Alk5 inhibitor conjugates and uses thereof

Abstract

The present disclosure relates to targeted drug conjugates comprising an ALK5 inhibitor and a targeting moiety that directs the ALK5 inhibitor to cells involved in fibrosis and cancer, such as myofibroblasts, activated fibroblasts and transitional fibroblasts, and their uses, in particular wherein the ALK5 inhibitor is N-methyl-2-(4-(4-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethan-1-amine.

Description

[0001] 1. Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 958,461, filed January 8, 2020, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] 2.1 Fibrosis

[0004] Idiopathic pulmonary fibrosis (IPF) is a destructive chronic lung disease characterized by progressive hardening and scarring of the lung tissue (Lederer et al., 2018, NEJM, 378:1811-23; Barratt et al., 2018, J Clin Med 7(8):201). Approximately 130,000 patients are diagnosed annually in the United States, with a 5-year mortality rate of 80%. To date, there is no cure for this disease, only options to slow its progression (Somogyi et al., 2019, Eur Respir Rev, 28(153):190021). IPF begins with repeated damage to the alveolar epithelium from external irritants such as smoking, followed by persistent fibroblast activation, one of the driving factors of fibrosis. Due to the continuous lung damage, fibrosis is essentially an unhealable wound.

[0005] The differentiation of lung fibroblasts into myofibroblasts is a major step in the development of tissue fibrosis (Yazdani et al., 2017, Adv Drug Deliv Rev 121:101-116; Huang et al., 2014, Austin J Pulm Resp, 1(1):3-9). Myofibroblasts are responsible for fibrosis and are primarily found and active in fibrotic areas. There are three possible sources of myofibroblasts in IPF: 1) resident lung epithelial cells that transform into myofibroblasts during a process called epithelial-mesenchymal transition (EMT); 2) resident lung fibroblasts that transform into myofibroblasts (FMT); and / or 3) myofibroblasts recruited to the lungs to drive fibrosis and scar formation (Pardali et al., 2017, Int J Mol Sci, 18(10)). The combination of these three pathways leads to an increase in resident lung myofibroblasts, thereby driving fibrotic disease. Inhibiting myofibroblasts will be an important step in reversing fibrotic lung disease.

[0006] The pleiotropic cytokine transforming growth factor-β (TGF-β) is responsible for the development, maintenance, and homeostasis of most tissues in the body. TGF-β initiates signal transduction by binding to TGF-β receptor II and TGF-β receptor I / ALK5. ALK5 is a serine-threonine kinase receptor that phosphorylates downstream signal transduction mediators Smad2 and Smad3. Activated Smad2 / 3 form a complex with Smad4 and translocate to the nucleus to regulate gene expression, which is determined by the cellular environment (Derynck et al., 2003, Nature, 425(6958):577-84). In the lung, TGF-β is produced by a wide variety of cell types, including alveolar macrophages, neutrophils, activated alveolar epithelial cells, endothelial cells, fibroblasts, and myofibroblasts (Caja et al., 2018, Int J Mol Sci, 19(5)). TGF-β is one of the most potent inducers of extracellular matrix (ECM) production, which includes α-smooth muscle actin (αSMA), collagen, and fibronectin (Pohlers et al., 2009, Biochim Biophys Acta, 1792(8):746-56; Kim et al., 2018, Cold Spring Harb PerspectBiol, 10(4)). During the progression of IPF, TGF-β increases collagen expression and ECM deposition, myofibroblast expansion, fibroblast-to-myofibroblast transformation, and epithelial-mesenchymal transition (EMT) (Pardali et al., 2017, Int JMol Sci, 18(10); Yue et al., 2010, Curr Enzym Inhib, 6(2)). Furthermore, TGF-β expression was elevated in both animal models of pulmonary fibrosis and fibrotic human lungs (Tashiro et al., 2017, Front Med (Lausanne), 4:118). In animal models of pulmonary fibrosis, elevated TGF-β levels preceded collagen synthesis and deposition. As further evidence of TGF-β's role as a driver of pulmonary fibrosis in vivo, adenovirus expressing TGF-β1 in the lungs or transgenic lungs specifically expressing TGF-β1 were sufficient to drive pulmonary fibrosis (Lee et al., Korean J Intern Med, 29:281).In the classic mouse model of bleomycin-induced IPF, TGF-β levels in the lungs are elevated. Blocking TGF-β signaling by Smad 3 knockout mice or TGFbRII in fibroblasts with specific dominant-negative expression leads to a reduction in disease severity (Fernandez et al., 2012, Proc Am Thorac Soc, 9(3): 111-116; Degryse et al., 2011, Am J Physiol Lung Cell Mol Physiol, 300(6): 887-897; Li et al., 2011, J ClinInvest, 121(1): 277-87). In terms of treatment, small molecule TGF-β receptor inhibitors or anti-TGF-β antibodies have also suppressed the disease in bleomycin and radiation-induced fibrosis (Giri et al., 1993, Thorax, 48: 959-66; Flechsiget al., 2012, Clin Cancer Res, 18(13): 3616-27).

[0007] Due to the prominent role of TGF-β in driving IPF, therapies targeting the TGF-β pathway have been investigated for the treatment of IPF. However, the risk of host tissue toxicity due to the widespread expression of TGF-β and its receptor in vivo makes the development of safe and effective therapies difficult (Anderton et al., 2011, Toxicologic Path, 39:916-24; Stauber et al., 2014, Clinical Tox, 4(3):1-10; Lonning et al., 2011, Curr Pharma Biotech, 12:2176-89). For example, a phase 2 trial (BG00011) of an αvβ6 integrin antibody that systemically blocks TGF-β activity was recently terminated due to safety concerns (Arefayene, et al., 2018, European Respiratory Journal, 52(suppl 62) PA596). As with most drugs, toxicity and therapeutic window must be balanced; for broad-acting TGF-β inhibitors, safety and toxicity risks are paramount. Selective and potent TGF-β inhibitors that can safely reverse fibrosis may stop disease progression and potentially improve patient survival.

[0008] In 2014, two IPF drugs were approved: pirfenidone (an antifibrotic molecule) and nintedanib (a tyrosine kinase inhibitor). Both drugs may partially block TGF-β signaling and other pathways (Gan et al., 2011, Ther Clin Risk Manag, 7:39-47; Margaritopoulos et al., 2016, Core Evid, 11:11-22; Lunardi et al., 2018, Arch Pathol Lab Med, 142:1090-1097). Generally, pirfenidone and nintedanib treatment can reduce the risk of IPF progression in patients with mild to moderate disease by 50% (Ren et al., 2017, Saudi Med J, 38(9):889-894; Case et al., 2017, BMJ OpenResp Res, 4:e000192). However, patients with IPF whose lung function has declined by <50% (measured by FVC, forced vital capacity, and total exhaled air), elderly patients with comorbidities, or patients not formally diagnosed with IPF were excluded from these trials. While both medications can slow the disease, they cannot completely stop or reverse its progression. Trials of IPF have generated considerable interest due to the significant unmet needs among fibrosis patients.

[0009] In the treatment of IPF, other therapies, such as IFN-γ inhibitors, angiogenesis inhibitors, and TNF-α blockers, have proven unsuccessful (Yazdani et al., 2017, Adv Drug Deliv Rev, 121:101-116; Somogyi et al., 2019, Eur Respir Rev, 28(153):190021). Ongoing IPF trials involve serum amyloid P (Pentraxin; PTX-2), a circulating protein that binds to monocytes and inhibits their differentiation into profibrotic fibroblasts, thereby promoting epithelial healing and fibrosis regression. IPF patients have low levels of Pentraxin, and an ongoing phase 2 trial has shown improvements in lung function and the 6-minute walk test. In a phase 2 trial, pamrevlumab, a fully recombinant human monoclonal antibody against connective tissue growth factor (CTGF), reduced fibrosis and decreased lung function (FVC) in patients with IPF (Somogyi et al., 2019, Eur Respir Rev, 28(153):190021). In contrast, tralokinumab, an IL-13 antibody that reduces TGF-β and CCl2 expression, and simtuzumab, an anti-LOXL2 antibody that reduces ECM crosslinking, both failed in phase 2 trials due to a lack of improvement in respiratory function (Raghu, 2017, European Respiratory Review, 26:170071). Many therapies directly or indirectly alter TGF-β function. However, despite these efforts, the need for improved treatment for patients with IPF remains unmet, particularly for therapies that can selectively and safely alter the disease.

[0010] Fibrosis is driven by TGF-β in a variety of diseases other than IPF, including other types of pulmonary fibrosis (e.g., associated with systemic sclerosis), liver fibrosis (e.g., associated with nonalcoholic steatohepatitis (NASH)), renal fibrosis, and cardiac fibrosis (Meng et al., 2016, Nat Rev Nephrol. 12(6):325-38; Biernacka et al., 2011, growth Factors, 29(5):196–202; gyorfi et al., 2017, Matrix Biology, 68-69:8-27). Therefore, there is an unmet need for therapies that can reverse TGF-β-driven myofibroblast activation and reduce fibrosis in patients, particularly those with pulmonary fibrosis (e.g., IPF), liver fibrosis (e.g., associated with NASH), renal fibrosis, cardiac fibrosis, and systemic sclerosis.

[0011] 2.2 Cancer

[0012] TGF-β signaling is also associated with tumor progression, and there has long been interest in inhibiting the TGF-β pathway as a cancer therapy (Syed, 2016, J Cell Biochem. 117(6): 1279-87). However, due to concerns about host toxicity, as TGF-β receptors are ubiquitous and there are worries that they might inadvertently promote tumor growth, most TGF-β inhibitors remain in the preclinical discovery stage.

[0013] TGF-β is secreted by tumor cells, cancer-associated fibroblasts (CAFs), and / or surrounding tumor microenvironment (TME) cells. CAFs are most abundant in the stromal cells of the TME and are closely associated with cancer progression (Pure and Blomberg, 2018, Oncogene, 37(32):4343-4357; Calon et al., 2014, Seminars in CancerBio, 25:15-22; Chen and Song, 2019, Nat Rev Drug Disc. 18:90). TGF-β is a key driver of CAF activation, recruitment, and activity, driving CAF differentiation from tissue-resident fibroblasts and epithelial cells and supporting their survival via epithelial-mesenchymal transition (EMT). Subsequently, CAFs influence tumor growth, angiogenesis, cancer stemness, ECM remodeling, tissue invasion, metastasis, and even chemoresistance (Harryvan and van der Burg, 2019, J Clin Med, 8:1989). CAF is a complex and often heterogeneous cell population, identified using a combination of various intracellular and cell surface markers, including increased expression of intracellular α-smooth muscle actin (SMA) and cell surface fibroblast activation protein (FAP) (Pure and Blomberg, 2018, Oncogene, 37(32):4343-4357). In patients with bladder and colorectal cancer, TGF-β signaling can promote immune rejection or “cold” tumors, in which CAF keeps T cells trapped outside the tumor, thus physically preventing them from infiltrating the tumor (Hegde, 2020, Immunity, 52:17-35; gajewski, 2015, Semin Oncol, 42:663-671; Mariathasan and Powles, 2018, Nature, 554:544-48).

[0014] Although TGF-β therapy is of interest in treating cancer, it has historically not reached its full therapeutic potential due to the widespread expression of TGF-β and its receptors and their roles in the development, maintenance, and homeostasis of tissues, including the heart and bone. Furthermore, TGF-β is an early tumor suppressor responsible for controlling the growth of early-stage tumors, and systemic TGF-β therapy has been shown to cause tissue toxicity and increase early-stage tumor growth (Anderton and Heier, 2011, Toxicologic Path, 39:916; Stauber et al., 2014, Clinical Tox, 4(3):1-10; Lonning and McPherson, 2011, Curr Pharma Biotech, 12:2176-89).

[0015] Therefore, there is a need to target TGF-β inhibitors to cell types where inhibition of TGF-β signaling is therapeutically useful, such as cancer-associated fibroblasts (“CAF”), while minimizing host tissue toxicity. 3. Overview of the Invention

[0017] This disclosure relates to compositions and methods for treating fibrosis and cancer. By primarily and preferably only targeting those cells in which the TGF-β inhibitor will confer therapeutic benefit, thereby avoiding pleiotropic off-target effects, the compositions and methods advantageously avoid intermediate-target host toxicity associated with systemic administration of TGF-β inhibitors.

[0018] Specifically, the composition and method deliver an ALK5 inhibitor to myofibroblasts, activated fibroblasts (e.g., cancer-associated fibroblasts (“CAF”)), and fibroblasts transforming into myofibroblasts (each cell type is designated as a “target cell”) via a targeting portion that binds to molecules on the surface of target cells. Without being bound by theory, it is assumed that the use of the targeting portion allows the ALK5 inhibitor to localize to and be internalized within the target cells, thereby inhibiting the TGFβ pathway in the target cells while limiting systemic toxicity. Inhibition of the TGFβ pathway in, for example, myofibroblasts or fibroblasts transforming into myofibroblasts can lead to inhibition of fibrosis (in the case of subjects with fibrosis or fibrosis-related diseases). Inhibition of the TGFβ pathway in CAFs can lead to inhibition of tumor progression (in the case of subjects with cancer). Unbound by theory, it is believed that selectively blocking TGF-β signaling in CAF can 1) eliminate CAF-mediated blockade of immune cell infiltration, and / or 2) drive tumor clearance, and / or 3) reduce CAF activity and / or 4) address bypass toxicity issues associated with systemic TGF-β inhibitors.

[0019] Therefore, this disclosure provides targeted drug conjugates (TDCs) wherein the drug is an ALK5 inhibitor. The TDCs of this disclosure comprise a targeting component, such as an antibody or antibody fragment that binds to cell surface molecules of target cells (e.g., human myofibroblast surface molecules). Alternatively, the targeting portion may comprise a non-immunoglobulin-based peptide or polypeptide that binds to the cell surface of target cell surface molecules. Without being bound by theory, it is believed that the TDCs of this disclosure can provide therapeutic effects by promoting the dedifferentiation of target cells into quiescent fibroblasts and / or by promoting apoptosis of target cells. Section 5.2 describes exemplary targeting portions that can be used in the TDCs of this disclosure. In some embodiments, the ALK5 inhibitor is an imidazole-benzodioxol compound, an imidazole-quinoxaline compound, a pyrazole-pyrrolo compound, or a thiazole compound. Exemplary ALK5 inhibitors are described in Section 5.3 and Tables 1-3. In some implementations, the ALK5 inhibitor is N-methyl-2-(4-(4-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethane-1-amine (referred to herein as "Compound C").

[0020] ALK5 inhibitors can be directly conjugated to the target moiety or attached to it via a connector. The connector can be an uncuttable connector, or preferably a cuttable connector. Exemplary uncuttable and cuttable connectors are described in Section 5.4. The average number of ALK5 inhibitor molecules attached to each target moiety can vary, and typically ranges from 2 to 8 ALK5 inhibitor molecules per target moiety. Drug loading is described in detail in Section 5.5.

[0021] This disclosure further provides pharmaceutical compositions comprising the TDC of this disclosure. Exemplary pharmaceutical excipients, described in Section 5.6, can be used to formulate pharmaceutical compositions comprising the TDC of this disclosure.

[0022] This disclosure further provides methods for treating fibrosis and cancer by administering the TDC or pharmaceutical composition of this disclosure to a subject in need. The TDC and pharmaceutical composition of this disclosure may be administered as a monotherapy or as part of a combination therapy, for example, in combination with another therapeutic agent, such as pirfenidone or nintedanib (when treating a subject with fibrosis or a fibrosis-related disease) or a chemotherapeutic agent (when treating a subject with cancer). As another example, the TDC and pharmaceutical composition may be administered in combination with a checkpoint inhibitor when treating a subject with cancer. Exemplary disease types that can be treated with the TDC and pharmaceutical composition of this disclosure and exemplary combination therapies are described in Section 5.7. Attached Figure Description

[0023] Figure 1A-1D The study demonstrated that compound AD inhibited TGF-β-induced luciferase activity in HEK293T cells. Figure 1A Compound A; Figure 1B Compound B; Figure 1C Compound C; Figure 1D Compound D.

[0024] Figure 2A-2C The results showed that anti-FAP antibodies only bind to HEK cells when human FAP cDNA is transfected and expressed on the cell surface. Figure 2A Unstained HEK cells. Figure 2B HEK cells stained with anti-FAP antibody. Figure 2C HEK cells transfected with FAP cDNA and stained with anti-FAP antibody.

[0025] Figures 3A-3B The targeted drug conjugate SYN-301 was shown to be effective. Figure 3A ) and SYN-302 ( Figure 3B The connectors and payload used in ).

[0026] Figures 4A-4B The study showed that SYN-301 inhibits TGF-β signaling in HEK cells expressing human FAP protein. Figure 4A : Relative luciferase reporter gene expression in HEK cells expressing human FAP protein. Figure 4B : Relative luciferase reporter gene expression in untransfected HEK cells.

[0027] Figures 5A-5E It was shown that 50-60% of WI-38 cells expressed FAP. Figure 5A Unstained WI-38 cells. Figure 5B WI-38 cells stained with anti-FAP antibody. Figure 5CWI-38 cells stained with SYN-301. Figure 5D WI-38 cells stained with SYN-302. Figure 5E WI-38 cells stained with isotype control ADC.

[0028] Figure 6 The percentage of FAP internalization induced by anti-FAP antibody (63%), SYN-301 (63%), and SYN-302 (52%) is shown.

