1-10 equivalents of TCEP. TCEP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to proceed at reduced temperature for 1-18 h. After reduction, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker-payload stocks (6-25 equivalents, 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0-25 °C for 1-3 h, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was subjected to buffer exchange (spin desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween® 20, 60, 80, etc.).

[0571] Example 4 Preparation of ADC3-antibody drug conjugates Antibody in conjugation buffer (concentration 0.5-25 mg / mL, PBS buffer (pH 6.0-7.0)) was incubated at reduced temperature (0-5 °C) for 10 min and 1-4 equiv. TCEP solution (5 mM stock in PBS buffer) was added to the reaction mixture, and the reduction reaction was allowed to proceed at reduced temperature for 1-18 h. After reduction, organic solvents (e.g., DMSO, DMF, DMA, PG, acetonitrile, 0-25% v / v) and linker-payload stock (10-25 equiv., 10 mM stock in organic solvent) were added stepwise. The conjugation solution was allowed to stand at 0-25 °C for 1-3 h, and the reaction was quenched with N-acetylcysteine ​​(1 mM stock). The solution was subjected to buffer exchange (spin desalting column, ultrafiltration, and dialysis) into a storage buffer (e.g., pH 5.5-6.5 histidine acetate buffer with optional additives such as sucrose, trehalose, Tween® 20, 60, or 80). The resulting ADC3-A,D4 species were in the range of 40-50% (Figure 30).

[0572] ADC3-A is a biosimilar ADC of DS7300a (benchmark). The average DAR was 3.99, as determined by HIC method 2 below.

[0573] Example 5 Maleimide Hydrolysis Process of Ring-Opened ADCs (Examples ADC3-2, ADC3-4, ADC3-6, ADC3-8, ADC3-9, ADC3-10, ADC3-11, ADC3-16)

[0574] After the linker-payload conjugation step, the resulting ADC underwent buffer exchange either by Amicon® ultrafiltration or desalting into a basic buffer (pH 8.5-9.5, Tris-acetate buffer or borate-acetate buffer). The reaction mixture was left for 18-24 hours, and the maleimide process was monitored by LCMS. After the maleimide hydrolysis rate reached >90%, the resulting ADC underwent buffer exchange into a formulation buffer (histidine acetate buffer, pH 5.5-6.5, containing optional additives such as sucrose, trehalose, and Tween® 20, 60, or 80).

[0575] Example 6 ADC characterization The disclosed ADC examples were prepared by following the procedures described above using the DAR8 profile. All ADCs were characterized by the following analytical methods:

[0576] The drug-to-antibody ratio (DAR) of the disclosed ADCs was determined by LCMS or HIC (hydrophobic interaction column) methods.

[0577] The SEC purity of the disclosed ADCs is all >95% pure.

[0578] LCMS method for DAR determination LC-MS analysis was carried out under the following measurement conditions: LC-MS system: Vanquish™ Flex UHPLC and Orbitrap Exploris 240 mass spectrometer Column: MAbPac(TM) RP, 2.1*50mm, 4μm, 1,500Å, Thermo Scientific(TM) Column temperature: 80℃ Mobile phase A: 0.1% formic acid (FA) in water Mobile phase B: Acetonitrile solution containing 0.1% formic acid (FA) Gradient program: 25% B to 25% B (0 min to 2 min), 25% B to 50% B (2 min to 18 min), 50% B to 90% B (18 min to 18.1 min), 90% B to 90% B (18.1 min to 20 min), 90% B to 25% B (20 min to 20.1 min), 25% B to 25% B (20.1 min to 25 min) Injected sample amount: 1 μg MS parameters: Intact and denatured MS data were acquired in HMR mode with R = 15k and deconvoluted using the ReSpect™ algorithm and sliding window integration in Thermo Scientific™ BioPharma Finder™ 4.0 software.

[0579] HIC method for determining DAR HPLC analysis was carried out under the following measurement conditions: HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID x 3.5 cm, 2.5 μm butyl non-porous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0 Gradient program: 0%B to 0%B (0 min to 2 min), 0%B to 100%B (2 min to 15 min), 100%B to 100%B (15 min to 16 min), 100%B to 0%B (16 min to 17 min), 0%B to 0%B (17 min to 20 min) Injected sample amount: 20 μg

[0580] SEC method for determining the purity of ADCs HPLC analysis was carried out under the following measurement conditions: HPLC system: Waters H-Class UPLC system Detector: Measurement wavelength: 280 nm Column: ACQUITY UPLC BEH200 SEC 1.7 μm 4.6 × 150 mm, Waters Column temperature: room temperature Mobile phase A: 200 mM phosphate buffer, 250 mM potassium chloride, 15% isopropyl alcohol, pH 7.0 Gradient program: 10 min isocratic elution at a flow rate of 0.3 mL / min Injected sample amount: 20 μg