[0029] Figures 7A-7B The effects of SYN-301 and SYN-302 on collagen and fibronectin in WI-38 cells were demonstrated. Figure 7A ) and LRRC15 ( Figure 7B The effect of RNA expression on ). 5. Detailed Description of the Invention

[0031] This disclosure provides targeted drug conjugates (TDCs) for the treatment of fibrosis and cancer, comprising a targeting moiety that is covalently bound to an ALK5 inhibitor, either directly or via a linker. Section 5.1 provides an overview of the TDCs of this disclosure. The targeting moiety of a TDC may include, for example, an intact antibody or a fragment thereof. Targeting moieties that may be used in the TDCs of this disclosure are described in detail in Section 5.2. ALK5 inhibitors that may be used in the TDCs of this disclosure are described in Section 5.3. The TDCs of this disclosure typically contain a linker between the targeting moiety and the ALK5 inhibitor. Exemplary linkers that may be used in the TDCs of this disclosure are described in Section 5.4. The TDCs of this disclosure may contain a different number of ALK5 inhibitor moieties for each targeting moiety. Drug loading is discussed in detail in Section 5.5. This disclosure further provides pharmaceutical formulations comprising the TDCs of this disclosure. Pharmaceutical formulations comprising TDCs are described in Section 5.6. This disclosure further provides methods for treating fibrosis and methods for treating cancer using the TDCs of this disclosure. Methods of using the TDC disclosed herein as a monotherapy or as part of a combination therapy for the treatment of fibrosis or cancer are described in Section 5.7.

[0032] 5.1. Drug conjugates

[0033] The TDC disclosed herein typically consists of an ALK5 inhibitor, which is usually covalently linked to a target moiety (such as an antibody or antibody fragment) via a linker, such that the covalent link does not interfere with the binding of the target moiety to the target.

[0034] Techniques for conjugating drugs to target moieties, such as antibodies and antibody fragments, are well known in the art (see, for example, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., at pp. 623-53 (Robinson et al., eds., 1987); Thorpe et al., 1982, Immunol. Rev. 62: 119-58; Dubowchik et al., 1999, Pharmacology and Therapeutics 83: 67-123; and Zhou, 2017, Biomedicines 5(4): E64)). ALK5 inhibitors are preferably conjugated to the target moieties in the TDC of this disclosure via site-specific conjugation. For example, ALK5 inhibitors can be conjugated to the target moiety via one or more natural or engineered cysteine, lysine, or glutamine residues, one or more non-natural amino acids (e.g., p-acetylphenylalanine (pAcF), p-azidomethyl-L-phenylalanine (pAMF), or selenocysteine ​​(Sec)), one or more polysaccharides (e.g., fucose, 6-thiofucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), or sialic acid (SA)), or one or more short peptide tags of four to six amino acids. See, for example, Zhou, 2017, Biomedicines 5(4):E64, the contents of which are incorporated herein by reference in their entirety.

[0035] In one instance, the targeting moiety is fused to the amino acid sequence of another protein (or a portion thereof; for example, a portion of a protein containing at least 10, 20, or 50 amino acids) via a covalent bond (e.g., a peptide bond), through the N-terminus or C-terminus of the targeting moiety, or internally. The targeting moiety may be linked to other proteins at the N-terminus, such as antibodies or antibody fragments linked to the N-terminus of an antibody constant domain. Recombinant DNA procedures can be used to generate such fusions, as described, for example, in WO 86 / 01533 and EP0392745. In another instance, the effector molecule may increase the in vivo half-life and / or enhance the delivery of TDCs to target cells. Examples of suitable effector molecules of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in PCT Publication WO 2005 / 117984.

[0036] Metabolic processes or reactions can be enzymatic, such as the proteolytic cleavage of the peptide linker of TDC, or the hydrolysis of functional groups such as hydrazones, esters, or amides. Intracellular metabolites include, but are not limited to, peptides and free drugs, which undergo intracellular cleavage after entering, diffusing, taking up, or being transported into the cell.

[0037] The terms "intracellularly cleaved" and "intracellular cleavage" refer to intracellular metabolic processes or reactions on a drug conjugate that disrupt the covalent bond, such as a linker, between the drug moiety (D) and the target moiety, resulting in free drug dissociating from the target moiety within the cell. Therefore, the cleaved portion of a TDC is an intracellular metabolite.

[0038] 5.2. Targeted Part

[0039] This disclosure provides drug conjugates in which a targeting moiety binds to molecules on the surface of target cells. The targeting moiety typically comprises an antibody or antibody fragment (such conjugates are sometimes referred to herein as “antibody-drug conjugates” or “ADCs”). Alternatively, the targeting moiety can be non-immunoglobulin-based, such as a non-immunoglobulin-based peptide or polypeptide (e.g., a ligand of a receptor expressed on the surface of a target cell). Therefore, the term “targeting moiety” should be understood to encompass peptides (e.g., peptides of ten to forty amino acids in length), single-chain polypeptides (e.g., polypeptides of more than forty amino acids in length, such as single-chain variable regions or scFvs), and molecules comprising multiple polypeptide chains (e.g., multimeric immunoglobulin molecules).

[0040] Unless otherwise stated, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or reacts with a specific antigen, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugated antibodies (e.g., bispecific antibodies, biantibodies, triantibodies, and tetraantibodies), and antibody fragments, including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments. Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) includes both the complete molecule and antibody fragments (e.g., Fab and F(ab')2 fragments) that can specifically bind to proteins. Fab and F(ab')2 fragments lack the Fc fragment of the complete antibody, are cleared more quickly from circulation in animals or plants, and may have less nonspecific tissue binding than the complete antibody (Wahlet al., 1983, J. Nucl. Med. 24:316).

[0041] The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, which includes the Fv, scFv, or Fab heavy chain. The term "VL" refers to the variable region of the immunoglobulin light chain, which includes the Fv, scFv, dsFv, or Fab light chain. Antibodies and immunoglobulins (Ig) are glycoproteins with similar structural features. While antibodies exhibit binding specificity to a particular target, immunoglobulins include antibodies and other antibody-like molecules lacking target specificity. Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at the amino terminus, followed by multiple constant domains. Each light chain has a variable domain at the amino terminus (VL) and a constant domain at the carboxyl terminus.

[0042] For optimal delivery of intracellular ALK5 inhibitors, the targeting moiety is preferably internalized, such as an internalizing antibody. After the internalizing targeting moiety binds to the target molecule on the cell surface, the cell internalizes the internalizing targeting moiety due to the binding. The effect of this is that the cell takes up TDC. Methods that allow for determining internalization, such as the internalization of an antibody after binding to its antigen, are known to those skilled in the art and are described, for example, on page 80 of PCT Publication WO 2007 / 070538. Once internalized, if the ALK5 inhibitor is attached to the targeting moiety using a cleavable linker, as described in Section 5.4, the ALK5 inhibitor can be released from the targeting moiety by cleavage in the lysosome or through other cellular mechanisms.

[0043] The term "antibody fragment" refers to a portion of a full-length antibody, typically the target-binding region or variable region. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, dsFv fragments, or single-domain antibodies.

[0044] The “Fv” fragment is the smallest antibody fragment containing a complete target recognition and binding site. This region consists of a dimer of a heavy chain and a light chain variable domain, forming a tight non-covalent bond (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer antibody target binding specificity. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can have the ability to recognize and bind to the target.

[0045] A “single-chain Fv” or “scFv” antibody fragment comprises the VH and VL domains of an antibody within a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker located between the VH and VL domains, which enables the scFv to form the structure required for target binding. Various scFv linkers have been described in the art. See, for example, Shen et al., 2008, Anal Chem. 80(6):1910–1917; Yusakul, et al., 2016, Biosci Biotechnol Biochem. 80(7):1306-12. An exemplary scFv linker comprises the sequence (GGGGS)n, where n is between 1 and 10.

[0046] "Disulfide-stabilized Fv" or "dsFv" antibody fragments contain the VH and VL domains of the disulfide-stabilized antibody via interdomain connections. See Brinkmann U., 2010, Disulfide-Stabilized Fv Fragments. In: Kontermann R., Dübel S. (eds) Antibody Engineering. Springer, Berlin, Heidelberg.

[0047] A "single-domain antibody" consists of a single VH or VL domain (e.g., of a human or mouse antibody) that exhibits sufficient affinity for a target (e.g., FAP). In a specific embodiment, the single-domain antibody is a camel V... H H antibody fragments (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38). The use of single-domain antibodies in the TDC of this disclosure may be advantageous because they are smaller in size, have higher solubility, higher stability, and better in vivo tissue penetration compared to full-length antibodies. Various methods for preparing single-domain antibodies have been described. See, for example, U.S. Patent 10,030,068, US 2006 / 0246058, U.S. Patent 7,371,849, Vincke et al, 2008, JBC, 284(5):3273-3284.

[0048] The Fab fragment contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. The F(ab') fragment is generated by cleaving the disulfide bond at the hinge cysteine ​​residue of the F(ab')2 pepsin digestion product. Additional chemical conjugation of antibody fragments is known to those skilled in the art.

[0049] In some embodiments, the antibodies of this disclosure are monoclonal antibodies. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, which includes any eukaryotic, prokaryotic, or phage clone, rather than its production method. Monoclonal antibodies related to this disclosure can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display technologies, or combinations thereof. The antibodies of this disclosure include chimeric, primate-derived, humanized, or human antibodies.

[0050] The antibodies of the present invention may be chimeric antibodies. As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin (such as a rat or mouse antibody) and a human immunoglobulin constant region typically selected from a human immunoglobulin template. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patents 5,807,715; 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety.

[0051] The antibodies disclosed herein can be humanized. "Humanized" forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of the antibody) containing minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies will contain substantially all, at least one, and typically two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins and all or substantially all of the FR regions are those of human immunoglobulin sequences. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically a portion of the common sequence of human immunoglobulins. Methods for antibody humanization are known in the art. See, for example, Riechmann et al., 1988, Nature 332:323-7; U.S. Patents 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; Queen et al.; European Patent Publication EP239400; PCT Publication WO 91 / 09967; U.S. Patent 5,225,539; European Patent Publication EP592106; European Patent Publication EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al. al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332, all of which are incorporated herein by reference in their entirety.

[0052] The antibodies of this invention may be human antibodies. For therapeutic treatment of human patients, fully "human" antibodies may be required. As used herein, "human antibody" includes antibodies having the amino acid sequence of human immunoglobulins, and includes antibodies isolated from a human immunoglobulin library or from animals genetically modified with one or more human immunoglobulins that do not express endogenous immunoglobulins. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patents 4,444,887 and 4,716,111; and PCT Publications WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO 96 / 33735; and WO 91 / 10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be generated using transgenic mice that do not express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT Publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598 and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), Amgen (Thousand Oaks, Calif.), and Regeneron (Tarrytown, NY) can use similar techniques to provide human antibodies against selected antigens. A technique called “guided selection” can be used to generate fully human antibodies that recognize selected epitopes. In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).

[0053] The antibodies disclosed herein can be primate-derived. The term "primate-derived antibody" refers to an antibody containing both a monkey variable region and a human constant region. Methods for generating primate-derived antibodies are known in the art. See, for example, U.S. Patents 5,658,570; 5,681,722; 5,693,780 and 5,693,780, which are incorporated herein by reference in their entirety.

[0054] The antibodies disclosed herein include derivatized antibodies. For example, but not limited to, derivatized antibodies are typically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, or linking to cellular ligands or other proteins (see Section 5.1, Discussion of Antibody Conjugates). Any of many chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tunicamycin. Additionally, the derivatives may contain one or more non-natural amino acids, for example, using the ambrx technique (see, for example, Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0055] In another embodiment of this disclosure, the antibody or a fragment thereof may be an antibody or antibody fragment whose sequence has been modified to alter the biological effector function mediated by at least one constant region relative to the corresponding wild-type sequence. For example, in some embodiments, the antibody of this disclosure may be modified to reduce the biological effector function mediated by at least one constant region relative to an unmodified antibody, such as reducing binding to the Fc receptor (FcγR) or C1q. The binding of FcγR and C1q can be reduced by mutating the immunoglobulin constant region fragment of the antibody in a specific region necessary for FcγR or C1q interaction (see, for example, Canfield and Morrison, 1991, J.Exp.Med.173:1483-1491; Lund et al., 1991, J.Immunol.147:2657-2662; Lo. et al., 2017, J Biol Chem 292:3900-08; Wang et al., 2018, Protein Cell 9:63-73).

[0056] A decrease in antibody binding to FcγR can also reduce other effector functions dependent on FcγR interactions, such as opsonization, phagocytosis, and antibody-dependent cytotoxicity (“ADCC”), while a reduction in C1q binding can reduce complement-dependent cytotoxicity (“CDCC”). Therefore, the reduction or elimination of effector functions can prevent the destruction of target cells targeted by the drug conjugates of this disclosure via ADCC or CDCC. Thus, in some embodiments, the effector function of the antibody is modified by selective mutation of the Fc portion of the antibody to maintain its antigen specificity and internalization capacity but eliminate ADCC / CDCC function. In other embodiments, the effector function of the antibody is not modified to reduce or eliminate ADCC / CDCC function. Without being bound by theory, it is believed that the TDCs of the present invention comprising antibodies or antibody fragments with ADCC / CDCC function can enhance the therapeutic effect of inhibiting TGFβ signaling by promoting target cell apoptosis, thereby further inhibiting fibrosis.

[0057] Numerous mutations have been described in the art to reduce FcγR and Clq binding, and such mutations can be included in the pharmaceutical conjugates disclosed herein. For example, U.S. Patent 6,737,056 discloses single-position Fc region amino acid modifications at positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438, or 439, resulting in reduced binding to FcγRII and FcγRII. U.S. Patent 9,790,268 discloses that an asparagine residue at amino acid position 298 and a serine or threonine residue at amino acid position 300 reduce FcγR binding. PCT disclosure WO Document 2014 / 190441 describes Fc domains modified with reduced FcγR binding having L234D / L235E:L234R / L235R / E233K, L234D / L235E / D265S:E233K / L234R / L235R / D265S, L234D / L235E / E269K:E233K / L234R / L235R / E269K, L 234D / L235E / K322A:E233K / L234R / L235R / K322A, L234D / L235E / P329W:E233K / L234R / L23 5R / P329W, L234D / L235E / E269K / D265S / K322A: E233K / L234R / L235R / E269K / D265S / K322A,

[0058] The L234D / L235E / E269K / D265S / K322E / E333K:E233K / L234R / L235R / E269K / D265S / K322E / E333K mutation occurs in the first Fc polypeptide, while the mutation after the semicolon occurs in the second Fc polypeptide of the Fc dimer.

[0059] Mutations that can reduce FcγR receptor binding and C1q binding include N297A, N297Q, N297G, D265A / N297A, D265A / N297G, L235E, L234A / L235A, and L234A / L235A / P329A (Lo. et al., 2017, J BiolChem 292:3900-08; Wang et al., 2018, Protein Cell 9:63-73).

[0060] As a mutation constant region to reduce effector function, for example, as described above, the substitution of the mutated Fc domain can be used to eliminate effector function by utilizing antibody fragments (e.g., Fab, Fab', or F(ab')2).

[0061] In other embodiments of this disclosure, antibodies or fragments thereof may be modified to acquire or improve the function of at least one constant region-mediated biological effector relative to unmodified antibodies, for example, to enhance FcγR interactions (see, for example, US2006 / 0134709). For example, antibodies of this disclosure may have constant regions of FcγRIIA, FcγRIIB, and / or FcγRIIIA with higher affinity than the corresponding wild-type constant regions.

[0062] Therefore, the antibodies of this disclosure may have alterations in biological activity that result in opsonization, phagocytosis, or reduced ADCC. Such alterations are known in the art. For example, antibody modifications that reduce ADCC activity are described in U.S. Patent 5,834,597.

[0063] On the other hand, antibodies or fragments thereof may be antibodies or antibody fragments modified to increase or decrease their binding affinity to the fetal Fc receptor FcRn, for example, by mutation of immunoglobulin constant region fragments involving specific regions of FcRn interaction (see, for example, WO 2005 / 123780). Such mutations can increase antibody binding to FcRn, thereby protecting the antibody from degradation and increasing its half-life.

[0064] In other respects, the antibody has one or more amino acids inserted into one or more hypervariable regions therein, for example, as described in Jung and Plückthun, 1997, Protein Engineering 10(9):959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9; and U.S. Patent Publication 2007 / 0280931.

[0065] The target of the targeting moiety will depend on the desired therapeutic application of the TDC. Typically, the target is a molecule present on the surface of cells wishing to receive ALK5 inhibitor delivery, such as myofibroblasts or cancer-associated fibroblasts, and the targeting moiety preferably internalizes upon binding to the target. Internalizing targeting moieties, such as antibodies, are described, for example, in Franke et al., 2000, Cancer Biother. Radiopharm. 15:459 76; Murray, 2000, Semin. Oncol. 27:64 70; Breitling et al., Recombinant Antibodies, John Wiley, and Sons, New York, 1998). In some embodiments, the targeting moiety does not significantly block the activity of target cell surface molecules. For example, an agonist antibody or a fragment thereof, or a non-antagonist antibody or a fragment thereof, can be used as the targeting moiety, for example, when the target molecule is FAP or αvβ6.