[0581] HIC method for assessing the hydrophobicity of ADCs HIC method 1 HPLC analysis was carried out under the following measurement conditions: HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: Tosoh Bioscience 4.6 μm ID x 3.5 cm, 2.5 μm butyl non-porous resin column Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM phosphate buffer, pH 7.0 Mobile phase B: 50 mM phosphate buffer, 25% (V / V) isopropanol, pH 7.0 Gradient program: 0%B to 0%B (0 min to 2 min), 0%B to 100%B (2 min to 15 min), 100%B to 100%B (15 min to 16 min), 100%B to 0%B (16 min to 17 min), 0%B to 0%B (17 min to 20 min) Injected sample amount: 20 μg

[0582] HIC method 2 HPLC analysis was carried out under the following measurement conditions: HPLC system: Waters ACQUITY ARC HPLC system Detector: Measurement wavelength: 280 nm Column: MABPac HIC-10, 5 μm, 4.6 × 10 mm (Thermo) Column temperature: 25℃ Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 Mobile phase B: 50 mM sodium phosphate, pH 7.0 Gradient program: 20%B to 20%B (0 to 1 min), 0%B to 0%B (1 to 35 min), 20%B to 20%B (35 to 40 min) Flow rate: 0.5mL / min Sample preparation: Samples were diluted to 0.5 mg / mL in the initial mobile phase.

[0583] DAR8 HIC retention time (min) is an indicator of the relative hydrophobicity of an ADC. The higher the DAR8 HIC retention time, the more hydrophobic the ADC. As shown in Table 3 below, the disclosed ADCs are more hydrophilic than the reference ADCs ADC3-A and ADC3-B. [Table 3-1] [Table 3-2] [Table 3-3]

[0584] Example 7. Antibody Information

[0585] The antibodies and antigen-binding fragments thereof provided herein can be prepared by methods known in the art. The sequences of exemplary antibodies and antigen-binding fragments are shown in the table below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11]

[0586] Example 8. Assays and in vitro data Cell line information NCI-H1650 (ATCC, CRL-5883) NCI-H1650 is a cell line exhibiting epithelial morphology isolated from the lung tissue of a 27-year-old male smoker with stage 3B bronchoalveolar carcinoma in 1987. NCI-H1650 was purchased from ATCC. The basal medium for NCI-H1650 is ATCC-formulated RPMI-1640 medium, ATCC30-2001. To create complete growth medium, fetal bovine serum was added to the basal medium to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0587] NCI-H1048 (ATCC, CRL-5853) NCI-H1048 is a cell line exhibiting epithelial cell morphology, and was purchased from ATCC. The basal medium for NCI-H1048 is ATCC-formulated DMEM:F12 medium, catalog number 30-2006. To create complete growth medium, fetal bovine serum was added to the basal medium to a final concentration of 10% (Gibco, 10099-141C). Cell lines were grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0588] Capan-1 (ATCC, HTB-79) Capan-1 is an epithelial cell line isolated from the pancreas of a 40-year-old Caucasian male with pancreatic adenocarcinoma. Capan-1 was purchased from ATCC. The basal medium for Capan-1 is ATCC-formulated Iscove's Modified Dulbecco's Medium (catalog number 30-2005). To create complete growth medium, fetal bovine serum was added to the basal medium to a final concentration of 20% (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO2 atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710).

[0589] MDA-MB-453(SIBS) MDA-MB-453 was obtained from the effusion of a 48-year-old female patient with metastatic breast cancer, including lymph nodes, brain, and both the pleural and pericardial cavities. MDA-MB-453 was purchased from SIBS. The basal medium for MDA-MB-453 was RPMI 1640 medium, HEPES (Gibco, 22400105). To create complete growth medium, fetal bovine serum was added to the basal medium to a final concentration of 10% (Gibco, 10099-141C). The cell line was grown at 37°C in a humidified 5% CO atmosphere and periodically tested for the presence of mycoplasma using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, LT07-710). [Table 5]

[0590] Example 8a. ADC Direct Killing in NCI-H1650, Capan-1, NCI-H1048, and MDA-MB-453 Cancer Lines Method: ADC direct kill NCI-H1650, NCI-H1048, MDA-MB-453 (2E3 / well) or Capan-1 (4E3 / well) cells were seeded at 80 μl / well in 3D-96-well plates (Corning: 4520) and incubated overnight at 37°C, 5% CO2. Fresh growth medium containing various concentrations of ADC was added at 40 μl / well. Cells were incubated at 37° C., 5% CO for 6 days. Cell viability was detected with 100 μl / well of 3D reagent (Promega, G9683). The plate was incubated at room temperature for 30 minutes to stabilize the luminescence signal. Then, it was analyzed with a microplate reader.

[0591] Cell killing by anti-B7H3 ADCs is shown in Tables 6 to 10 and Figures 1 to 15. [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

[0592] Example 8b. ADC Bystander Killing in Co-culture of NCI-H358 with MDA-MB-453-nanoLuc Methods: Construction of MDA-MB-453-nanoLuc cell line PT67-nanoLuc cells were cultured, and then the cell culture medium (containing the virus (nano-Luc gene)) was collected and filtered. MDA-MB-453 cells were seeded into a 6-well plate at 1E5 cells / well and incubated overnight at 37°C, 5% CO2. PT67-nanoLuc cell medium and 8 μg / ml of polybrene were added. Infection was repeated three times, one day at a time. Next, MDA-MB-453-nanoLuc cells were cultured with 1 mg / ml of Geneticin for 5 days. MDA-MB-453-nanoLuc cells were harvested and Nano-Glo reagent (Promega: N1120) was added to test the nano-Luc transfection efficiency.

[0593] Method: ADC bystander killing NCI-H358 and MDA-MB-453-nanoLuc (10:1), or MDA-MB-453-nanoLuc cells alone, were seeded into 3D 96-well plates (Corning:4520) at 80 μl / well and incubated overnight at 37°C and 5% CO2. Fresh growth medium containing various concentrations of ADC was added at 40 μl / well. The cells were incubated at 37°C and 5% CO2 for 6 days. The 3D plates were centrifuged at 1500 rpm at 25°C for 5 minutes, after which the supernatant was discarded. Calu-6-nanoLuc cell viability was detected by Nano-Glo reagent (Promega: N1120), 150 μl / well. The 3D plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Then, it was analyzed by a microplate reader.

[0594] The bystander killing activity of B7H3ADC is shown in Tables 11 to 14 and Figures 16 to 23. [Table 11] [Table 12] [Table 13] [Table 14]

[0595] Example 8c. ADC Plasma Stability Evaluation Incubation of ADC with plasma The ADC was diluted in mouse or human plasma to give a final solution of 100 μg / mL of ADC in plasma. Samples were incubated at 37°C. Aliquots (100 μL) were taken at six time points (0, 4, 24, 72, 96, or 168 h). Samples were frozen at -80°C until analysis.

[0596] The plasma payload concentration was measured under the following conditions. Equipment: LC-MS / MS (Triple Quad 6500 plus) Monitor: MRM Column: Advanced Materials Technology, HALO AQ-C18 2.7μm 90Å, 50*2.1mm Column temperature: 40℃ Mobile phase A: HO-0.1% FA Mobile phase B: ACN-0.1%FA Gradient program for DXd (1-A) and compounds 1-1 to 1-9: 2% B to 2% B (0 min to 0.2 min), 2% B to 98% B (0.2 min to 1.2 min), 98% B to 98% B (1.2 min to 2.0 min), 98% B to 2% B (2.0 min to 2.01 min), 2% B to 2% B (2.01 min to 4.0 min). Injection volume: 10 μL (DXd and other payloads)

[0597] Plasma ADC and total Ab (Tab) concentrations were measured under the following conditions: Assay: Ligand binding assay (ELISA) Capture reagent: B7H3 extracellular domain (ECD) Detection reagents: anti-payload Ab for ADC, anti-human IgG polyclonal Ab for total Ab.

[0598] Changes in ADC DAR in human / mouse plasma stability study samples Methods: Human B7H3 ECD was biotinylated and immobilized on Dynabeads M-280 streptavidin. ADCs were captured from plasma samples using the ECD bead system for 2 hours at room temperature. The captured ADCs were then washed with HBS-EP buffer (10 mM Hepes [pH 7.4], 150 mM NaCl, 3.4 mM ethylenediaminetetraacetic acid [EDTA], 0.005% surfactant P20) and digested with IdeS enzyme for 1 hour at 37°C. After extensive washing of the beads with HBS-EP, water, and 10% acetonitrile, the ADC analytes were eluted using 30% acetonitrile containing 1% formic acid. Finally, a reduction treatment with 100 mM TCEP was performed for 45 minutes. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to analyze the DAR of ADCs.

[0599] result: 1. Stability of ADC3-1 in mouse and human plasma Although the ADC3-1 total mAb showed good stability, the conjugated ADC decreased to approximately 40% after 168 hours of incubation in mouse plasma, with a recovery of approximately 0.3% of free payload in plasma at the end of incubation (Figure 24).

[0600] Although the total mAb of ADC3-1 showed good stability, the conjugated ADC decreased to approximately 25% after 168 hours of incubation in human plasma, with approximately 0.5% recovery of free payload in plasma at the end of incubation. The mean DAR of the ADC decreased from 7.34 to 3.40 at the end of incubation (Figure 25).

[0601] 2. Stability of ADC3-2 in mouse and human plasma The ADC3-2 total mAb and conjugated ADC showed good stability after 168 hours of incubation in mouse plasma; plasma free payload recovery at the end of the incubation was approximately 0.3% (Figure 26).