[0066] Preferably, the target selectively binds to myofibroblasts, activated fibroblasts, fibroblasts converting to myofibroblasts, or combinations thereof, rather than other cell types, such as quiescent fibroblasts, lung epithelial cells, hepatocytes, T cells, cells that do not express collagen, and / or cells that do not express α-smooth muscle actin (αSMA). Quiescent or dormant fibroblasts can be identified as spindle-shaped single cells, while activated fibroblasts acquire αSMA and vimentin expression and become star-shaped. Selective binding can be achieved by targeting cell surface molecules that are expressed on the surface of one or more target cells but are expressed at low levels or not at all on other cell types. Selectivity can be measured by various assays known in the art, such as flow cytometry. In some embodiments, the targeting portion of the TDC of this disclosure has at least 2 to 3 times selectivity for myofibroblasts relative to resting fibroblasts, for example, as measured by FACS (e.g., 2 to 1000 times, 2 to 100 times, 2 to 50 times, 2 to 10 times, 3 to 1000 times, 3 to 100 times, 3 to 50 times, 3 to 10 times, 5 to 1000 times, 5 to 100 times, 5 to 50 times, 5 to 10 times, 20 to 1000 times, 20 to 100 times, 20 to 50 times, 50 to 1000 times, 50 to 100 times, 100 to 1000 times or more than 1000 times). In some embodiments, the targeting portion of the TDC of this disclosure has at least 2-fold or at least 3-fold selectivity for activated fibroblasts (e.g., CAFs) relative to quiescent fibroblasts, as measured by FACS (e.g., 2 to 1000-fold, 2 to 100-fold, 2 to 50-fold, 2 to 10-fold, 3 to 1000-fold, 3 to 100-fold, 3 to 50-fold, 3 to 10-fold, 5 to 1000-fold, 5 to 100-fold, 5 to 50-fold, 5 to 10-fold, 20 to 1000-fold, 20 to 100-fold, 20 to 50-fold, 50 to 1000-fold, 50 to 100-fold, 100 to 1000-fold, or more than 1000-fold). In some embodiments, the targeting portion of the TDC of this disclosure has at least 2-fold or at least 3-fold selectivity for fibroblasts transforming into myofibroblasts relative to quiescent fibroblasts, for example, as measured by FACS (e.g., 2 to 1000-fold, 2 to 100-fold, 2 to 50-fold, 2 to 10-fold, 3 to 1000-fold, 3 to 100-fold, 3 to 50-fold, 3 to 10-fold, 5 to 1000-fold, 5 to 100-fold, 5 to 50-fold, 5 to 10-fold, 20 to 1000-fold, 20 to 100-fold, 20 to 50-fold, 50 to 1000-fold, 50 to 100-fold, 100 to 1000-fold, or more than 1000-fold).Examples of cell surface molecules suitable for partial targeting include, but are not limited to, fibroblast activation protein (FAP), platelet-derived growth factor receptor β (PDGFR-β), fibroblast growth factor receptor 1 (FGFR1), peroxisome proliferator-activated receptor γ (PPAR-γ), fibroblast-specific protein 1 (FSP1), glial fibrillary acidic protein (GFAP), myofascitis, CD147, CXC chemokine receptor type 4 (CXCR4), αVβ6, AXL, and MERTK. AXL and MERTK are members of the TAM receptor kinase family. A further example of a cell surface molecule suitable for partial targeting is leucine-rich repeat 15 (LRRC15).

[0067] In some embodiments, the targeting portion of the TDC of this disclosure binds to FAP. In other embodiments, the targeting portion of the TDC of this disclosure binds to PDGFR-β. In other embodiments, the targeting portion of the TDC of this disclosure binds to FGFR1. In other embodiments, the targeting portion of the TDC of this disclosure binds to PPAR-γ. In other embodiments, the targeting portion of the TDC of this disclosure binds to FSP1. In other embodiments, the targeting portion of the TDC of this disclosure binds to GFAP. In other embodiments, the targeting portion of the TDC of this disclosure binds to myofascitis. In other embodiments, the targeting portion of the TDC of this disclosure binds to CD147. In other embodiments, the targeting portion of the TDC of this disclosure binds to CXCR4. In other embodiments, the targeting portion of the TDC of this disclosure binds to αvβ6. In other embodiments, the targeting portion of the TDC of this disclosure binds to AXL. In other embodiments, the targeting portion of the TDC of this disclosure binds to MERTK. In other embodiments, the targeting portion of the TDC of this disclosure binds to LRRC15.

[0068] Fibroblast activating protein (FAP) is a member of the dipeptidyl peptidase (DPP) family, expressed as a type II integrated membrane protein. Membrane-bound FAP contains a short cytoplasmic tail (residues 1-4), a transmembrane region (residues 5-25), and an extracellular domain (residues 26-760) (www.uniprot.org / uniprot / Q12884). FAP is active on the cell surface as a 170 kDa dimer, but its extracellular domain is also active after cleavage from the membrane. FAP exhibits both dipeptidase and endopeptidase activity. Like other members of the DPP family, FAP is a prolyl-specific serine protease, but it also possesses gelatinase activity, allowing it to degrade denatured collagen I and III, human fibroblast growth factor 21 (FGF-21), and human α2 antifibrinolytic enzyme. FAP is expressed during development but is rarely expressed in healthy adult tissues. However, elevated FAP expression in activated fibroblasts is observed at sites of inflammation and activated tissue remodeling, including wound healing, fibrosis, and cancer. Because FAP is a marker of activated fibroblasts and myofibroblasts in a variety of disease settings, FAP-targeted therapies are not limited to cancer patients but are broadly applicable for treating IPF and other fibrotic diseases such as NASH (liver), cardiac, and / or renal fibrosis. Furthermore, TGF-β can increase FAP expression, where the FAP promoter has an Smad3 binding element. Due to this limited tissue expression and localized expression in fibrotic tissues, FAP has been used for imaging and therapeutic targeting of diseased tissues. In cancer, FAP is highly and selectively expressed on cancer-associated fibroblasts (CAFs) that support cancer growth and metastasis. FAP is readily internalized into cells, making it an excellent cell-targeting and delivery agent. Anti-FAP antibodies conjugated with cytotoxic payloads in combination with chemotherapy have demonstrated tumor clearance, while anti-FAP conjugated with radioactive nucleotides has improved survival in in vivo mouse tumor models (Ostermann et al., 2008, Clin Cancer Res, 2008.14(14):4584-92; Fang et al., 2016, Int J Cancer, 138(4):1013-23; Fischer et al., Clin Cancer Res 18(22):6208-18). Although sibroluzumab is an unconjugated humanized anti-FAP antibody, it has not shown monotherapy efficacy in patients with metastatic FAP+ cancer, but it does accumulate specifically in tumors rather than normal tissues and is well-tolerated in patients with limited adverse events. The soluble form of FAP, also known as anti-plasmin cleavage enzyme (APCE), lacks the cytoplasmic tail and transmembrane region of membrane-bound FAP.Soluble FAP has been shown to be elevated in patients with cirrhosis, with levels increasing with disease severity (eWillige et al., 2013, J Thromb Haemost. 11(11): 2029-36). Preferably, the targeting portion of FAP preferentially binds to the membrane-bound form of FAP rather than the soluble form. Without being bound by theory, it is considered that soluble FAP can act as a precipitate, with TDCs that preferentially bind to the membrane-bound form of FAP exhibiting reduced in vivo activity compared to TDCs that bind to the soluble form of FAP.

[0069] Examples of antibodies that bind to FAP are described in WO 2012 / 020006, WO 2016 / 116399 (e.g., antibody F5) and WO 2016 / 110598, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-FAP antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2012 / 020006, WO 2016 / 116399, or WO 2016 / 110598. In some embodiments, the targeting portion of the TDC of this disclosure comprises sibroluzumab (Boehringer Ingelheim) or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0070] Examples of antibodies binding to PDGFR-β are described in WO 2017 / 106609 and WO 2014 / 109999, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-PDGFR-β antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2017 / 106609 or WO 2014 / 109999. In some embodiments, the targeting portion of the TDC of this disclosure comprises IMC-2C5 (ImClone) or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0071] Examples of antibodies binding to FGFR1 are described in WO 2018 / 095932 and WO 2012 / 125124, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-FGFR1 antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2018 / 095932 or WO 2012 / 125124. In some embodiments, the targeting portion of the TDC of this disclosure comprises an antibody comprising a heavy chain having an amino acid sequence of SEQ ID NO: 45 having WO 2012 / 125124 and a light chain having an amino acid sequence of EQ ID NO: 50 having WO2012 / 125124S, or a fragment thereof (e.g., a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a scFv fragment, a dsFv fragment, or a single-domain antibody).

[0072] Examples of antibodies binding to PPAR-γ are described in WO 2005 / 026336, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-PPAR-γ antibody or a fragment thereof (e.g., a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2005 / 026336. In some embodiments, the targeting portion of the TDC of this disclosure comprises an antibody or a fragment thereof (e.g., a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) designated as Pγ48.34A in WO 2005 / 026336.

[0073] Examples of antibodies binding to FSP1 are described in WO 2011 / 157724, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-FSP1 antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2011 / 157724. In some embodiments, the targeting portion of the TDC of this disclosure comprises antibody MAB4137 (R&D Systems) or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0074] Examples of antibodies binding to GFAP are described in WO 2018 / 081649, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-GFAP antibody or a fragment thereof (e.g., a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or a single-domain antibody) as described in WO 2018 / 081649. In some embodiments, the targeting portion of the TDC of this disclosure comprises one of the antibodies specified in WO 2018 / 081649 as gFAP-1 to GFAP-19 or a fragment thereof (e.g., a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or a single-domain antibody).

[0075] Examples of antibodies that bind to myofascitis include FCN01 (ThermoFisher), ab126772 (Abcam), and ab183891 (Abcam). In some embodiments, the targeting portion of the TDC disclosed herein comprises one of FCN01, ab126772, or ab183891 or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0076] Examples of antibodies binding to CD147 are described in WO 2015 / 160853, WO 2018 / 121578, and WO 2018 / 165619, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure includes the anti-CD147 antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) described in WO 2015 / 160853, WO 2018 / 121578, or WO 2018 / 165619. In some embodiments, the targeting portion of the TDC of this disclosure includes an antibody designated as 3A11 in WO 2018 / 165619 or a humanized variant or fragment of such an antibody as described in WO 2018 / 165619 (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0077] Examples of antibodies binding to CXCR4 are described in WO 2011 / 098762, WO 2008 / 060367, and WO 2006 / 089141, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure includes the anti-CXCR4 antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) described in WO 2011 / 098762, WO 2008 / 060367, or WO 2006 / 089141. In some embodiments, the targeting portion of the TDC of this disclosure comprises the antibody C-9P21, B-1M22, C-1I24, D-1K21 or 9N10 or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment or single-domain antibody) described in WO 2011 / 098762.

[0078] Examples of antibodies binding to αvβ6 are described in WO 2008 / 112004 and WO 2013 / 123152, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-αvβ6 antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2008 / 112004 or WO 2013 / 123152. In some embodiments, the targeting portion of the TDC of this disclosure comprises antibody STX-100 (Biogen) or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0079] Examples of antibodies targeting AXL are described in WO 2009 / 062690, WO 2010 / 130751, WO 2015 / 193430 and WO 2016 / 005593, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure includes the anti-AXL antibody described in WO 2009 / 062690, WO 2010 / 130751, WO 2015 / 193430 or WO 2016 / 005593, or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment or single-domain antibody). In some embodiments, the targeting portion of the TDC disclosed herein comprises an antibody or fragment of ADCT-601 (ADCTherapeutics) or thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0080] Examples of antibodies binding to MERTK are described in WO 2016 / 106221, WO 2019 / 005756, and WO 2019 / 084307, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the targeting portion of the TDC of this disclosure comprises an anti-MERTK antibody or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody) as described in WO 2016 / 106221, WO 2019 / 005756, or WO 2019 / 084307. In some embodiments, the targeting portion of the TDC of this disclosure comprises antibody RGX-019 (Rgenix) or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0081] LRRC15 is expressed on cancer-associated fibroblasts in many cancers (e.g., breast cancer, head and neck cancer, lung cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, esophageal cancer, gastric adenocarcinoma, and bladder cancer) (Purcell et al., 2018, CancerRes. 78(14):4059-4072; Dominguez et al., 2019, Cancer Discovery 10(2):232-253). Therefore, in some embodiments, the TDC of this disclosure targets LRRC15. Examples of antibodies that bind to LRRC15 are described in WO2017 / 095805, the contents of which are incorporated herein by reference in their entirety. Antibodies that bind to LRRC15 are also commercially available, for example, in the Abcam catalog #ab150376 and the Creative Biolabs catalog #TAB-0709CL. ABBV-085 (Abbvie) is an ADC containing MMAE targeting LRRC15 (Purcell et al., 2018, Cancer Res. 78(14):4059-4072). In some embodiments, the targeting portion of the TDC disclosed herein comprises the antibody described in WO 2017 / 095805, an antibody against ABBV-085, one of the commercially available antibodies described in this paragraph, or a fragment thereof (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, scFv fragment, dsFv fragment, or single-domain antibody).

[0082] 5.3 ALK5 Inhibitors

[0083] The ALK5 inhibitor disclosed herein is preferably a small molecule that competitively and reversibly binds to the ATP-binding site in the cytoplasmic kinase domain of the ALK5 receptor.

[0084] ALK5 inhibitors may, but need not, be specific and selective for ALK5 relative to other TGF-β family receptors, such as ALK4 and / or ALK7 and / or TGF-β receptor II. In some embodiments, ALK5 inhibitors are active against both ALK5 and TGF-β receptor II. In some embodiments, ALK5 inhibitors have limited inhibitory activity against BMP II receptors.

[0085] When measured in an in vitro cell assay using HEK293T cells, the ALK5 inhibitor disclosed herein preferably has an IC50 of 100 nM or less, more preferably 50 nM or less, and most preferably 20 nM or less. 50 An exemplary cell assay is described in Section 6.6 below.

[0086] Explanatory examples of ALK5 inhibitors applicable to the TDC of this disclosure include imidazole-benzodioxane compounds, imidazole-quinoxaline compounds, pyrazole-pyrrolo compounds, and thiazole compounds.

[0087] According to one aspect of this disclosure, an imidazole-benzodioxane-pentene type ALK5 inhibitor has the following formula:

[0088]

[0089] Where R 1 It is hydrogen or a lower alkyl group having 1 to about 5 carbon atoms, R 2 It is hydrogen or a lower alkyl group having 1 to about 5 carbon atoms and R 3 It is an amide, nitrile, alkynyl, carboxyl, or alkane group having 1 to 3 carbon atoms, A is a direct bond or an alkyl group having 1 to 5 carbon atoms, and B is a direct bond or an alkyl group having 1 to 5 carbon atoms. In a separate preferred embodiment of this disclosure, R 2 It is either hydrogen or methyl, A has 1 carbon atom and B is a direct bond with a benzyl group and R 3 It is an amide. In the preferred embodiment of the combination disclosed herein, R 2 It is either hydrogen or methyl, A has 1 carbon atom and B is a direct bond with benzyl.

[0090] According to another aspect of this disclosure, imidazole-quinoxaline type ALK5 inhibitors have the following formula:

[0091]

[0092] Where R 1 It is hydrogen or a lower alkyl group having 1 to about 5 carbon atoms, R 2 It is hydrogen, halogen, or a lower alkyl group having 1 to 5 carbon atoms, R 3 It is an amide, nitrile, alkynyl group having 1 to 3 carbon atoms, carboxyl group, or alkanoyl group having 1 to 5 carbon atoms, A is a direct bond or an alkyl group having 1 to 5 carbon atoms, and B is a direct bond or an alkyl group having 1 to 5 carbon atoms. In a separate preferred embodiment of this disclosure, R 2 It is hydrogen or methyl, halogens include fluorine or chlorine, A has 1 carbon atom and B is a direct bond with benzyl and R 3 It is an amide. In the preferred embodiment of the combination disclosed herein, R 2 It is either hydrogen or methyl, A has 1 carbon atom and B is a direct bond with a benzyl group.

[0093] According to another aspect of this disclosure, pyrazole-type ALK5 inhibitors have the following formula:

[0094]

[0095] Where R 2 It is hydrogen, halogen, or a lower alkyl group having 1 to 5 carbon atoms, R 4 It is hydrogen, halogen, lower alkyl group having 1 to 5 carbon atoms, alkoxy group having 1 to 5 carbon atoms, haloalkyl group, carboxyl group, carboxylalkyl ester, nitrile group, alkylamine group or group having the following formula.

[0096]

[0097] Where R 5 It is a lower alkyl, halogenated, or morpholino group having 1 to 5 carbon atoms, and R 6 It is pyrrole, cyclohexyl, morpholino, pyrazole, pyran, imidazole, oxane, pyrrolidinyl or alkylamine, and A is a direct bond or an alkyl group having 1 to about 5 carbon atoms.

[0098] According to another aspect of this disclosure, pyrazole-pyrrolo-type ALK5 inhibitors have the following formula:

[0099]

[0100] Where R 7 It is hydrogen, halogen, lower alkyl, alkanol, morpholino or alkylamine having 1 to 5 carbon atoms, R 2 It is hydrogen, halogen, or a lower alkyl group having 1 to 5 carbon atoms and R 8 It is hydrogen, hydroxyl, amino, halogen, or a group having the following formula.

[0101]

[0102] Where R 5 It is piperazine group, R 6 It is morpholino, piperidinyl, piperazine, alkoxy, hydroxyl, oxane, halogen, thioalkyl or alkylamine, and A is a lower alkyl group having 1 to about 5 carbon atoms.

[0103] According to another aspect of this disclosure, thiazole-type ALK5 inhibitors have the following formula:

[0104]

[0105] Where R 9 It is hydrogen, halogen, or a lower alkyl group having 1 to 5 carbon atoms, and R 10 It is hydrogen or a lower alkyl group having 1 to 5 carbon atoms.

[0106] In some implementations, the ALK5 inhibitor is selected from any compound designated A through N in Table 1 below:

[0107]

[0108]

[0109]

[0110]

[0111] In a further specific embodiment, the ALK5 inhibitor is selected from any compound designated as 1 to 283 in Table 2 below:

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] The preparation and use of ALK5 inhibitors are well known and well documented in scientific and patent literature. PCT Publication WO 2000 / 61576 and US Patent Publication US 2003 / 0149277 disclose triarylimidazolium derivatives and their use as ALK5 inhibitors. PCT Publication WO 2001 / 62756 discloses pyridylimidazolium derivatives and their use as ALK5 inhibitors. PCT Publication WO 2002 / 055077 discloses imidazolium cyclic acetal derivatives as ALK5 inhibitors. PCT Publication WO 2003 / 087304 discloses trisubstituted heteroaryl derivatives and their use as ALK5 and / or ALK4 inhibitors. WO 2005 / 103028, US Patent Publication No. 2008 / 0319012, and US Patent No. 7,407,958 disclose 2-pyridyl-substituted imidazoles as ALK5 and / or ALK4 inhibitors. One representative compound, IN-1130, has shown ALK5 and / or ALK4 inhibitory activity in several animal models. The following patents and patent publications provide additional examples of ALK5 inhibitors and provide illustrative synthetic schemes and methods of using ALK5 inhibitors: US Patents 6,465,493; 6,906,089; 7,365,066; 7,087,626; 7,368,445; 7,265,225; 7,405,299; 7,407,958; 7,511,056; 7,612,094; 7,691,865; 7,863,288; 8,410,146; 8,410,146; 8,420,685; 8,513,2228,614,226; 8,791,113; 8,815,893; 8,846,931; 8,912,216; 8,987,301; 9,051,307; 9,051,318; 9,073,918 and PCT Publications WO 2004 / 065392; WO 2009 / 050183; WO 2009 / 133070; WO2011 / 146287; and WO 2013 / 009140. The aforementioned patents and patent publications are incorporated herein by reference in their entirety.

[0128] Several ALK5 inhibitors are commercially available, including SB-525334 (CAS 356559-20-1), SB-505124 (CAS 694433-59-5), SB-431542 (CAS 301836-41-9), SB-202474 (EMD4 Biosciences Merck KGaA, Darmstadt, Germany), LY-364947 (CAS 396129-53-6), IN-1130, GW-788388, and D4476 (EMD4 Biosciences Merck KGaA, Darmstadt, Germany).

[0129] The structures and names of ALK5 inhibitors described in this article refer to the molecules preceding the antibody and / or linker.

[0130] Preferred ALK5 inhibitors are those that can be attached to a free NH or NH2 group, preferably an NH or NH2 group or a portion thereof, via an alkyl, heteroaryl, or aryl group (e.g., compounds 1-23, 26-29, 31, 35, 37, 39, 40, 42, 43, 45-48, 50-85, 87-90, 93, 96, 98-104, 106, 108, 109, 111, 112, 114, 116-120, 132, 146, 149, 156, 184, 187, 193, 218, 260-277, 282, and 283). ALK5 inhibitors can be derivatized by adding a free NH or NH2 group. The design of derivatized ALK5 inhibitors should prioritize the structure-activity relationship (SAR) of the inhibitor to reduce the possibility of eliminating inhibitory activity upon addition of an NH or NH2 group, although the activity can also be determined empirically. Exemplary derivatives of the compounds shown in Table 1 are shown in Table 3 below.

[0131]

[0132]

[0133] 5.4. Connector

[0134] Typically, a TDC contains a linker between the ALK5 inhibitor and the target moiety. The linker is a moiety containing a covalent bond or chain of atoms that covalently links the target moiety to the drug moiety. In various embodiments, the linker includes divalent radicals such as alkyldiyl, aryldiyl, heteroaryldiyl, and moieties such as -(CR2). n O(CR2) n- Repeating units of alkoxy groups (e.g., polyvinyloxy, PEG, polymethyleneoxy) and alkylamino groups (e.g., polyvinylamino, Jeffamine) TM ); and esters and amides (including succinates, succinamides, diglycolates, malonates and hexamethylenetetramine). For example, various PEG-containing connectors are known in the art and are commercially available (e.g., from BroadPharm (broadpharm.com). Exemplary PEG-containing connectors include Mal-PEG2-Val-Cit-PAB-OH (BroadPharm cat.no.BP-23203), Mal-PEG4-Val-Cit-PAB-OH (BroadPharm cat.no.BP-23204), Mal-PEG4-Val-Cit-PAB-PNP (BroadPharm cat.no.BP-23668), Mal-amido-PEG2-Val-Cit-PAB-PNP (BroadPharm cat.no.BP-23675), Azido-PEG3-Val-Cit-PAB-OH (BroadPharm cat.no.BP-23206), and Azido-PEG4-Val-Cit-PAB-OH (BroadPharm...). cat.no.BP-23207), Azido-PEG3-Val-Cit-PAB-PNP (BroadPharm cat.no.BP-23368), Fmoc-PEG4-Ala-Ala-Asn-PAB (BP-23328), Azido-PEG5-Ala-Ala-Asn-PAB (BroadPharm cat.no.BP-23368) cat.no.BP-23329), Fmoc-PEG3-Ala-Ala-Asn(Trt)-PAB(BroadPharm cat no.BP-23285), Azido-PEG4-Ala-Ala-Asn(Trt)-PAB(BroadPharm cat.no.BP-23285) catno.BP-23284) and Fmoc-PEG3-Ala-Ala-Asn(Trt)-PAB-PNP (BroadPharm cat (No. BP-23297). In some embodiments, the TDC linker comprises PEG and a peptide, such as one of the dipeptides described in this section, such as Val-Cit.

[0135] A linker may comprise one or more linker components, such as an extension portion and a spacer portion. For example, a peptide linker may comprise a peptide component of two or more amino acids and optionally one or more extension portions and / or spacer portions. Various linker components are known in the art, some of which are described below.

[0136] The adapter can be a “cutterable adapter” that facilitates drug release in cells. For example, acid-labile adapters (e.g., hydrazones), protease-sensitive adapters (e.g., peptidase-sensitive adapters), photostable adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., 1992, Cancer Research 52:127-131; US ​​patent no. 5, 208, 020).

[0137] Examples of connectors and connector components known in the art include maleimide hexanoyl (MC); maleimide hexanoyl-p-aminobenzyl carbamate; maleimide hexanoyl-peptide-aminobenzyl carbamate connectors, such as maleimide hexanoyl-L-phenylalanine-L-lysine-p-aminobenzyl carbamate and maleimide hexanoyl-L-valine-L-citrulline-p-aminobenzyl carbamate (VC); N-succinimide 3-(2-pyridyldithio)propionate (also known as N-succinimide 4-(2-pyridyldithio)valerate or SPP); 4-succinimide-oxycarbonyl-2-methyl-2-(2-pyridyldithio)-toluene (SMPT); N-Succinimidyl 3-(2-pyridyl dithio)propionate (SPDP); N-Succinimidyl 4-(2-pyridyl dithio)butyrate (SPDB); 2-Iminothione; S-acetylsuccinic anhydride; benzyl carbamate disulfide; carbonate; hydrazone linker; N-(α-maleiminoacetoxy)succinimidate; N-[4-(p-azidosalicylic acid amino)butyl]-3'-(2'-pyridyl dithio)propionamide (AMAS); N[β-maleimidepropoxy]succinimidate (BMPS); [N-ε-maleimidehexanoyloxy]succinimidate (EMCS); N-[γ-maleimidebutyryloxy]succinimidate (GMBS); Succinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxy-[6-aminohexanoate] (LC-SMCC); Succinimide-6-(3-[2-pyridyldithio]propamido)hexanoate (LC-SPDP); m-maleimidebenzoyl-N-hydroxysuccinimide (MBS); N-succinimide[4-iodoacetyl]aminobenzoate (SIAB); Succinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylate (SMCC); N-succinimide-3-[2-pyridyldithio]propamido (SPDP); [N-ε-maleimidehexanoyloxy]sulfosuccinimide (Sulfo-EMC) S); N-[γ-maleimide butyryloxy]sulfosuccinimide ester (sulfon-GMBS); 4-sulfosuccinimide-6-methyl-α-(2-pyridyldithio)toluidine]hexanoate (sulfon-LC-SMPT); sulfosuccinimide-6-(3'-[2-pyridinyldithio]-propionamide)hexanoic acid (Sulfo-LC-SPDP); m-maleimide benzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS); N-sulfosuccinimide-[4-iodoacetyl]aminobenzoate (sulfon-SIAB); sulfosuccinimide-4-[N-maleimide methyl]cyclohexane-1-carboxylic acid ester (Sulfo-SMCC);Sulfosuccinimide-4-[p-maleimide phenyl]butyrate (Sulfo-SMPB); Ethylene glycol-bis(N-hydroxysuccinimide succinate) (EGS); Disuccinimide tartrate (DST); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); Diethylenetriamine-pentaacetic acid (DTPA); thiourea connectors; and oxime-containing connectors.

[0138] In some embodiments, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker releases the ALK5 inhibitor from the target site in a suitable environment. In other embodiments, the linker is uncleavable and releases the drug, for example, by targeting partial degradation in lysosomes (see U.S. Patent Publication 2005 / 0238649, which is incorporated herein by reference in its entirety and for all purposes).

[0139] Examples of uncuttable connectors that can be used in the TDC of this disclosure include N-maleimide methylcyclohexane 1-carboxylate, maleimide hexanoyl, or mercaptoacetamide hexanoyl connectors.

[0140] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., in lysosomes, endosomes, or caveoles). The linker can be a peptide linker cleaved, for example, by an intracellular peptidase or protease, including but not limited to lysosomal or endosomal proteases. In some embodiments, the peptide linker comprises a peptide component that is at least two amino acids long or at least three amino acids long or longer.

[0141] Cleavage agents can include, but are not limited to, cathepsins B and D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug within the target cell (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, peptide linkers can be cleaved by the thiol-dependent protease cathepsin-B (e.g., Phe-Leu or Gly-Phe-Leu-Gly linkers). Other examples of such linkers are described, for example, in U.S. Patent 6,214,345, the contents of which are incorporated herein by reference in their entirety and for all purposes.

[0142] In some embodiments, the peptide linker that can be cleaved by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Patent 6,214,345, which describes the synthesis of doxorubicin using a val-cit linker).

[0143] In other embodiments, the cleavable adapter is pH-sensitive, meaning it is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive adapters are hydrolyzable under acidic conditions. For example, acid-labile adapters that are hydrolyzable in lysosomes (e.g., hydrazones, aminoureas, thioureas, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used. (See, for example, U.S. Patents 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.). Such adapters are relatively stable under neutral pH conditions, such as in blood, but unstable at pH values ​​below 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable connector is a thioether connector (e.g., a thioether linked to the therapeutic agent via an acylhydrazone bond (see, for example, U.S. Patent No. 5,622,929).

[0144] In other embodiments, the joint is cuttable under reducing conditions (e.g., a disulfide joint). Various disulfide joints are known in the art, including, for example, those formed using: SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987. See also, for example, U.S. Patent 4,880,935.)

[0145] In other embodiments, the connector is a malonic acid ester connector (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimide benzoyl connector (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0146] In some implementations, the linker is a multivalent linker that can be used to link a number of drug molecules to a single target moiety molecule (e.g., a single antibody molecule). For example, the Fleximer linker technology developed by Mersana is based on binding drug molecules to a solubilized polyacetal backbone via a series of ester bonds. This method enables high TDC loading (e.g., with a drug-to-antibody ratio (DAR) up to 20) while maintaining favorable physicochemical properties. Exemplary multivalent connectors are described in, for example, WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; and WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety.

[0147] Typically, adapters are essentially insensitive to the extracellular environment. As used herein, “essentially insensitive to the extracellular environment” in the context of adapters means that, when TDCs are present in the extracellular environment (e.g., in plasma), no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the adapter is cleaved in a sample containing TDCs.

[0148] It can be determined whether the adapter is substantially insensitive to the extracellular environment, for example, by incubating TDC with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0149] In other non-mutually exclusive embodiments, the adapter can promote cell internalization. In some embodiments, the adapter promotes cell internalization when conjugated with a therapeutic agent (i.e., in the context of the adapter-therapeutic agent portion of a TDC as described herein). In other embodiments, the adapter promotes cell internalization when conjugated with both an ALK5 inhibitor and an antibody.

[0150] In many embodiments, the linker is self-sacrificing. As used herein, the term "self-sacrificing" refers to a bifunctional chemical moiety capable of covalently linking two separated chemical moieties to a stable terad. If its bond with the first moiety is cleaved, it will spontaneously dissociate from the second chemical moiety. See, for example, PCT Publications WO 2007 / 059404, WO 2006 / 110476, WO 2005 / 112919, WO 2010 / 062171, WO 2009 / 017394, WO 2007 / 089149, WO 2007 / 018431, WO 2004 / 043493 and WO 2002 / 083180, which pertain to drug-cleavable substrate conjugates in which the drug and cleavable substrate are optionally linked by a self-sacrificing linker and are all explicitly incorporated herein by reference. Instances of self-sacrificing interval units that can be used to generate self-sacrificing joints are described in Equation I below.

[0151] Various exemplary connectors that may be used with the present compositions and methods are described in PCT Publication WO 2004 / 010957, U.S. Patent Publication US 2006 / 0074008, U.S. Patent Publication US 2005 / 0238649 and U.S. Patent Publication US2006 / 0024317 (each of which is incorporated herein by reference in its entirety and for all purposes).

[0152] The TDC of this disclosure may have the following formula I, wherein an antibody or other targeting moiety (represented as "Ab" in Formula I) is conjugated to one or more ALK5 inhibitor drug moieties (D) via an optional linker (L).

[0153] Ab-(LD) p I

[0154] Therefore, the target moiety can be conjugated to the drug directly or via a connector. In Formula I, p is the average number of drug (i.e., ALK5 inhibitor) moieties per target moiety, which can range from about 1 to about 20 drug moieties per target moiety, and in some embodiments, from 2 to about 8 drug moieties per target moiety. Further details regarding drug loading are described in Section 5.5 below.

[0155] In some embodiments, the adapter component may include a "stretcher" that links the targeting portion (e.g., via cysteine ​​residues) to another adapter component or drug portion. Exemplary stretchers are shown below (where the wavy line on the left indicates a site covalently linked to the targeting portion, and the wavy line on the right indicates a site covalently linked to another adapter component or drug portion):

[0156]

[0157]

[0158] See U.S. Patent No. 9,109,035; Ducry et al., 2010, Bioconjugate Chem. 21:5-13.

[0159] In some embodiments, the linker component may comprise an amino acid unit. In one such embodiment, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating drug release from the TDC upon exposure to intracellular proteases, such as lysosomal enzymes. See, for example, Doronina et al., 2003, Nat. Biotechnol. 21:778-784. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline (VC or val-cit), alanine-phenylalanine (AF or ala-phe); phenylalanine-lysine (FK or phe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-gly (gly-gly-gly). Exemplary tetrapeptides include glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly). Amino acid units can contain naturally occurring amino acid residues, as well as small amounts of amino acids and non-naturally occurring amino acid analogs. For example, the selectivity of citrulline amino acid units for enzymatic cleavage by specific enzymes (e.g., cathepsins B, C, and D, or plasminase) can be designed and optimized.

[0160] In some embodiments, the adapter component may comprise a “spacer” unit that links the targeting moiety to the drug moiety directly or via an extended and / or amino acid unit. The spacer unit may be “self-sacrificing” or “non-self-sacrificing.” A “non-self-sacrificing” spacer unit is one in which part or all of the spacer unit remains bound to the drug moiety upon enzymatic (e.g., proteolytic) cleavage of the TDC. Examples of non-self-sacrificing spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. A “self-sacrificing” spacer unit allows for the release of the drug moiety without a separate hydrolysis step. In some embodiments, the spacer unit of the adapter comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is linked to the amino acid unit via an amide bond and is prepared as a carbamate, methyl carbamate, or carbonate between benzyl alcohol and the cytotoxic agent. See, for example, Hamann et al., 2005, Expert Opin. Ther. Patents 15:1087-1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In some embodiments, the phenylene moiety of the p-aminobenzyl unit is replaced with Qm, where Q is a -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; and m is an integer in the range of 0-4. Examples of self-sacrificing spacer units further include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, for example, U.S. Patent Publication No. US 2005 / 0256030), such as 2-aminoimidazolium-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetal. Intervals that undergo cyclization upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223); appropriately substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems (Storm et al., 1972, Amer. Chem. Soc. 94:5815); and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs with α-substitution at the glycine position (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-sacrificing interval that can be used for TDCs.

[0161] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as described below, which can be used for incorporation and release of a variety of drugs.

[0162]

[0163] Where Ab and D are defined as in Formula I above; A is the extension segment, a is an integer from 0 to 1; W is an amino acid unit, w is an integer from 0 to 12; Q is -C1-C8 alkyl, -O--(-C1-C8 alkyl), -halogen, -nitro or -cyano; m is an integer from 0 to 4; n is 0 or 1; p ranges from 1 to about 20.

[0164] The connector may comprise any one or more of the connector components described above. In some embodiments, the connector is as shown in parentheses in the following TDC formula:

[0165] Ab–(–[Aa-Ww-Yy]-D) p II

[0166] Where Ab, A, a, W, w, D, and p are as defined in the preceding paragraph; Y is a spacer unit, where y is 0, 1, or 2; exemplary embodiments of such connectors are described in U.S. Patent Publication 2005 / 0238649A1, which is incorporated herein by reference.

[0167] Exemplary connector components and their combinations are shown below in the context of the TDC of Formula II:

[0168]

[0169] The linker components, including extension segments, spacer segments, and amino acid units, can be synthesized by methods known in the art, such as those described in U.S. Patent Publication 2005 / 0238649.

[0170] 5.5. Drug Loading

[0171] Drug loading is denoted by p and is the average number of ALK5 inhibitor portions per target moiety (e.g., per antibody) in the molecule. Drug loading (“p”) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more portions (D) per target moiety, although the average is usually a fraction or decimal. Typically, ALK5 inhibitors are loaded with an average of 2 to 8 drug portions per target moiety, more preferably 2 to 4 drug portions per antibody or 5 to 7 drug portions per target moiety.

[0172] As those skilled in the art will understand, in many cases, the reference to TDC is an abbreviation for a group or set of TDC molecules (sometimes in the context of a pharmaceutical composition), each molecule consisting of a target moiety covalently linked to one or more ALK5 inhibitor moieties. The drug loading rate represents the average drug loading in the group or set, although the ratio based on individual molecules may vary from one TDC molecule to another within the group. In some embodiments, the group or set contains TDC molecules comprising antibodies covalently linked to any position between 1 and 30 pharmaceutical moieties, or in some embodiments, antibodies at any position between 1 and 20, 1 and 15, 2 and 12, 2 and 8, 4 and 15, or 6 and 12 pharmaceutical moieties. Preferably, the average value in the group is as described in the preceding paragraph, for example, 2 to 8 pharmaceutical moieties per target moiety, more preferably 4 to 8 pharmaceutical moieties per target moiety, or 5 to 7 pharmaceutical moieties per target moiety.

[0173] Some TDC groups can be in the form of compositions containing TDCs as described herein and targeting moieties lacking the drug moiety, such as antibodies that fail to link ALK5 inhibitors.

[0174] The average number of ALK5 inhibitor portions of each target moiety in the TDC formulation derived from the conjugation reaction can be characterized by conventional methods such as mass spectrometry, hydrophobic interaction chromatography (HIC), and ELISA.