[0602] The ADC3-2 total mAb and conjugated ADC showed good stability after 168 hours of incubation in human plasma; plasma free payload recovery at the end of incubation was approximately 0.1%. The mean DAR of the ADC decreased from 7.86 to 7.78 at the end of incubation (Figure 27).

[0603] 3. Stability of ADC3-4 in mouse and human plasma The total mAb and conjugated ADC of ADC3-4 showed good stability after 168 hours of incubation in mouse plasma; plasma free payload recovery at the end of the incubation was approximately 0.2% (Figure 28).

[0604] The total mAb and conjugated ADC of ADC3-4 showed good stability after 168 hours of incubation in human plasma; plasma free payload recovery at the end of the incubation was approximately 0.4% (Figure 29).

[0605] Example 9. In vivo efficacy studies of ADCs method Female BALB / c nude mice were injected with 3 x 10 cells per 200 μL PBS / Matrigel into the right flank. 6 H1650 cells were subcutaneously implanted. After inoculation, tumor volume was measured twice a week in two dimensions using calipers and calculated using the formula: V = 0.5(a × b 2 ) to mm 3 (where a and b are the long and short diameters of the tumor, respectively). The tumors had an average volume of approximately 200 mm 3 At that time, mice were randomly assigned to groups of 8 animals and treated intravenously on day 1 with vehicle or ADC as follows: (1) ADC3-A, ADC3-1, ADC3-3, ADC3-14, or ADC3-15 at 3 / 10 mg / kg; (2) ADC3-A at 10 mg / kg, or ADC3-2, ADC3-4, ADC3-5, or ADC3-6 at 3 mg / kg. ADC3-6, (3) ADC3-A at 10 mg / kg or ADC3-8 or ADC3-9 at 3 mg / kg, (4) ADC3-A at 10 mg / kg or ADC3-10, ADC3-11, ADC3-12, or ADC3-13 at 3 mg / kg, (5) ADC3-A, ADC3-1, ADC3-3, ADC3-14, or ADC3-15 at 3 / 10 mg / kg.

[0606] Partial regression (PR) was defined as a tumor volume less than 50% of the starting tumor volume on the first day of treatment in three consecutive measurements, and complete regression (CR) was defined as a tumor volume less than 14 mm in three consecutive measurements. 3 Tumor growth inhibition (TGI) was defined as a tumor volume less than 0.05% of the control group. Data are presented as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGI) is calculated using the formula (TGI):

number

[0607] Results: (1) ADC3-A, ADC3-1, ADC3-3, ADC3-14, and ADC3-15 The in vivo efficacy of ADC3-A, ADC3-1, ADC3-3, ADC3-14, and ADC3-15 was compared in H1650 xenografts (B7H3IHC3+) grown subcutaneously in BALB / c nude mice. Treatment with 3 / 10 mg / kg ADC3-A, 3 / 10 mg / kg ADC3-1, 3 / 10 mg / kg ADC3-3, 3 / 10 mg / kg ADC3-14, or 3 / 10 mg / kg ADC3-15 resulted in TGI of 53% / 86%, 90% / 104%, 84% / 103%, 73% / 98%, and 68% / 96%, respectively, at day 28. ADC3-1 demonstrated superior efficacy to ADC3-A at both 3 mg / kg and 10 mg / kg. ADC3-3 showed comparable efficacy to ADC3-A at 3 mg / kg, but superior efficacy at 10 mg / kg. ADC3-14 and ADC3-15 showed comparable efficacy to ADC3-A at both 3 mg / kg and 10 mg / kg. All ADCs were well tolerated without any signs of toxicity or significant weight loss (Figures 31A-31L and Table 15). [Table 15]

[0608] Results: (2) ADC3-A, ADC3-2, ADC3-4, ADC3-5, and ADC3-6 The in vivo efficacy of ADC3-A was compared with that of ADC3-2, ADC3-4, ADC3-5, and ADC3-6 in H1650 xenografts (B7H3 IHC3+) grown subcutaneously in BALB / c nude mice. Treatment with 10 mg / kg ADC3-A or 3 mg / kg ADC3-2, ADC3-4, ADC3-5, or ADC3-6 resulted in 85%, 104%, 103%, 85%, and 96% TGI at day 30, respectively. In terms of tumor growth inhibition in individual animals, ADC3-2, ADC3-4, and ADC3-6 induced 2 / 8 PR, 1 / 8 PR, and 1 / 8 PR, respectively. In summary, ADC3-2 and ADC3-4 at 3 mg / kg demonstrated superior efficacy compared with 10 mg / kg ADC3-A. ADC3-5 and ADC3-6 at 3 mg / kg demonstrated efficacy comparable to that of ADC3-A at 10 mg / kg. All ADCs were well tolerated without any signs of toxicity or significant weight loss (Figures 32A-32G and Table 16). [Table 16]