[0175] The quantitative distribution of TDC with respect to p can also be determined. In some cases, homogeneous TDCs with specific p values ​​can be separated, purified, and characterized from TDCs loaded with other ALK5 inhibitors by means such as electrophoresis.

[0176] For some drug conjugates, p may be limited by the number of linker sites on the target moiety. For example, when the linker is a cysteine ​​thiol, as in the exemplary embodiments described above, the target moiety (e.g., antibody) may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker can be attached. In some embodiments, higher drug loading, such as p > 5, may lead to aggregation, insolubility, toxicity, or loss of cell permeability of some drug conjugates. In some embodiments, the drug loading of the TDC of this disclosure is in the range of 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. Indeed, it has been shown that for some TDCs, the optimal ratio of drug moiety per antibody may be less than 8, and may be about 2 to about 5. See U.S. Patent Publication US 2005 / 0238649 (incorporated herein by reference in its entirety).

[0177] In some embodiments, during the conjugation reaction, less than the theoretical maximum amount of the drug moiety conjugates with the target moiety. As described below, the target moiety may contain, for example, lysine residues that do not react with drug-connector intermediates or connector reagents. Typically, antibodies do not contain numerous free and reactive cysteine ​​thiols that could potentially bind to the drug moiety; in fact, most cysteine ​​thiols in antibodies exist as disulfide bonds. In some embodiments, the antibody or other target moiety can be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiols. In some embodiments, the antibody or other target moiety is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0178] The loading (drug / antibody ratio) of TDC can be controlled in various ways, for example, by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the target moiety, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reduction conditions for cysteine ​​thiol modification, and (iv) modifying the amino acid sequence of the target moiety by recombinant technology to modify the number and position of cysteine ​​residues to control the number and / or position of the linker-drug conjugate (e.g., thioMab or thioFab prepared as disclosed in PCT Publication WO 2006 / 034488 (incorporated herein by reference in its entirety)).

[0179] It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, and then with a drug moiety reagent, the resulting product is a mixture of TDC compounds in which one or more drug moieties are distributed in conjunction with the target moieties. The average amount of drug in each target moiety can be calculated from the mixture using a dual ELISA antibody assay that is specific to both the target moieties and the drug. Individual TDC molecules in the mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography.

[0180] In some implementations, homogeneous TDCs with a single loading value can be separated from conjugated mixtures by electrophoresis or chromatography.

[0181] 5.6. Formulation and Application

[0182] Suitable routes of administration for TDC include, but are not limited to, oral, parenteral, rectal, transmucosal, enteral, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intracavitary, intraperitoneal, or intratumoral injection. The preferred route of administration is parenteral, more preferably intravenous. Alternatively, the compound can be administered locally rather than systemically, for example, by injecting the compound directly into the fibrotic area or by injecting the compound directly into the solid tumor.

[0183] Immunoconjugates can be formulated according to known methods to prepare pharmaceutically useful compositions, thereby combining TDC with a pharmaceutically suitable excipient in a mixture. Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. For example, see Ansel et al., Pharmaceutical Dosage Forms And Drug Delivery Systems, 5th Edition (Lea & Febbiger 1990) and Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990) and their revisions.

[0184] In a preferred embodiment, the TDC is prepared in Good's biological buffer (pH 6-7) using a buffer solution selected from the group consisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES); N-(2-acetamido)iminodiacetic acid (ADA); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES); 2-(N-morpholino)ethanesulfonic acid (MES); 3-(N-morpholino)propanesulfonic acid (MOPS); 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO); and piperazine-N,N'-bis(2-ethanesulfonic acid). A more preferred buffer solution is MES or MOPS, preferably in the concentration range of 20 to 100 mM, more preferably about 25 mM. Most preferably, 25 mM MES at pH 6.5 is used. The formulation may further contain 25 mM trehalose and 0.01% v / v polysorbate 80 as excipients, and the final buffer concentration is modified to 22.25 mM due to the addition of excipients. The preferred storage method is as a lyophilized formulation of the conjugate, stored in a temperature range of -20°C to 2°C, with the most preferred storage range being 2°C to 8°C.

[0185] TDC can be formulated for intravenous administration via, for example, bolus, slow infusion, or continuous infusion. Preferably, the TDC is infused over a period of less than about 4 hours, more preferably over a period of less than about 3 hours. For example, the first 25-50 mg can be infused over 30 minutes, preferably even 15 minutes, with the remainder infused over the next 2-3 hours. The formulation for injection can be in a single dosage form, for example, in an ampoule or in a multi-dose container, and with added preservatives. The composition can be in the form of a suspension, solution, or emulsion, such as in an oily or aqueous medium, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the active ingredient can be in powder form for mixing with a suitable medium, such as sterile pyrogen-free water, prior to use.

[0186] Additional pharmaceutical methods can be employed to control the duration of action of TDCs. Controlled-release formulations can be prepared by using polymer complexes or adsorbing TDCs. For example, biocompatible polymers include poly(ethylene-co-vinyl acetate) matrices and stearic acid dimer and sebacic acid polyanhydride copolymer matrices. Sherwood et al., 1992, Bio / Technology 10:1446. The rate of TDC release from such matrices depends on the molecular weight of the TDC, the amount of TDC in the matrix, and the size of the dispersed particles. Saltzman et al., 1989, Biophys.J.55:163; Sherwood et al., ibid. Other solid dosage forms are described in Ansel et al., Pharmaceutical Dosage Forms And Drug Delivery Systems, 5th Edition (Lea & Febbiger 1990) and Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990) and their revisions.

[0187] Generally, the dosage of TDC administered to humans will vary depending on factors such as the patient's age, weight, height, sex, general health condition, and medical history. A single intravenous infusion of approximately 0.3 mg / kg to 5 mg / kg may be necessary, although lower or higher doses may be administered depending on the circumstances. For example, for a 70 kg patient, a dose of 0.3–5 mg / kg would be 21–350 mg, or for a 1.7-m patient, a dose of 12–20 mg / kg would be 21–350 mg. 6 mg / m 2The dosage is 21-350 mg. The dosage can be repeated as needed, for example, once weekly for 2-10 weeks, once weekly for 8 weeks, or once weekly for 4 weeks. It can also be given less frequently, such as every other week for several months, or monthly or quarterly for several months, depending on the need for maintenance therapy. Preferred dosages may include, but are not limited to, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1.0 mg / kg, 1.2 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg. A more preferred dosage is 0.6 mg / kg weekly and less frequently administered at 1.2 mg / kg. Any amount in the range of 0.3 to 5 mg / kg can be used. This dosage is preferably administered once weekly multiple times. The minimum dose can be administered according to a schedule of 4 weeks, more preferably 8 weeks, more preferably 16 weeks or longer, with the frequency of administration depending on the toxic side effects and recovery from them, primarily related to hematologic toxicity. The administration schedule may include administration once or twice weekly, for example, in a cycle selected from the following groups: (i) weekly; (ii) every other week; (iii) one week of treatment followed by two, three, or four weeks of rest; (iv) two weeks of treatment followed by one, two, three, or four weeks of rest; (v) three weeks of treatment followed by one, two, three, four, or five weeks of rest; (vi) four weeks of treatment followed by one, two, three, four, or five weeks of rest; (vii) five weeks of treatment followed by one, two, three, four, or five weeks of rest; (viii) once a month. This cycle may be repeated 2, 4, 6, 8, 10, or 12 or more times.

[0188] Alternatively, TDC can be administered as a dose every 2 or 3 weeks, repeated for a total of at least 3 doses. Alternatively, it can be administered twice weekly for 4–6 weeks. This dose can be administered every other week, or even less frequently, so that the patient can recover from any drug-related toxicities. Alternatively, the dosing schedule can be reduced to once every 2 or 3 weeks for 2–3 months. The dosing regimen can optionally be repeated at other intervals, and the dose can be administered via various parenteral routes, with appropriate adjustments to the dose and schedule.

[0189] 5.7. Treatment Methods

[0190] 5.7.1. Fibrosis

[0191] The TDC disclosed herein can be used to treat various fibrotic conditions, such as fibrosis associated with systemic sclerosis (also known as scleroderma) or NASH. Patients with systemic sclerosis often suffer from pulmonary fibrosis, skin fibrosis, and esophageal fibrosis, although fibrosis can occur in almost any organ. Patients with NASH often suffer from liver fibrosis. The TDC can be used as a monotherapy or as part of a combination therapy regimen, such as with standard care agents or regimens. In some embodiments, the combination therapy includes administration of the TDC in combination with pirfenidone, nintedanib, pentraxin-2, pamrevlumab, prednisone, cortisone, cyclophosphamide, azathioprine, or combinations thereof. In some embodiments, the combination therapy includes administration of the TDC in combination with pirfenidone and / or nintedanib.

[0192] Examples of conditions for which the TDC of this disclosure can be used for treatment include, but are not limited to, pulmonary fibrosis (e.g., IPF), liver fibrosis (e.g., NASH-related), renal fibrosis, cardiac fibrosis, skin fibrosis, esophageal fibrosis, and systemic sclerosis. The TDC of this disclosure can be administered to subjects suffering from, for example, a diagnosed fibrosis-related disease (e.g., systemic sclerosis or NASH) before the development of signs and / or symptoms of fibrosis. Alternatively, or otherwise, the TDC can be administered to subjects suffering from, for example, a diagnosed fibrosis-related disease, after the observation of signs and / or symptoms of fibrosis.

[0193] The use of the TDC disclosed herein in combination with one or more therapies does not limit the order in which the therapies are administered. For example, the TDC of this disclosure may be administered before, during, or after treatment of a subject with one or more therapies. In some embodiments, the TDC of this disclosure is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) treatment with another therapy (e.g., a second therapeutic agent as described above). In some embodiments, the TDC of this disclosure is incorporated into the same regimen as the second therapeutic agent.

[0194] 5.7.2. Cancer

[0195] The TDCs disclosed herein (e.g., FAP-targeting TDCs) can be used to treat a variety of cancers. TDCs can be used as a monotherapy or as part of a combination therapy regimen, such as with a standard care agent or regimen. In some embodiments, the combination therapy includes administration of TDCs in combination with immunotherapies (e.g., checkpoint inhibitor therapy), chimeric antigen receptor (CAR) therapy, adoptive T-cell therapy (e.g., autologous T-cell therapy), oncolytic virus therapy, dendritic cell vaccine therapy, interferon gene stimulator (STING) agonist therapy, toll-like receptor (TLR) agonist therapy, intratumoral CpG therapy, cytokine therapy (e.g., IL2, IL12, IFN-α, or INF-γ therapy), or combinations thereof. In some embodiments, the combination therapy includes administration of TDCs in combination with an ADC having a cytotoxic payload, such as an FAP-targeting ADC (e.g., OMTX705 (Oncomatryx)). In some implementations, combination therapy includes TDC administered in combination with immunoprotective chemotherapy (e.g., antimetabolites, such as 5-fluorouracil, gemcitabine, or methotrexate), alkylating agents (such as cyclophosphamide, dacarbazine, nitrogen mustard, diacinone, or temozolomide), anthracyclines (such as doxorubicin or epirubicin), antimicrotubule agents (such as vincristine), platinum compounds (such as cisplatin or oxaliplatin), taxanes (such as paclitaxel or docetaxel), or topoisomerase inhibitors (such as etoposide or mitoxantrone), or vinca alkaloids (such as vincristine).

[0196] Examples of cancers that can be treated with the TDC disclosed herein include, but are not limited to, urothelial carcinoma (e.g., bladder cancer, urethral cancer, and ureteral cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC) (such as adenocarcinoma, squamous cell carcinoma, large cell carcinoma) and small cell lung cancer), breast cancer, colorectal cancer (e.g., adenocarcinoma, carcinoid tumor, gastrointestinal stromal tumor, and colorectal lymphoma), pancreatic cancer, prostate cancer, and esophageal cancer. Other examples of cancers that can be treated with the TDC disclosed herein include head and neck cancer, ovarian cancer, kidney cancer, and gastric adenocarcinoma.

[0197] The TDC disclosed herein can be used in combination with checkpoint inhibitors, such as those targeting PD1, PDL1, CTLA4, TIGIT, LAG3, OX40, CD40, or VISTA. Checkpoint inhibitors include antibodies and small molecules. Exemplary checkpoint inhibitors targeting PD1 include pembrolizumab, nivolumab, cemiplimab, and dostarlimab. Exemplary checkpoint inhibitors targeting PDL1 include atezolizumab, avelumab, durvalumab, BMS-1001, and BMS-1166. An exemplary checkpoint inhibitor targeting CTLA4 is ipilimumab. Exemplary checkpoint inhibitors targeting TIGIT include etigilimab, tiragolumab, and AB154. Exemplary checkpoint inhibitors targeting LAG3 include LAG525, Sym022, relatlimab, and TSR-033. Exemplary checkpoint inhibitors targeting OX40 include MEDI6469, PF-04518600, and BMS986178. Exemplary checkpoint inhibitors targeting CD40 include selicrelumab, CP-870,893, and APX005M. An exemplary checkpoint inhibitor targeting VISTA is HMBD-002. For the treatment of urothelial carcinoma, the TDC of this disclosure can be used in combination with standard of care, including but not limited to cisplatin, mitomycin C, carboplatin, docetaxel, paclitaxel, doxorubicin, 5-FU, methotrexate, vincristine, ifosfamide, and pemetrexed. Furthermore, the TDC can be used in combination with checkpoint inhibitors such as ipilimumab.

[0198] For the treatment of non-small cell lung cancer (NSCLC), the TDC disclosed herein can be used in combination with standard-of-care chemotherapy, including drugs such as cisplatin, carboplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, etoposide, or vincaine. Additionally, the TDC can be used in combination with targeted therapies such as bevacizumab or erbitux. Furthermore, the TDC can be used in combination with checkpoint inhibitors such as pembrolizumab, nivolumab, cimiprimab, dotalipramab, atezolizumab, acitumab, durvalumab, or ipilimumab.

[0199] For the treatment of breast cancer, the TDC disclosed herein can be used in combination with standard-of-care chemotherapy agents such as anthracyclines (doxorubicin or epirubicin) and taxanes (paclitaxel or docetaxel), as well as fluorouracil, cyclophosphamide, and carboplatin. Furthermore, the TDC disclosed herein can be used in combination with targeted therapies. Targeted therapies for HER2 / neu-positive tumors include trastuzumab and pertuzumab, and targeted therapies for estrogen receptor (ER)-positive tumors include tamoxifen, toremifene, and fulvestrant. Additionally, the TDC can be used in combination with checkpoint inhibitors such as atezolizumab.

[0200] For the treatment of colorectal cancer, the TDC disclosed herein can be used in combination with standard of care, including but not limited to 5-FU, capecitabine, irinotecan, oxaliplatin, trifluridine, and tipiracil. Furthermore, the TDC disclosed herein can be used in combination with targeted therapies, including bevacizumab, ramucirumab, and ziv-aflibercept. Additionally, the TDC can be used in combination with checkpoint inhibitors such as pembrolizumab, nivolumab, or ipilimumab.

[0201] For pancreatic cancer, the TDC disclosed herein can be used in combination with standard-of-care chemotherapy agents such as gemcitabine, 5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin, or docetaxel. In addition, the TDC can be used in combination with targeted therapies, such as erlotinib, which inhibits EGFR.

[0202] For prostate cancer, the TDC disclosed herein can be used in combination with standard-of-care chemotherapy agents, including docetaxel, and optionally in combination with the steroids prednisone or cabazitaxel. Additionally, the TDC can be used in combination with checkpoint inhibitors, such as ipilimumab.

[0203] For esophageal cancer, the TDC disclosed herein can be used in combination with standard-of-care chemotherapy agents such as carboplatin and paclitaxel, cisplatin and 5-FU, epirubicin, cisplatin and 5-FU, docetaxel, cisplatin and 5-FU, cisplatin with capecitabine, oxaliplatin and 5-FU or capecitabine, irinotecan or trifluuridine and tipyrimidine. Furthermore, the TDC can be used in combination with targeted therapies such as trastuzumab or ramucirumab. Additionally, the TDC can be used in combination with checkpoint inhibitors such as pembrolizumab.

[0204] The use of the TDC disclosed herein in combination with one or more therapies does not limit the order of administration. For example, the TDC of this disclosure may be administered before, during, or after treatment of a subject with one or more therapies. In some embodiments, the TDC of this disclosure is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) treatment with another therapy (e.g., after). In some embodiments, the TDC of this disclosure is incorporated into the same regimen as the second therapy.

[0205] Example

[0206] The following abbreviations can be found throughout the embodiments section:

[0207] Boc—tert-butyloxycarbonyl

[0208] DCM—Dichloromethane

[0209] DMA-dimethylamine

[0210] DMF—Dimethylformamide

[0211] DIPEA—N,N-Diisopropylethylamine

[0212] EtOAc—ethyl acetate

[0213] EtOH—ethanol

[0214] Fmoc—fluorenylmethoxycarbonyl

[0215] HOBt—hydroxybenzotriazole

[0216] MeOH—Methanol

[0217] NaHMDS—Sodium hexamethyldisilazide

[0218] RT—Room temperature, approximately 21°C

[0219] TBTU—O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0220] TEA—Triethylamine

[0221] THF—Tetrahydrofuran

[0222] TFA—Trifluoroacetic acid

[0223] TMS-Imidazole-1-(trimethylsilyl)imidazolium

[0224] 6.1. Example 1: Synthesis of 4-(6-methylpyridin-2-yl)-5-(1,5-naphthidin-2-yl)-1,3-thiazolyl-2-amine (compound A)

[0225] Compound A was prepared according to the general method in Scheme 1 below:

[0226]

[0227] 6.1.1.2-Methyl-1,5-naphthidine (A1)

[0228] A mixture of concentrated sulfuric acid (2.5 ml), sodium m-nitrobenzenesulfonate (2.08 g, 9.24 mmol), boric acid (445 mg, 7.21 mmol), and ferrous sulfate heptahydrate (167 mg, 0.60 mmol) was stirred at room temperature. Glycerol (1.5 ml) was added to the reaction mixture, followed by 5-amino-2-methylpyridine (A-SM) (500 mg, 4.62 mmol) and water (2.5 ml), and the mixture was heated at 135 °C for 18 h. After the reaction was complete, as measured by TLC, the reaction mixture was cooled to approximately 21 °C, alkalized with 4N NaOH, and extracted with EtOAc (2 x 100 ml). The organic extracts were combined, washed with water (200 ml), dried over Na2SO4, and evaporated under reduced pressure to give crude compound A1. The crude product was purified by silica gel column chromatography (2% MeOH / CH2Cl2) to give compound A1, which is a light brown crystalline solid (200 mg, 30%).