[0609] Results: (3) ADC3-A, ADC3-8, and ADC3-9 The in vivo efficacy of ADC3-A, ADC3-8, and ADC3-9 was compared in H1650 xenografts (B7H3 IHC3+) grown subcutaneously in BALB / c nude mice. Treatment with 10 mg / kg ADC3-A, 3 mg / kg ADC3-8, or 3 mg / kg ADC3-9 resulted in 85%, 21%, and 69% TGI at day 30, respectively. 3 mg / kg ADC3-8 was less effective than 10 mg / kg ADC3-A. 3 mg / kg ADC3-9 demonstrated efficacy comparable to 10 mg / kg ADC3-A. All ADCs were well tolerated without any signs of toxicity or significant weight loss (Figures 33A-33E and Table 17). [Table 17]

[0610] Results: (4) ADC3-A, ADC3-10, ADC3-11, ADC3-12, and ADC3-13 The in vivo efficacy of ADC3-A, ADC3-10, ADC3-11, ADC3-12, and ADC3-13 was compared in H1650 xenografts (B7H3 IHC3+) grown subcutaneously in BALB / c nude mice. Treatment with 10 mg / kg ADC3-A or 3 mg / kg ADC3-10, ADC3-11, ADC3-12, or ADC3-13 resulted in 70%, 107%, 60%, 75%, and 56% TGI at day 28, respectively. Regarding tumor growth inhibition in individual animals, ADC3-10 induced 3 / 8 PR and 1 / 8 CR. In summary, ADC3-10 at 3 mg / kg demonstrated superior efficacy compared with ADC3-A at 10 mg / kg. ADC3-11, ADC3-12, and ADC3-13 at 3 mg / kg demonstrated efficacy comparable to that of ADC3-A at 10 mg / kg. All ADCs were well tolerated without any signs of toxicity or significant weight loss (Figures 34A-34G and Table 18). [Table 18]

[0611] Results: (5) ADC3-A, ADC3-1, ADC3-3, ADC3-14, and ADC3-15 The in vivo efficacy of ADC3-A, ADC3-1, ADC3-3, ADC3-14, and ADC3-15 was compared in H1975 xenografts (B7H3 IHC3+) grown subcutaneously in BALB / c nude mice. Treatment with 3 / 10 mg / kg ADC3-A, 3 / 10 mg / kg ADC3-1, 3 / 10 mg / kg ADC3-3, 3 / 10 mg / kg ADC3-14, or 3 / 10 mg / kg ADC3-15 resulted in TGI of 56% / 74%, 109% / 110%, 109% / 110%, 103% / 109%, and 101% / 109%, respectively, on day 31. ADC3-1, ADC3-3, ADC3-14, and ADC3-15 demonstrated superior efficacy to ADC3-A at both 3 mg / kg and 10 mg / kg. All ADCs were well tolerated without any signs of toxicity or significant weight loss (Figures 35A-35L and Table 19). [Table 19] Example 10. Humanization of anti-human B7H3 mAb BGA-3295 For humanization of BGA-3295, human germline IgG genes were searched for sequences sharing high homology with the cDNA sequences of the BGA-3295 variable regions by comparing them with the human immunoglobulin gene databases on the IMGT and NCBI websites. Human IGVH and IGVκ genes, which are frequently present in the human antibody repertoire (Glanville et al., 2009 PNAS 106:20216-20221) and share high homology with BGA-3295, were selected as templates for humanization.

[0612] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and a humanized antibody based on BGA-3295 was engineered in a human IgG1 variant (SEQ ID NO: 31) format using an in-house developed expression vector. In the first round of humanization, mutations from murine to human amino acid residues in the framework regions were guided by simulated 3D structures, and structurally important murine framework residues for maintaining the canonical structure of the CDRs were retained in the first version of the humanized antibody BGA-4348. Specifically, the CDRs of BGA-3295Vκ (SEQ ID NOs: 600-800) were substituted with two murine framework residues (S 49 and V 54 The CDRs of BGA-3295Vh (SEQ ID NOs: 300-500) were grafted onto the framework of the human germline variable gene IGVκ4-1 (SEQ ID NOs: 1400 and 1600), retaining the four mouse framework residues (I2, Y). 27 , A 68 , and K 71 ) was grafted into the framework of the human germline variable gene IGVH4-1 (SEQ ID NOs: 1300 and 1500).

[0613] Humanized antibody BGA-4348 was constructed in a full-length human antibody format using an in-house developed expression vector containing the constant regions of a human IgG1 variant (SEQ ID NO: 31) and a kappa chain, respectively, with adaptable subcloning sites. Expression and preparation of the humanized antibody can be achieved by cotransfection of the heavy chain construct and the corresponding light chain construct into 293G cells (developed in-house) and purification using a Protein A column. The purified antibody was concentrated to 0.5-5 mg / mL in PBS and stored in aliquots at -80°C in a freezer.