[0229] 1 H NMR (500MHz, CDCl3): δ8.92(d,J=3.0Hz,1H),8.35(d,J=9.0Hz,1H),8.31(d,J=5.9Hz,1H),7.62(dd,J=8.5,4.5Hz,1H),7.54(d,J=5.9Hz,1H),2.8(s,3H)

[0230] LC-MS(ESI): m / z 145 [M+H] +

[0231] 6.1.2.1-(6-methylpyridin-2-yl)-2-(1,5-naphthidin-2-yl)ethane-1-one (A2)

[0232] A solution of A1 (200 mg, 1.38 mmol) and methyl 6-methylpicolinate (209 mg, 1.38 mmol) in anhydrous THF (10 mL) was placed under a nitrogen atmosphere and cooled to -78 °C. Potassium bis(trimethylsilyl)amide (0.5 M in toluene, 6.9 mL, 3.47 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at -78 °C for 1 h, then heated to approximately 21 °C and maintained for 20 h. After the reaction was complete (as measured by TLC), the reaction mixture was quenched with a saturated ammonium chloride solution (20 mL). The aqueous layer was extracted with EtOAc (2 × 20 mL). The combined organic extracts were washed with water (100 mL), dried over Na2SO4, and evaporated to give crude compound A2. The crude material was purified by column chromatography (1% MeOH / CH2Cl2) to obtain compound A2, which was an orange-yellow solid (110 mg, 30.5%).

[0233] 1 H NMR (400MHz, CDCl3:Enol form): δ15.74(brs,-OH),8.69(t,J=3.6,1H),8.12(d,J=9.2Hz,1H),8.06(dd,J=8.4,4.4Hz,2H),7. 82(t,J=7.6Hz,1H),7.55(dd,J=8.4,4.8Hz,1H)7.45(d,J=9.6Hz,1H),7.3(dd,J=7.6,4.0Hz,1H),7.16(s,1H),2.75(s,3H)

[0234] LC-MS(ESI): m / z 264 [M+H] +

[0235] 6.1.3.4-(6-methylpyridin-2-yl)-5-(1,5-naphthidin-2-yl)-1,3-thiazolyl-2-amine (Compound A)

[0236] A solution of A2 (110 mg, 0.418 mmol) in 1,4-dioxane (10 mL) was treated with bromine (0.025 mL, 0.501 mmol). The resulting reaction mixture was stirred at about 21 °C for 1 h, then concentrated under reduced pressure to give crude A3 (120 mg), which could be used for the next step without further purification. Crude A3 (120 mg) was dissolved in ethanol (15 mL). Thiourea (3.5 mg, 0.046 mmol) was then added, and the reaction mixture was heated at 78 °C for 4 h (until the starting material was observed to be completely consumed by TLC). The reaction mixture was cooled to about 21 °C, and ammonia solution (25%, 1.5 mL) was added with gentle stirring. The solvent was evaporated, and the residue was then dissolved in CH2Cl2 (2 x 20 mL) and washed with water (50.0 mL). The separated organic layer was then washed with 1 N HCl (30 mL x 2). The combined aqueous layers were alkalized with 35% aqueous (aq.) sodium hydroxide (20 ml) and extracted with CH2Cl2 (2 x 20 ml). The organic layers were dried with sodium sulfate and evaporated to give crude compound A. Crude compound A was recrystallized from acetonitrile (2 ml) to give purified compound A as a yellow crystalline solid (35 mg after 2 steps, yield 49%).

[0237] 1 H NMR (400MHz, CDCl3): δ8.86(dd,J=4.4,1.6Hz,1H),8.29(t,J=8.4Hz,1H),8.06(d,J=9.2Hz,1H),7.64( t,J=7.6Hz,1H),7.60-7.55(m,2H),7.46(d,J=8Hz,1H),7.20(d,J=7.6,1H),5.32(brs,2H),2.57(s,3H)

[0238] LC-MS(ESI): m / z 320 [M+H] +

[0239] UPLC purity: 97.6%

[0240] 6.2. Example 2: Synthesis of N-methyl-2-(4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}phenoxy)ethane-1-amine (Compound B)

[0241] Compound B is prepared according to the general method in Scheme 2 below:

[0242]

[0243] 6.2.1. (2-Chloroethyl)(methyl)carbamate tert-butyl ester (B7)

[0244] A solution of B6 (1 g, 7.69 mmol) in water (4 ml) and a solution of TEA (1 ml, 7.69 mmol) in THF (4 ml) were simultaneously added to a stirred solution of Boc-anhydride (1.7 ml, 7.30 mmol) in THF (4 ml) over a period of 1 hour. The resulting mixture was stirred at approximately 21 °C for 16 hours. The reaction mixture was diluted with saturated NaCl solution (20 ml) and extracted with DCM (3 × 50 ml). The combined organic extracts were dried over Na2SO4 and concentrated under vacuum to give a crude compound, which was purified by silica gel column chromatography using 10% EtOAc / hexane to give compound B7 as a pale yellow liquid (1 g, 5.18 mmol, 71%).

[0245] 1 H NMR (400MHz, CDCl3): δ3.58-3.52(m,4H),2.93(s,3H),1.46(s,9H)

[0246] 6.2.2. Methyl (2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxoron-2-yl)phenoxy)ethyl)tert-butyl carbamate (Int-B)

[0247] Under an argon atmosphere, B7 (900 mg, 4.66 mmol), KI (18 mg, 0.10 mmol), and Cs₂CO₃ (2.57 g, 7.88 mmol) were added to a stirred solution of 4-hydroxyphenylboronic acid pinacol ester (789 mg, 3.58 mmol) in DMF (13 mL). The reaction mixture was heated to 65 °C and stirred for 16 hours. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography using 7% EtOAc / hexane to give Int-B as a pale yellow solid (580 mg, 1.53 mmol, 43%).

[0248] 1 H NMR (400MHz, CDCl3): δ7.74(d,J=8.4Hz,2H),6.87(d,J=8.8Hz,2H),4.16-4.06(m,2H),3.65-3.59(m,2H),2.97(s,3H),1.45(s,9H),1.33(s,12H)

[0249] 6.2.3.2-(2-bromopyridin-4-yl)-1-(pyridin-2-yl)ethane-1-one (B2)

[0250] A solution of NaHMDS (2M in THF, 12.7ml, 25.58mmol) was added dropwise to a solution of 2-bromo-4-methylpyridine (B1) (2g, 11.62mmol) in THF (30ml) under argon at -78°C. The yellow solution was stirred at -78°C for 30 minutes. Then, a solution of ethyl pyridinecarboxylate (1.72ml, 12.79mmol) in THF (10ml) was added, and the reaction mixture was heated to approximately 21°C and stirred for 16 hours. The solvent was evaporated under reduced pressure, and the solid residue was prepared with diethyl ether, filtered, and washed with diethyl ether. The solid was then diluted with saturated NH4Cl solution (30ml), and the aqueous phase was extracted with EtOAc (2 × 200ml). The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography using 10% EtOAc / hexane to give compound B2, which was a yellow solid (2.06 g, 7.46 mmol, 64.3%).

[0251] 1 H NMR (400MHz, CDCl3): δ8.75(d,J=5.2Hz,1H),8.32(d,J=5.2Hz,1H),8.08(d,J=8.0Hz,1H),7.89(t,J=7.6Hz 1H),7.56-7.51(m,2H),7.28-7.25(m,1H),4.55(s,2H)

[0252] LC-MS (ESI): m / z 277[M]+

[0253] 6.2.4.2-Bromo-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine (B3)

[0254] A solution of B2 (850 mg, 3.07 mmol) in dry DMF (3.4 mL) was treated with glacial acetic acid (0.45 mL, 7.39 mmol) in DMF under argon atmosphere. DMA (0.6 mL, 4.61 mmol) was added dropwise, and the mixture was stirred at approximately 21 °C for 2 hours under argon atmosphere. Hydrazine monohydrate (1.15 mL, 23.09 mmol) was added dropwise, and the resulting mixture was heated at 50 °C for 3 hours and then at approximately 21 °C for 16 hours. The reaction mixture was poured into water (30 mL) and extracted with CH2Cl2 (3 × 30 mL). The organic layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure to give the crude compound. The crude product was purified by silica gel column chromatography using 30% EtOAc / hexane to give compound B3 as a yellow solid (560 mg, 1.86 mmol, 60.6%).

[0255] 1H NMR (400MHz, CDCl3) δ8.74(brs,1H),8.34(d,J=5.0Hz,1H),7.83(brs,1H),7.81(t,J =6.0Hz,1H),7.56(s,1H),7.49(d,J=8.0Hz,1H),7.39-7.84(m,1H),7.31-7.26(m,1H)

[0256] LC-MS (ESI): m / z 301[M]+

[0257] 6.2.5.2-Bromo-4-(3-(pyridin-2-yl)-1-triphenylmethyl-1H-pyrazol-4-yl)pyridine (B4)

[0258] To a stirred solution of B3 (500 mg, 1.66 mmol) in acetone (10 mL), K₂CO₃ (1.37 g, 9.99 mmol) and triphenylmethyl chloride (464 mg, 2.49 mmol) were added. The reaction mixture was then heated to reflux and stirred for 24 hours. The reaction mixture was filtered and the filtrate was concentrated, then partitioned between CH₂Cl₂ (20 mL) and water (10 mL). The organic phase was dried over Na₂SO₄ and concentrated. The crude solid was purified by silica gel column chromatography using 2% MeOH / CH₂Cl₂ to give compound B4 as a pale yellow solid (402 mg, 0.74 mmol, 44%).

[0259] 1 H NMR (500MHz, CDCl3): δ8.53(d,J=4.5Hz,1H),8.20(d,J=5.5Hz,1H),7.75-7 .05(m,2H),7.56(s,1H),7.51(s,1H),7.35-7.32(m,9H),7.25-7.22(m,8H)

[0260] 6.2.6. Methyl(2-(4-(4-(3-(pyridin-2-yl)-1-triphenylmethyl-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)tert-butyl carbamate (B5)

[0261] Under an argon atmosphere, an inert-B solution (185 mg, 0.49 mmol) in EtOH (0.75 ml) was added to a stirred solution of B4 (100 mg, 0.18 mmol) in toluene (2 ml), followed by the addition of 2 M Na₂CO₃ solution (0.45 ml). The reaction mixture was degassed with argon for 20 min, then Pd(PPh₃)₄ (16 mg, 0.01 mmol) was added and refluxed for 3 h. After the starting material was completely consumed (as monitored by TLC), the reaction mixture was poured into water and extracted with toluene (3 × 15 ml). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography using 30% EtOAc / hexane to give compound B5 as a colorless solid (70 mg, 0.09 mmol, 53%).

[0262] 1 H NMR (400MHz, CDCl3): δ8.53(s,1H),8.49(d,J=4.8Hz,1H),7.82(d,J=8.8Hz,2H)7.74-7.76(m,3H),7.60(s,1H),7.40-7.34(s,8H),7.31- 7.30(m,2H),7.24-7.19(m,4H),7.12-7.10(m,1H),6.93(d,J=8.8Hz,2H),4.19-4.12(m,2H),3.66-3.58(m,2H),2.98(s,3H),1.46(s,9H).

[0263] 6.2.7. N-Methyl-2-(4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethane-1-amine hydrochloride (compound B)

[0264] At 0 °C, 4N HCl in 0.5 mL of 1,4-dioxane was added to a stirred solution B5 (70 mg, 0.09 mmol) in CH2Cl2 (6 mL). The reaction mixture was stirred for 1 hour under an argon atmosphere. After the starting material was completely consumed (as monitored by TLC), the solvent was evaporated under reduced pressure to give a crude compound, which was ground with n-pentane (2 x 1 mL) and dried to give compound B hydrochloride as a colorless solid (25 mg, 0.06 mmol, 69%).

[0265] 1H NMR (400MHz, DMSO-d6): δ8.94(brs,2H),8.62-8.56(m,3H),8.30(brs,1H),8.03-7.96(m,3H),7.86(d,J=7.6Hz,1H),7.69(brs,1H), 7.49(dd,J=7.2,5.6Hz,1H),7.29(d,J=7.6Hz,1H),7.20(d,J=8.4Hz,1H),4.36(t,J=4.8Hz,2H),3.39-3.35(m,2H),2.67-2.63(m,3H)

[0266] LC-MS (ESI): m / z 372 [M+H] +

[0267] 6.3. Example 3: Synthesis of N-methyl-2-(4-{4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}phenoxy)ethane-1-amine (Compound C)

[0268] Compound C was prepared according to the general method in Scheme 3 below:

[0269]

[0270] 6.3.1.2-(2-bromopyridin-4-yl)-1-(6-methylpyridin-2-yl)ethane-1-one (C2)

[0271] Under argon atmosphere at -78°C, NaHMDS (2M, 6.39 mL, 12.8 mmol in THF) was added dropwise to a stirred solution of 2-bromo-4-methylpyridine (B1) (1 g, 5.81 mmol) in 15 mL of THF. The yellow solution was stirred at -78°C for 30 minutes. Then, a solution of methyl 6-methylpyridinecarboxylate (1.19 mL, 8.72 mmol) in 7 mL of THF was added, and the reaction mixture was heated to approximately 21°C and stirred for 16 hours. The solvent was evaporated under reduced pressure, and the solid residue was prepared with diethyl ether, filtered, and washed with diethyl ether. The solid was then diluted with 20 mL of saturated NH4Cl solution, and the aqueous phase was extracted with EtOAc (2 × 150 mL). The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography using 10% EtOAc / hexane to give compound C2, which was a yellow solid (1.1 g, 3.79 mmol, 65.4%).

[0272] 1H NMR (500MHz, CDCl3): δ8.30(d,J=5.0Hz,1H),7.86(d,J=8Hz,1H),7.73(t,J=7.5Hz, 1H),7.51(s,1H),7.36(d,J=8Hz,1H),7.24(d,J=5Hz,1H),4.52(s,2H),2.64(s,3H)

[0273] LC-MS (ESI): m / z 291 [M] +

[0274] 6.3.2.2-Bromo-4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]pyridine (C3)

[0275] Under argon atmosphere, a C2 solution (300 mg, 1.03 mmol) in pure DMF (1 ml) was treated with glacial acetic acid (0.14 ml, 2.48 mmol) in DMF. DMA (0.2 ml, 1.55 mmol) was added dropwise, and the mixture was stirred at approximately 21 °C under argon atmosphere for 1 hour. Hydrazine monohydrate (0.37 ml, 7.75 mmol) was added dropwise, and the resulting mixture was heated at 50 °C for 3 hours and then at approximately 21 °C for 16 hours. The reaction mixture was poured into water (20 ml) and extracted with CH2Cl2 (3 × 20 ml). The organic layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure to give crude C3. Crude C3 was purified by silica gel column chromatography using 2% MeOH / DCM to give purified C3 as a yellow solid (172 mg, 0.54 mmol, 53%).

[0276] 1 H NMR (500MHz, CDCl3): δ11.40(brs,1H),8.37(d,J=5.0Hz,1H),7.74(s,1H),7.64(s,1H),7.58(t ,J=8.0Hz,1H),7.34(d,J=6.0Hz,1H),7.26(d,J=8.0Hz,1H),7.17(d,J=8.0Hz,1H),2.60(s,3H)

[0277] LC-MS (ESI): m / z 315 [M] +

[0278] 6.3.3.2-Bromo-4-(3-(6-methylpyridin-2-yl)-1-triphenylmethyl-1H-pyrazol-4-yl)pyridine (C4)

[0279] K₂CO₃ (53 mg, 0.38 mmol) and triphenylmethyl chloride (53 mg, 0.19 mmol) were added to a stirred solution of C₃ (40 mg, 0.12 mmol) in acetone (2 mL). The reaction mixture was then heated to reflux and stirred for 24 hours. The reaction mixture was filtered and the filtrate was concentrated, then partitioned between CH₂Cl₂ (5 mL) and water (5 mL). The organic phase was dried over Na₂SO₄ and concentrated. The crude solid was purified by silica gel column chromatography using 2% MeOH / CH₂Cl₂ to give compound C₄ as a pale yellow solid (30 mg, 0.05 mmol, 41%).

[0280] 1 H NMR (400MHz, CDCl3): δ8.22(d,J=4.8Hz,1H),7.73(s,1H),7.59(s,3H),7.39- 7.35(m,9H),7.31(s,1H),7.28-7.25(m,6H),7.24(d,J=12Hz,1H),2.53(s,3H)

[0281] LC-MS (ESI): m / z 558 [M] +

[0282] 6.3.4. Methyl(2-(4-(4-(3-(6-methylpyridin-2-yl)-1-triphenylmethyl-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)tert-butyl carbamate (C5)

[0283] Under an argon atmosphere, a solution of Int-B (152 mg, 0.40 mmol) in EtOH (1 ml) was added to a stirred solution of C4 (150 mg, 0.26 mmol) in toluene (5 ml), followed by the addition of 2 M Na₂CO₃ solution (0.7 ml). The reaction mixture was degassed with argon for 20 min, then Pd(PPh₃)₄ (25 mg, 0.02 mmol) was added and refluxed for 6 h. After the starting material was completely consumed (monitored by TLC), the reaction mixture was poured into water and extracted with toluene (3 × 10 ml). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give crude C₅, which was purified by silica gel column chromatography using 30% EtOAc / hexane to give purified C₅ as a brown solid (51 mg, 0.07 mmol, 26%).