[0614] Based on BGA-4348, several single mutations were introduced to convert retained murine residues in the framework regions to the corresponding human germline residues. Humanized antibodies were also engineered by introducing mutations into the CDR regions to remove potential post-translational modification (PTM) sites and improve stability in therapeutic applications in humans. All humanized mutations were generated using primers containing mutations at specific positions and a site-directed mutagenesis kit (catalog number FM111-02, TransGen, Beijing, China). The desired mutations were verified by sequencing analysis. These humanized antibodies were tested in binding assays described elsewhere.

[0615] In summary, humanized monoclonal antibodies, BGA-4348 (see Table 4) and BGA-5063 (see Table 4), were obtained from the above-described mutation process and were extensively characterized.

[0616] For affinity measurement, the antibodies were captured by anti-human Fc surface and used in affinity assays based on surface plasmon resonance (SPR) technology. The results of SPR-determined binding profiles of the humanized antibodies to the ECD of human 4Ig-B7H3 (Sinobiological, Cat. No. 11188-H08H) are summarized in Table 20. BGA-4348 (see Table 4) and BGA-5063 (see Table 4) had dissociation constants of 0.6 nM and 0.9 nM, respectively, and had binding affinities comparable to those of BGA-3295. [Table 20]

[0617] To assess the binding activity of humanized antibodies to native B7H3 in live cells, NK92mi cells were engineered to overexpress human 4Ig-B7H3. Live NK92mi / B7H3 cells were seeded into 96-well plates and incubated with serial dilutions of chimeric or humanized antibodies. Goat anti-human IgG was used as a secondary antibody to detect antibody binding to the cell surface. EC2 values ​​for dose-dependent binding to human native B7H3 were50 Values ​​were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in Table 21, the humanized antibodies maintained binding affinity for native B7H3. [Table 21]

[0618] Example 11. Epitope mapping of anti-B7H3 antibodies To study the binding epitope of the anti-B7H3 mAb BGA-6938, we generated domain-truncated human B7H3 by fusing each Ig-like domain from the extracellular region of human B7H3 (SEQ ID NO: 801), i.e., IgV1 (amino acids 29-139 of SEQ ID NO: 801), IgC1 (amino acids 145-238 of SEQ ID NO: 801), IgV2 (amino acids 243-357 of SEQ ID NO: 801), and IgC2 (amino acids 363-456 of SEQ ID NO: 801), to its transmembrane and intracellular domains. The truncated versions of B7H3 were also fused to an N-terminal FLAG tag. The resulting truncated human B7H3 construct contains an N-terminal FLAG tag, followed by the Ig-like domains, including the transmembrane and intracellular domains, and the B7H3 C-terminal domain. DNA encoding the truncated versions of B7H3 was cloned into the pcDNA3.4 vector.

[0619] Plasmids containing these truncated B7H3 constructs were used to transfect ExpiCHO™ cells for transient protein expression, followed by incubation with 100 nM purified BGA-6938 and the reference antibody DS-7300 (Daiichi Sankyo) (US2022 / 0064312A1). Binding of BGA-6938 and DS-7300 to various B7H3 truncated forms was assessed by detection with Alexa Fluor 647 rabbit anti-human IgG (catalog: 309-605-008 Jackson ImmunoResearch). Expression of each construct was verified by detection of the FLAG tag at the N-terminus after incubation of transfected cells with anti-FLAG mAb (catalog: A01809, Genscript). BGA-6938 specifically binds to the B7H3 V1 and V2 domains, but not to the B7H3 C1 and C2 domains. In contrast, the reference antibody DS-7300 binds to the C1 and C2 domains of B7H3, but not to the V1 and V2 domains. Thus, BGA-6938 and DS-7300 have non-overlapping epitopes.

[0620] The invention is generally disclosed herein using affirmative language to describe many embodiments. The invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses, etc., is excluded, in whole or in part. Thus, the invention is generally not expressed herein in terms of what the invention does not include, but aspects not expressly included in the invention are nevertheless disclosed herein.

[0621] Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain minor changes and modifications may be practiced. Accordingly, the descriptions and examples should not be construed as limiting the scope of the invention.

[0622] Where any publication is referred to herein, it is to be understood that such reference is not an admission that the publication forms part of the common general knowledge in the art in any country.

[0623] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein by an identifying citation are hereby incorporated by reference in their entirety.

Claims

1. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof capable of specifically binding to human B7H3, and a cytotoxic agent.