[0284] 1H NMR (400MHz, CDCl3): δ8.48 (d, J = 5.2Hz, 1H), 7.82 (d, J = 8.8Hz, 3H), 7.74 (s, 1H), 7.60 (s, 1H), 7.56 (d, J = 15.2Hz, J = 7.6Hz, 2H), 7.35-7.33 (m, 8 H),7.28-7.27(m,6H),7.08(d,J=6.8Hz,2H),6.93(d,J=8.8Hz,2H),4.1 6-4.08(m,2H),3.63-3.58(m,2H),2.98(s,3H),2.41(s,3H),1.46(s,9H)

[0285] 6.3.5. N-Methyl-2-(4-{4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}phenoxy)ethane-1-amine (Compound C)

[0286] 4N HCl in 1,4-dioxane (0.3 ml) was added to a stirred solution of C5 (51 mg, 0.07 mmol) in CH2Cl2 (5 ml) at 0 °C. The reaction mixture was then stirred under an argon atmosphere for 1 hour. After the starting material was completely consumed (as monitored by TLC), the solvent was evaporated under reduced pressure to obtain crude compound C. Crude compound C was then ground together with n-pentane (2 x 1 ml) and dried to give compound C, which is an HCl salt, as a brown solid (20 mg, 0.05 mmol, 74%).

[0287] 1 H NMR (400MHz, DMSO-d6): δ8.93(brs,2H),8.61(d,J=5.6Hz,1H),8.56(brs,1H ),8.33(brs,1H),8.03(d,J=8.8Hz,2H),7.88(t,J=7.6Hz,1H),7.78-7.74(m, 1H),7.65(d,J=7.2Hz,1H),7.38(d,J=7.6Hz,1H),7.20(d,J=8.4Hz,2H),4.36 (t,J=5.2Hz,2H),3.36(t,J=5.2Hz,2H),2.66-2.63(m,3H),2.50-2.46(m,3H)

[0288] LC-MS (ESI): m / z 386 [M] +

[0289] 6.4. Example 4: Synthesis of (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide ((Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide)(Compound D)

[0290] Compound D is prepared according to the general method in Scheme 4 below:

[0291]

[0292] 6.4.1.1-Acetyl-2-oxoindoline-6-carboxylic acid methyl ester (D2)

[0293] A stirred solution of methyl 2-oxoindoline-6-carboxylic acid (D1) (2.0 g, 10.47 mmol) in acetic anhydride (16 mL) was heated to 130 °C for 6 h under an inert atmosphere. After the starting material was completely consumed (as monitored by TLC), the reaction mixture was cooled to approximately 21 °C. The precipitate was filtered, washed with n-hexane (2 x 50 mL), and dried under vacuum to give compound D2 as a yellow solid (1.5 g, 61.5%).

[0294] 1 H NMR (400MHz, DMSO-d6): δ8.66(s,1H),7.82(d,J=8.0Hz,1H),7.48(d,J=8.0Hz,1H),3.91(s,2H),3.87(s,3H),2.57(s,3H)

[0295] 6.4.2. (Z)-1-acetyl-3-(hydroxy(phenyl)methylene)-2-oxoindoline-6-carboxylic acid methyl ester (D3)

[0296] TBTU (2.69 g, 8.36 mmol), benzoic acid (903 mg, 7.40 mmol), and triethylamine (2.2 ml) were added to a stirred solution of compound D2 (1.5 g, 6.43 mmol) in DMF (10 ml) at 0 °C under an inert atmosphere. The reaction mixture was heated to approximately 21 °C and stirred for 16 hours. After the starting material was completely consumed (as monitored by TLC), the reaction mixture was quenched with ice-cold water (30 ml) and extracted with EtOAc (2 × 40 ml). The combined organic extracts were dried over Na2SO4, filtered, and concentrated under vacuum to give crude product D3, which was purified by silica gel column chromatography using 80% EtOAc / hexane to give compound D3 (900 mg, 42%) as a yellow solid.

[0297] 1 H NMR (400MHz, CDCl3): δ14.01(brs,1H),8.93(s,1H),7.76-7.70(m,3H),7.67-7 .63(m,1H),7.59-7.56(m,2H),7.12(d,J=8.0Hz,1H),3.90(s,3H),2.83(s,3H)

[0298] LC-MS (ESI): m / z 338.3 [M+H] +

[0299] 6.4.3. (Z)-3-(hydroxy(phenyl)methylene)-2-oxoindoline-6-carboxylic acid (D4)

[0300] A 1N aqueous NaOH solution (15 mL) was added to a stirred solution of compound D3 (900 mg, 2.67 mmol) in MeOH (15 mL) at approximately 21 °C. The mixture was heated to 100 °C and stirred for 6 hours. After the starting material was completely consumed (as monitored by TLC), the reaction mixture was cooled to approximately 21 °C, quenched with a 1N aqueous HCl solution (13 mL), and stirred for 30 minutes. The precipitated solid was filtered and washed with 20% EtOAc / hexane to give compound D4 (580 mg, 77%), which was a grayish-white solid and could be used for the next step without further purification.

[0301] 1 H NMR (400MHz, DMSO-d6): δ12.76(brs,1H),11.61(brs,1H),7.77-7.50(m,8H),7.13(brs,1H)

[0302] 6.4.4. (Z)-N-ethyl-3-(hydroxy(phenyl)methylene)-2-oxoindoline-6-carboxamidelate (Fragment A)

[0303] At approximately 21 °C under an inert atmosphere, TBTU (729 mg, 2.27 mmol), HOBt (306 mg, 2.27 mmol), and N,N-diisopropylethylamine (1.9 mL, 10.32 mmol) were added to a stirred solution of compound D4 (580 mg, 2.06 mmol) in DMF (10 mL). After 30 minutes, 2N ethylamine in THF (2.1 mL, 4.12 mmol) was added at 0 °C and the mixture was stirred for 1 hour. The reaction mixture was then heated to approximately 21 °C and stirred for another 16 hours. After the starting material was completely consumed (as monitored by TLC), the volatiles were removed under vacuum. The residue was diluted with water (15 mL), filtered, and washed with 20% EtOAc / hexane (2 x 10 mL) to obtain a crude product, which was purified by silica gel column chromatography using 10% MeOH / CH2Cl2 to give fragment A (410 mg, 64.5%) as a grayish-white solid.

[0304] 1 H NMR (400MHz, DMSO-d6): δ13.62(brs,1H),11.39(brs,1H),8.35-8.33(m,1H), 7.76-7.52(m,5H),7.44-7.36(m,3H),3.29-3.22(m,2H),1.10(t,J=7.2Hz,3H)

[0305] LC-MS (ESI): m / z 307.1 (MH) + )

[0306] 6.4.5. N-(2-(dimethylamino)ethyl)-N-(4-nitrophenyl)methanesulfonamide (D8)

[0307] Potassium carbonate (1.32 g, 9.62 mmol), sodium iodide (110 mg, 0.74 mmol), and compound B6 (799 mg, 5.55 mmol) were added to a stirred solution of compound D7 (800 mg, 3.70 mmol) in acetone (15 mL) at 0 °C under an inert atmosphere. The reaction mixture was heated to 50 °C and stirred for 20 hours. After the starting material was completely consumed (as monitored by TLC), volatiles were removed under vacuum. The residue was diluted with water (20 mL) and extracted with EtOAc (2 × 40 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated under vacuum to give a crude product, which was purified by silica gel column chromatography using 5% MeOH / CH₂Cl₂ to give compound D8 (460 mg, 43%) as a pale yellow solid.

[0308] 1 H NMR (500MHz, DMSO-d6): δ8.27(d,J=9.5Hz,2H),7.68(d,J=9.5Hz,2H),3.85(t,J=6.5Hz,2H),3.13(s,3H),2.31(t,J=6.5Hz,2H),2.12(s,6H)

[0309] LC-MS (ESI): m / z 288.3 (MH) + )

[0310] 6.4.6. N-(4-aminophenyl)-N-(2-(dimethylamino)ethyl)methanesulfonamide (fragment B)

[0311] 10% Pd / C (40 mg) was added to a stirred solution of compound D8 (460 mg, 1.60 mmol) in MeOH (10 mL) and stirred for 3 hours at approximately 21 °C under a hydrogen atmosphere (balloon pressure). After the starting material was completely consumed (as monitored by TLC), the reaction mixture was... The sample was filtered and washed with MeOH (10 ml). The filtrate was concentrated under vacuum to obtain a crude product, which was purified by silica gel column chromatography using 10% MeOH / / CH2Cl2 to obtain fragment B (300 mg 73%), which was a pale yellow solid.

[0312] 1 H NMR (400MHz, DMSO-d6): δ6.99(d,J=8.8Hz,2H),6.54(d,J=8.8Hz,2H),5.25( s,2H),3.55(t,J=7.2Hz,2H),2.91(s,3H),2.24(t,J=7.2Hz,2H),2.12(s,6H)

[0313] LC-MS (ESI): m / z 258.2 (MH) + )

[0314] 6.4.7. (Z)-3-(((4-(N-(2-(dimethylamino)ethyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-N-ethyl-2-oxoindoline-6-carboxamide (D5)

[0315] A solution of fragment A (200 mg, 0.64 mmol), fragment B (500 mg, 1.94 mmol), and TMS-imidazole (455 mg, 3.24 mmol) in THF (5 mL) was heated to 170 °C for 1 hour in a microwave oven. After the starting material was consumed (monitored by TLC and LC-MS), volatiles were removed under vacuum. The residue was diluted with water (10 mL) and extracted with EtOAc (3 × 25 mL) to give a crude product, which was purified by preparative HPLC to give compound D5 (150 mg, 42%) as a pale yellow solid.

[0316] 1 H NMR (400MHz, DMSO-d6): δ12.14(s,1H),10.91(s,1H),8.17(t,J=5.6Hz,1H),7.64- 7.57(m,3H),7.53-7.51(m,2H),7.34(s,1H),7.17(d,J=8.8Hz,2H),7.06(d,J=8.4 Hz,1H),6.84(d,J=8.8Hz,2H),5.73(d,J=8.4Hz,1H),3.58(t,J=6.8Hz,2H),3.23- 3.20(m,2H),2.93(s,3H),2.13(t,J=6.8Hz,2H),1.90(s,6H),1.06(t,J=7.2Hz,3H)

[0317] LC-MS (ESI): m / z 548.6 (MH) + )

[0318] 6.4.8. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (Compound D)

[0319] At approximately 21 °C under an inert atmosphere, 2,2,2-trichloroethoxycarbonyl chloride (0.04 mL, 0.19 mmol) was added to a stirred solution of compound D5 (70 mg, 0.12 mmol) in pure toluene (3 mL). The reaction mixture was heated to reflux temperature (120 °C) and maintained for 16 hours. After the starting material was consumed (monitored by TLC), the reaction mixture was cooled to approximately 21 °C, diluted with EtOAc (30 mL), and washed with 1 N aqueous HCl solution (15 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under vacuum to give a monodemethylated, di-troc-protected compound (40 mg).

[0320] The crude product from the above reaction was dissolved in acetic acid (3 ml) and zinc powder (9 mg, 0.13 mmol) was added under an inert atmosphere at approximately 21 °C. The reaction mixture was heated to 50 °C and stirred for 8 hours. After the starting material was completely consumed (as monitored by TLC), the reaction mixture was cooled to approximately 21 °C and volatiles were removed under vacuum. The residue was diluted with water (20 ml) and extracted with EtOAc (2 × 25 ml). The combined organic extracts were washed with a saturated NaHCO3 solution (20 ml), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude compound D, which was purified by silica gel column chromatography using 5–6% MeOH / CH2Cl2 to give 12 mg of compound D with an HPLC purity of 83%.

[0321] The reaction was repeated at a scale of 60 mg. The crude product obtained was combined with the above batch and purified by preparative HPLC to obtain compound D (8.0 mg, 6.3%), which was a pale yellow solid.

[0322] 1 H NMR (400MHz, CD3OD): δ7.65-7.59(m,3H),7.52.7.50(m,2H),7.40(s,1H),7.31(d,J=8.8Hz,2H),7.07(d,J=8.4Hz,1H),6.90(d,J=8.8Hz,2 H),5.95(d,J=8.4Hz,1H),3.95(t,J=5.6Hz,2H),3.39-3.32(m,2H),3.05(t,J=5.6Hz,2H),2.93(s,3H),2.71(s,3H),1.19(t,J=7.2Hz,3H)

[0323] LC-MS (ESI): m / z 534.6 (MH) + )

[0324] UPLC purity: 99.18%

[0325] 6.5. Example 5: Substitute synthesis of (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (compound D)

[0326] Compound D was also prepared according to the general method in Scheme 5 below:

[0327]

[0328] 6.5.1. N-(2-Bromoethyl)-N-(4-Nitrophenyl)methanesulfonamide (D9)

[0329] Sodium hydride (60% in mineral oil; 320 mg, 7.99 mmol) was added to a stirred solution of compound D7 (1.0 g, 4.65 mmol) in DMF (10 mL) at 0 °C under an inert atmosphere, and the mixture was stirred at approximately 21 °C for 30 min. 1,2-Dibromoethane (2.18 g, 11.60 mmol) was added to the mixture at approximately 21 °C. The mixture was heated to 90 °C and stirred for 24 h. The reaction was monitored by TLC. The reaction mixture was cooled to approximately 21 °C, quenched with ice-cold water (30 mL), and extracted with EtOAc (2 x 40 mL). The combined organic extracts were dried over Na₂SO₄, filtered, and concentrated under vacuum to give a crude product, which was purified by silica gel column chromatography using 5% MeOH / CH₂Cl₂ to give 1.2 g of D9, a mixture containing 40% unreacted starting material. The resulting mixture was used directly in the next reaction without further purification.

[0330] 1 H NMR (500MHz, CDCl3): δ8.29(d,J=8.5Hz,2H),7.56(d,J=8.5Hz,2H),4.12(t,J=7.0Hz,2H),3.44(t,J=7.0Hz,2H),3.01(s,3H)

[0331] 6.5.2. N-(2-(methylamino)ethyl)-N-(4-nitrophenyl)methanesulfonamide (D10)

[0332] Triethylamine (1.6 ml) and methylamine (2 M in THF; 9.3 ml, 18.63 mmol) were added to a mixed solution of compound D9 (1.2 g, impure) in THF (10 ml) in a sealed tube under an inert atmosphere at approximately 21 °C. The reaction mixture was heated to 80 °C and maintained for 16 hours. After the starting material was completely consumed (as monitored by TLC), the reaction mixture was cooled to approximately 21 °C and concentrated under reduced pressure to obtain crude D10. Crude D10 was purified by silica gel column chromatography using 15% MeOH / CH2Cl2 to give compound D10 as a yellow solid (500 mg, overall yield of 39% in both steps).

[0333] 1 H NMR(500MHz,DMSO-d6)δ8.94(brs,1H),8.31(d,J=9.0Hz,2H),7.80(d,J=8.5H z,2H),4.06(t,J=6.0Hz,2H),3.15(s,3H),3.00(t,J=6.0Hz,2H),2.55(s,3H)

[0334] 6.5.3. Methyl (2-(N-(4-nitrophenyl)methylsulfonamido)ethyl)tert-butyl carbamate (D11)

[0335] At approximately 21°C under an inert atmosphere, triethylamine (0.4 mL, 2.61 mmol) and Boc-anhydride (659 mg, 3.02 mmol) were added to a stirred solution of D10 (500 mg, 1.83 mmol) in 10 mL of CH2Cl2, and the mixture was kept in this state for 5 hours. After the starting material was completely consumed (as monitored by TLC), the volatiles were removed under vacuum to obtain the crude product, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 to give D11, a colorless, viscous syrup (320 mg, 47%).

[0336] 1 H NMR (400MHz, DMSO-d6): δ8.27(d,J=8.4Hz,2H),7.68(d,J=8.4Hz,2H),3.91(t,J= 6.4Hz,2H),3.28-3.25(m,2H),3.07(s,3H),2.72-2.70(m,3H),1.33-1.27(m,9H)

[0337] LC-MS (ESI): m / z 274.2 (M + -B℃)

[0338] 6.5.4. (2-(N-(4-aminophenyl)methylsulfonamido)ethyl)(methyl)carbamate tert-butyl ester (Boc- variant of fragment B)

[0339] To a solution of compound D11 (250 mg, 0.67 mmol) in EtOH (10 mL), Raney-Ni (40 mg) was added and the mixture was stirred for 1 hour at approximately 21 °C under a hydrogen atmosphere (balloon pressure). After the starting material was completely consumed (as monitored by TLC), the reaction mixture was passed through... The mixture was filtered through a filter and washed with EtOH (10 ml). The combined filtrates were concentrated under vacuum to obtain a crude product, which was purified by silica gel column chromatography using 10% MeOH / CH2Cl2 to give the Boc- variant of fragment B as a pale yellow solid (180 mg, 77%).

[0340] 1 H NMR (400MHz, DMSO-d6): δ7.01(d,J=8.4Hz,2H),6.53(d,J=8.4HZ,2H),5.24(s,2H),3.60( t,J=6.4Hz,2H),3.18(t,J=6.4HZ,2H),2.88(s,3H),2.75-2.71(m,3H),1.36-1.33(m,9H)

[0341] LC-MS (ESI): m / z 244.2 (M + -B℃)

[0342] 6.5.5.(Z)-(2-(N-(4-(((6-(ethylcarbamoyl)-2-oxoindoline-3-ylidene)(phenyl)methyl)amino)phenyl)methylsulfonamide)ethyl)(methyl)carbamate tert-butyl

[0343] (Z)-(2-(N-(4-(((6-(ethylcarbamoyl)-2-oxoindolin-3-ylidene)(phenyl)methyl)amino)phenyl)methylsulfonamido)ethyl)(methyl)carbamate)(D10)

[0344] A solution of fragment A (70 mg, 0.22 mmol), the Boc-variant of fragment B (155 mg, 0.45 mmol), and TMS-imidazole (159 mg, 1.13 mmol) in THF (3 mL) was heated to 170 °C for 160 min in a microwave oven. After the starting material was consumed (monitored by TLC and LC-MS), the volatiles were removed under vacuum to obtain the residue, which was purified by preparative HPLC to give compound D10 (50 mg, 36%) as a pale yellow solid.