2. 2. The antibody drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof is: (i) a heavy chain variable region (VH) comprising (a) HCDR1 (heavy chain complementarity determining region 1) of SEQ ID NO: 11, (b) HCDR2 of SEQ ID NO: 2, and (c) HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) LCDR1 (light chain complementarity determining region 1) of SEQ ID NO: 23, (e) LCDR2 of SEQ ID NO: 5, and (f) LCDR3 of SEQ ID NO: 6; (ii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (iv) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 14, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (v) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 4, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vi) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 20, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (vii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 1, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (viii) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 11, (b) an HCDR2 of SEQ ID NO: 2, and (c) an HCDR3 of SEQ ID NO: 28, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 23, (e) an LCDR2 of SEQ ID NO: 5, and (f) an LCDR3 of SEQ ID NO: 6; (ix) a heavy chain variable region (VH) comprising (a) an HCDR1 of SEQ ID NO: 300, (b) an HCDR2 of SEQ ID NO: 1700, and (c) an HCDR3 of SEQ ID NO: 500, and a light chain variable region (VL) comprising (d) an LCDR1 of SEQ ID NO: 600, (e) an LCDR2 of SEQ ID NO: 700, and (f) an LCDR3 of SEQ ID NO: 800; or (x) The antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to (1) an epitope comprising or consisting of amino acid residues 29 to 139 of human 4Ig-B7H3 (SEQ ID NO: 801), and / or (2) an epitope comprising or consisting of amino acid residues 243 to 357 of human 4Ig-B7H3 (SEQ ID NO: 801), and / or an epitope comprising or consisting of amino acid residues 145 to 238 of human 4Ig-B7H3 (SEQ ID NO: 801), and / or (4) an epitope comprising or consisting of amino acid residues 363 to 456 of human 4Ig-B7H3 (SEQ ID NO: 80).

3. 3. The antibody drug conjugate of claim 1 or 2, having the formula: Ab-(L-(D)m)n, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is the antibody or antigen-binding fragment thereof; L is a linker, D is a residue of the cytotoxic agent; m is an integer from 1 to 8, and The antibody-drug conjugate, wherein n is 1 to 10.

4. 4. The antibody drug conjugate of claim 3, wherein m is 1.

5. The antibody-drug conjugate of claim 3 or 4, wherein n is 3 to 10.

6. 6. The antibody drug conjugate of claim 5, wherein n is about 8.

7. Formula (II): 【Chemistry 1】 or a tautomer, stereoisomer, pharmaceutically acceptable salt or solvate thereof, and wherein Su is a hydrophilic residue.

8. Su, 【Chemistry 2】 8. The antibody-drug conjugate of claim 7, wherein:

9. Formula (III): 【Transformation 3】 or Formula (IIIo): 【Chemistry 4】 or Formula (IIIoo): 【Transformation 5】 or a tautomer, stereoisomer, pharmaceutically acceptable salt, or solvate thereof, wherein Su is a hydrophilic residue.

10. Su, 【Transformation 6】 10. The antibody-drug conjugate of claim 9, wherein:

11. The antibody drug conjugate of any one of claims 3 to 10, wherein D is 【Transformation 7】 wherein: Y is -ABC'-D'-*, where * represents the bond connecting D to the antibody drug conjugate; A is a bond, CR １ R ２ , or N-R １ and B is a bond, —C(═O)—, or —C(═O)O—; C' is a bond or a divalent group, wherein the divalent group is an unsubstituted or substituted C １－８ alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, or unsubstituted or substituted heteroaryl; D' is a bond, NH, or O; R １ and R ２ are each independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R １ and R ２ together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl; R ３ and R ４ are each independently hydrogen, halogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxyl, or R ３ and R ４ together with the atom to which they are attached form an unsubstituted or substituted cycloalkyl, an unsubstituted or substituted heterocyclyl, an unsubstituted or substituted aryl, or an unsubstituted or substituted heteroaryl.

12. D is, 【Transformation 8】 and In the formula, R ７ and R ８ The antibody drug conjugate of any one of claims 3 to 11, wherein each is independently hydrogen, halogen, or alkyl.

13. D is, 【Chemistry 9】 The antibody-drug conjugate of any one of claims 3 to 10, wherein

14. D is, 【Chemistry 10】 14. The antibody-drug conjugate of claim 13, wherein:

15. 7. The antibody drug conjugate of any one of claims 3 to 6, having one of the following formulas, or a tautomer, stereoisomer, pharmaceutically acceptable salt, or solvate thereof: 【Chemistry 11-1】 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】 【Chemistry 11-5】

16. 7. The antibody drug conjugate of any one of claims 3 to 6, having one of the following formulas, or a tautomer, stereoisomer, pharmaceutically acceptable salt, or solvate thereof: 【Chemistry 12-1】 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 【Chemistry 12-5】

17. 17. The antibody drug conjugate of any one of claims 1 and 3 to 16, wherein the antibody or antigen-binding fragment is: (i) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (ii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8; (iii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (iv) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (v) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 18, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; (vi) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21; (vii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; (viii) a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; or (ix) the antibody drug conjugate, comprising: a heavy chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1800; and a light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1400.

18. 18. The antibody drug conjugate of claim 17, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids within each of SEQ ID NOs: 26 and 24, each of SEQ ID NOs: 7 and 8, each of SEQ ID NOs: 12 and 8, each of SEQ ID NOs: 15 and 8, each of SEQ ID NOs: 18 and 8, each of SEQ ID NOs: 7 and 21, each of SEQ ID NOs: 7 and 24, each of SEQ ID NOs: 29 and 24, each of SEQ ID NOs: 1800 and 1400 have been inserted, deleted, or substituted into the antibody or antigen-binding fragment.

19. 18. The antibody drug conjugate of any one of claims 1 and 3 to 17, wherein the antibody or antigen-binding fragment is: (i) a heavy chain variable region comprising SEQ ID NO: 26, and a light chain variable region comprising SEQ ID NO: 24; (ii) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 8; (iii) a heavy chain variable region comprising SEQ ID NO: 12 and a light chain variable region comprising SEQ ID NO: 8; (iv) a heavy chain variable region comprising SEQ ID NO: 15, and a light chain variable region comprising SEQ ID NO: 8; (v) a heavy chain variable region comprising SEQ ID NO: 18, and a light chain variable region comprising SEQ ID NO: 8; (vi) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 21; (vii) a heavy chain variable region comprising SEQ ID NO: 7, and a light chain variable region comprising SEQ ID NO: 24; (viii) a heavy chain variable region comprising SEQ ID NO: 29 and a light chain variable region comprising SEQ ID NO: 24; or (ix) the antibody drug conjugate, comprising a heavy chain variable region comprising SEQ ID NO: 1800, and a light chain variable region comprising SEQ ID NO: 1400.

20. 20. The antibody drug conjugate of any one of claims 1 to 19, wherein the antibody or antigen-binding fragment is a monoclonal antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment, or a F(ab')2 fragment.

21. 21. The antibody drug conjugate of any one of claims 1 to 20, wherein the antibody or antigen-binding fragment comprises an scFv comprising a VH having the amino acid sequence of SEQ ID NO:26 and a VL having the amino acid sequence of SEQ ID NO:24, optionally wherein the VH and VL are connected via an amino acid linker, and optionally wherein the amino acid linker is any sequence from SEQ ID NO:35 to SEQ ID NO:

77.

22. 22. The antibody drug conjugate of claim 21, wherein the antibody or antigen-binding fragment comprises an scFv having the amino acid sequence of SEQ ID NO:

32.

23. 23. The antibody drug conjugate of any one of claims 1 to 22, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1, IgG2, IgG3, or IgG4 subclass, or a light chain constant region of the kappa or lambda type.

24. 24. The antibody drug conjugate of claim 23, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the IgG1 subclass and a light chain constant region of the kappa type.

25. 25. The antibody drug conjugate of any one of claims 1 to 24, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of amino acid residues 29 to 139 of human 4Ig-B7H3 (SEQ ID NO: 801), and / or amino acid residues 243 to 357 of human 4Ig-B7H3 (SEQ ID NO: 801), and / or amino acid residues 145 to 238 of human 4Ig-B7H3 (SEQ ID NO: 801), and / or amino acid residues 363 to 456 of human 4Ig-B7H3 (SEQ ID NO: 801).

26. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

27. 27. A method for treating 4Ig-B7H3 positive cancer, comprising administering to a patient in need thereof an effective amount of the antibody drug conjugate of any one of claims 1 to 25 or the pharmaceutical composition of claim 26.

28. 28. The method of claim 27, wherein the cancer is colorectal cancer, prostate cancer, breast cancer, lung cancer, or esophageal cancer.

29. 28. The method of claim 27, wherein the cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

30. 30. The method of claim 29, wherein the non-small cell lung cancer is squamous non-small cell lung cancer.

31. 28. The method of claim 27, wherein the cancer is esophageal squamous cell carcinoma.

32. The method of any one of claims 27 to 31, wherein the antibody drug conjugate is administered in combination with another therapeutic agent.

33. 33. The method of claim 32, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, or 5-azacytidine.

34. 34. The method of claim 32 or 33, wherein the therapeutic agent is an immune checkpoint inhibitor.

35. 35. The method of claim 34, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

36. 36. The method of claim 35, wherein the anti-PD-1 antibody is tislelizumab.

37. Formula (IIIa): 【Chemistry 13】 or a pharmaceutically acceptable salt thereof, wherein Su is 【Chemistry 14】 and D is a residue of a cytotoxic agent, or a pharmaceutically acceptable salt thereof.

38. D is, 【Chemistry 15】 38. The compound of claim 37, wherein:

39. 38. The compound of claim 37, comprising: 【Chemistry 16】 or a pharmaceutically acceptable salt thereof.

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