[0345] 1 H NMR (400MHz, CDCl3): δ12.13(brs,1H),8.01(brs,1H),7.61-7.51(m,3H),7.44-7.41(m,3H),7.13-7.11(m,2H),6.98(d,J=8.4HZ,1H),6.75(d, J=8.4HZ,2H),5.96-5.91(m,2H),3.74-3.71(m,2H),3.49-3.41(m,2H), 3.30-3.27(m,2H),2.80(s,6H),1.40-1.36(m,9H),1.19(t,J=7.2HZ,3H)

[0346] LC-MS (ESI): m / z 634.6 [M+H] +

[0347] 6.5.6. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide hydrochloride (compound D as HCl salt)

[0348] At 0 °C under an inert atmosphere, 4N HCl in 0.3 mL of 1,4-dioxane was added to a stirred solution of compound D10 (20 mg, 0.03 mmol) in diethyl ether (3 mL). The reaction mixture was stirred at approximately 21 °C for 1 hour. After the starting material was completely consumed (as monitored by TLC), the volatiles were removed under vacuum to obtain a crude product, which was then ground with n-pentane (2 x 4 mL) to give compound D (12 mg, 71%) in the form of an HCl salt, which was a pale yellow solid.

[0349] 1H NMR (400MHz, CD3OD): δ7.65-7.59(m,3H),7.52.7.50(m,2H),7.40(s,1H),7.31(d,J=8.8Hz,2H),7.07(d,J=8.4Hz,1H),6.90(d,J=8.8Hz,2 H), 5.95 (d, J = 8.4Hz, 1H), 3.95 (t, J = 5.6Hz, 2H), 3.39-3.32 (m, 2H), 3.05 (t, J = 5.6Hz, 2H), 2.93 (s, 3H), 2.71 (s, 3H), 1.19 (t, J = 7.2Hz, 3H).

[0350] LC-MS (ESI): m / z 534.7 [M+H] +

[0351] UPLC purity: 96.26%

[0352] 6.6. Example 6: In vitro determination of the activity of compound AD

[0353] Compound AD was tested to determine whether it could inhibit TGF-β-induced luciferase activity in HEK293T cells in vitro.

[0354] 30,000 HEK293T cells were seeded overnight in 96-well white flat-bottom plates. The next day, 100 ng of SMAD luciferase reporter plasmid per well was transfected into the cells using lipofectamine for 24 hours. The cells were then treated with compound AD and 100 pM TGF-β for 24 hours the following day. Dual-Globe technology was used to transfect the cells. Luciferase activity was measured using a luciferase assay kit (Promega). Compounds A, B, and D were measured twice, and compound C was measured three times. The results are shown in Table 4.

[0355]

[0356] The activity data from Experiment 1 are shown in Figure 1.

[0357] Compound AC exhibited the greatest inhibitory activity.

[0358] 6.7. Example 7: Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2H-pyrrolo-1(5H)-yl)hexaamido)-3-methylbutamido)-5-ureidopentamido)benzylmethyl(2-(4-(4-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)carbamate

[0359] Compound C is connected to the valine-citrulline linker according to the general method in scheme 6 below:

[0360]

[0361] L1 (122 mg, 0.165 mmol, 1.1 equivalents) and TEA (52 μl, 0.375 mmol, 2.5 equivalents) were added to a solution of compound C (58 mg, 0.150 mmol, 1.0 equivalents) in DMF (2 mL) at 0 °C. The reaction mixture was stirred at approximately 21 °C for 2 hours to give crude ADC-1. Crude ADC-1 was purified by preparative HPLC to give purified ADC-1 as a white solid (34 mg, 24% yield).

[0362] 6.8. Example 8: Generation of Antibody-Drug Conjugate 1 (ADC1)

[0363] Anti-human FAP antibody was dialyzed overnight in conjugation buffer (25 mM sodium borate / 25 mM NaCl, and 0.3 mM EDTA, final pH 7.4). The antibody was reduced for 2 hours using tris(2-carboxyethyl)phosphine (TCEP) at a reduction ratio of 10–30. ADC-1 was dissolved in DMSO to a final concentration of 10 mM and then conjugated with the antibody at a conjugation ratio of 5–30 in the presence of 15% DMSO. All reactions were performed at approximately 21°C. For some drug-antibody ratios (DARs), 50% propylene glycol was used as the organic solvent in the conjugation step. The final ADC was dialyzed overnight in PBS, filtered using a 0.22 μm filter, and analyzed by HPLC-HIC to determine the DAR and by HPLC-SEC to determine the aggregation level. For HPLC-HIC, the sample was analyzed in TSKgel. The butyl-NPR column was run at a flow rate of 0.5 mL / min. Phase A consisted of 25 mM sodium phosphate and 1.5 M ammonium sulfate at pH 6.95, while Phase B consisted of 75% 25 mM sodium phosphate and 25% isopropanol at pH 6.95. For HPLC-SEC analysis, the flow rate was 0.25 mL / min, and the solution was used at 280 nM. G3000SW column (Tosoh Bioscience), up to 25 minutes.

[0364] 6.9. Example 9: Synthesis of compound C connected with a disulfide junction (ADC-2)

[0365] Compound C is attached to the disulfide connector according to the general method in schemes 7A-B below:

[0366]

[0367]

[0368] 6.9.1. Synthesis of intermediate A

[0369] 2-Chlorotriphenylmethyl chloride resin (L2) (4 g, 4 mmol) was washed with DCM (2 x 40 ml), swollen in 50 ml of DCM for 10 minutes, and then drained. Fmoc-Cys(Trt)-OH (L3) (7.03 g, 12 mmol) was dissolved in 40 ml of DCM and added to the container containing the 2-chlorotriphenylmethyl chloride resin. 8.7 ml of DIPEA (6.8 ml, 40 mmol) was added to the container, and the mixture was vortexed for 2 hours at approximately 21°C. Then, 10 ml of methanol was added to the mixture, and the mixture was vortexed for 30 minutes. The resulting resin (L4) was then drained and washed five times with DMF. Resin L4 was then deprotected to provide resin L5 by adding approximately 40 ml of 20% piperidine in DMF, shaking the mixture, and then draining the liquid from the resin. Another 40 ml of 20% piperidine in DMF was added to the resin, and the mixture was shaken for 15 minutes. Then drain the liquid from resin L5 and wash with DMF (6×40ml).

[0370] Fmoc-amino acid solutions were prepared by combining Fmoc-Asp(OtBu)-OH (4.93 g, 12 mmol), Fmoc-Asp(OtBu)-OH (4.93 g, 12 mmol), Fmoc-Arg(Pbf)-OH (7.79 g, 12 mmol), Fmoc-Asp(OtBu)-OH (4.93 g, 12 mmol), and Fmoc-Glu-OtBu (5.1 g, 12 mmol) with HBTU / HOBT (4.55 g, 12 mmol / 1.62 g, 12 mmol) and DIPEA (2 ml, 12 mmol), respectively.

[0371] Fmoc-Asp(OtBu)-OH solution was added to resin L5 and shaken for 60 minutes to prepare resin L6. Resin L6 was washed with DMF (6 × 40 ml) and then deprotected with 20% piperidine in DMF as described above. Resins L7, L8, L9, and L10 were then prepared by sequential coupling using Fmoc-amino acid solution and the same procedure used to prepare resin L6 from resin L5.

[0372] In the exemplary synthesis, dry resin L10 (8 g) was added to a flask along with 80 mL of cleavage solution (TFA:TES:EDT:H2O = 90:5:3:2, v / v / v / v). The reaction was allowed to proceed for 1.5 h. The resin was then separated from the reaction mixture by pressure filtration. The resin was then washed twice with TFA. The filtrates were combined, and 10 volumes of cold MTBE were added dropwise. The precipitated peptide (intermediate A) was then centrifuged and washed four times with cold MTBE. Intermediate A was then dried under reduced pressure and purified by preparative HPLC to give 1.1 g of intermediate A as a white solid (yield: 37%). LC-MS (ESI) m / z: 752 [M+H] + .

[0373] 6.9.2. 2-(pyridin-2-yldithioalkyl)ethylmethyl(2-(4-(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)ethyl)carbamate (L12)

[0374] To a solution of compound C (40 mg, 0.1038 mmol) and ethyl 4-nitrophenyl 2-(pyridin-2-yldithioalkyl)carbonate in DMF (5 mL), DIPEA (0.5 mL) and HOBt (14 mg, 0.1038 mmol) were added. The mixture was stirred under N2 at approximately 21 °C for 16 hours to provide L12. Crude L12 was purified by preparative HPLC to give 35 mg of purified L12 as a white solid (yield 56%).

[0375] 6.9.3.(2R,5S,8S,11S,14S,19S)-19-amino-5,8,14-tris(carboxymethyl)-11-(3-guanidinopropyl)-2-(((2-(methyl(2-(4-(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)ethyl)carbamoyloxy)ethyl)dithioalkyl)methyl)-4,7,10,13,16-pentoxo-3,6,9,12,15-pentazaeicosano-1,20-diacid((2R,5S,8S,11S,14S,19S)-19-amino-5,8,14-tris(carboxymethyl) methyl)-11-(3-guanidinopropyl)-2-(((2-(methyl(2-(4-(4-(4-(6-methylpyridin-2-yl))-1H-pyrazol-3-yl)pyridin-2-yl)phenox y)ethyl)carbamoyloxy)ethyl)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaicosane-1,20-dioicacid)(L13)

[0376] Intermediate A (80 mg, 0.106 mmol) was added to L12 (35 mg, 0.058 mmol) in a THF / H2O (5 mL / 5 mL) solution under N2. The mixture was stirred at approximately 21 °C for 16 hours to provide L13. Crude L13 was purified by preparative HPLC to give 23 mg of purified L13 as a white solid (yield 31%).

[0377] 6.9.4.(2R,5S,8S,11S,14S,19S)-19-(2-(tert-butoxycarbonylaminooxy)acetamido)-5,8,14-tris(carboxymethyl)-11-(3-guanidinylpropyl)-2-(((2-(methyl(2-(4-(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin-2-1)phenoxy)ethyl)carbamoyloxy)ethyl)dithioalkyl)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentazaeicosane-1,20-diacid(L15)

[0378] To a solution of L13 (32 mg, 0.025 mmol) in DMF (3 mL), 2,5-dioxopyrrolidone-1-yl-2-(tert-butoxycarbonylaminooxy)acetate (L14) (28 mg, 0.097 mmol) was added, followed by TEA (0.5 mL). The reaction mixture was stirred at approximately 21 °C for 16 hours under a N2 atmosphere to provide L15. Crude L15 was purified by preparative HPLC to give 12 mg of purified L15 as a white solid (33% yield).

[0379] 6.9.5.(2R,5S,8S,11S,14S,19S)-19-(2-(aminooxy)acetamido)-5,8,14-tris(carboxymethyl)-11-(3-guanidinopropyl)-2-(((2-(methyl(2-(4-(4(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)ethyl)carbamoyloxy)ethyl)dithioalkyl)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentazaeicosano-1,20-diacid (ADC-2)

[0380] L15 (12 mg, 0.0085 mmol) was added to a mixture in DCM (5 ml) with TFA (1 ml). The mixture was stirred at approximately 21 °C for 30 min to provide ADC-2. The crude ADC-2 was concentrated and purified by preparative HPLC to give 3.5 mg of purified ADC-2 as a white solid (yield 31%).

[0381] 6.10. Example 10: Generation of Antibody-Drug Conjugate 2 (ADC2)

[0382] The ADC-2 is linked to the anti-human FAP antibody via antibody lysine residues according to the general method of scheme B below:

[0383]

[0384] Antibody was dialyzed into PBS at pH 7.4. S-4FB was added to the antibody in PBS at pH 7.4 at different molar ratios and incubated at approximately 21°C for 3 hours. The S-4FB-modified antibody solution was incubated with 0.5 mM 2-hydrazinopyridine solution (in 100 mM MES buffer, pH 5.0) at 37°C for 30 minutes at various conjugation ratios ranging from 5 to 50. The S4FB / Ab molar substitution ratio was determined by UV-Vis at A354. Zeba was used. TMThe modified antibody was purified using a rotary desalting column. The buffer was replaced with 50 mM phosphate buffer (pH 6.5, 150 mM NaCl) and then mixed with adapter-SS-drug ADC-2 (10 mM, in DMSO) at different molar ratios for 24 hours at 37 °C to provide ADC2. The next day, the ADC2 sample was dialyzed against PBS overnight. The sample was filtered and then analyzed by HPLC-SEC, SDS-PAGE, and LC-MS.

[0385] If HPLC-SEC detects more than 5% ADC2 aggregation, the aggregated components are separated by AKTA with an SEC column (GE Healthcare Life Sciences, Superdex 200 increase 10 / 300GL) and analyzed again by HPLC-SEC.

[0386] 6.11. Example 11: Synthesis and Characterization of Compound N

[0387] Compound N was synthesized according to the general method in Scheme 9 below:

[0388]

[0389] Compound N was compared with compound C in multiple in vitro assays. Their IC50 activities and their K+ levels in recombinant kinase assays were compared. i The values ​​are summarized in Table 5. Table 5 also shows the activity of compound C in inhibiting TGF-β signaling in human HEK cells. Compound C was found to be 10 times more effective than compound N in kinase assays.

[0390]

[0391] 6.12. Example 12: Anti-FAP antibody binds to HEK cells

[0392] The ability of an anti-FAP antibody (commercially obtained mouse IgG1, clone 427819) to bind to HEK293 cells expressing human FAP was assessed using FACS. The anti-FAP antibody bound to HEK293 cells transfected with human FAP cDNA and expressed on the cell surface. Figure 2C ), but does not bind to parental HEK cells that have not been transfected with human cDNA. Figure 2B ).

[0393] 6.13. Example 13: Production of targeted drug conjugates SYN-301 and SYN-302

[0394] Two targeted drug conjugates were synthesized. In the first, compound C was conjugated to an anti-FAP antibody (commercially available mouse IgG1, clone 427819) using an MC-Val-Cit-PABC cleavable linker. Figure 3A This targeted drug conjugate is referred to herein as SYN-301. In the second case, an incleivable (MC) linker is used to conjugate the compound with an anti-FAP antibody. Figure 3B This targeted drug conjugate is referred to as SYN-302 in this paper. A PLRP-S column was used (…). The drug-antibody ratio was determined by reversed-phase liquid chromatography (Particle size 5 μm, 1 x 50 mm). Aggregation percentage was determined using a g3000SWXL column by size exclusion chromatography. The drug-antibody ratio (DAR) for the SYN-301 formulation was 5.5% and 4% aggregation, while the DAR for the SYN-302 formulation was 5% and 3.9% aggregation.

[0395] 6.14. Evaluation of the inhibitory effect of SYN-301 and SYN-302 on TGF-β signaling in HEK cells expressing human FAP protein.

[0396] To evaluate the ability of SYN-301 and SYN-302 to inhibit TGF-β signaling, assays were performed using HEK293FT cells expressing human FAP.

[0397] HEK293FT cells were expressed using a construct encoding human FAP, a TGF-β responsive luciferase expression construct (containing three copies of the SMAD-binding element driving the expression of the luciferase reporter gene luc2P) (pGL4.48 [luc2P / SBE / Hygro]; Promega), and an expression control construct (encoding Renilla luciferase pGL4.74; Promega) (1 μg:1 μg:0.125 μg / ml cell culture) via Mirus. Transfected transiently with LT1 transfection reagent. Cells were seeded at 350,000 cells / ml in 96-well plates (100 μl per well). After 24 hours, cells were pretreated for 4 hours with SYN-301, SYN-302, unconjugated anti-FAP antibody, or unconjugated compound C. Then, 1 nM TGF-β was added and the cells were incubated for 3 hours. Luciferase expression was then measured using the Dual-Glo luciferase assay system (Promega).

[0398] The results are shown in Figures 4A-4B SYN-301 with a cleavable linker can inhibit TGF-β signaling in engineered HEK cells expressing FAP. Figure 4A), while no significant effect of SYN-301 on TGF-β signaling was observed in parental HEK cells that do not express FAP. Figure 4B In this test, it was observed that SYN-302, with its uncuttable joint, was less effective than SYN-301. Figure 4A ).

[0399] 6.15. Example 15: SYN-301 and SYN-302-induced FAP internalization

[0400] To evaluate the ability of SYN-301 and SYN-302 to internalize FAP on target cells that endogenously express FAP, an internalization assay was performed using WI-38 human lung fibroblasts.

[0401] WI-38 cells were incubated at 4°C for 30 min with anti-FAP antibody, SYN-301, SYN-302, or an isotype control ADC (a non-specific antibody control conjugated to the cleavable ValCit-ALK5 inhibitor compound C) to detect cell surface FAP expression. Cells were then washed twice with cold PBS to remove residual antibody / antibody conjugates from the supernatant, and incubated at 37°C for 3 h to induce receptor internalization. After 3 hours of incubation, cells were washed and incubated with PE-conjugated rat anti-mouse secondary antibody to detect remaining cell surface FAP expression. Relative FAP expression was compared with WI-38 cells incubated at 4°C with anti-FAP antibody or conjugates (as a measure of total FAP expression).

[0402] The results are shown in Figures 5A-5E and Figure 6 50-60% of WI-38 cells express FAP ( Figures 5A-5E Furthermore, the anti-FAP antibodies SYN-301 and SYN-302 were able to bind to and internalize FAP in WI-38 cells quite well (63%, 63%, and 52%, respectively). Figure 6 ).

[0403] 6.16. Example 16: Functional characterization of SYN-301 and SYN-302 in WI-38 cells

[0404] Increased expression of type IV collagen (COL4A1), fibronectin (FN1), and leucine-rich repeat 15 (LRRC15) is a marker of increased fibrosis. WI-38 human lung fibroblasts were used to assess the ability of SYN-301 and SYN-302 cells to reduce the expression of COL4A1, FN1, and LRRC15.

[0405] WI-38 human lung fibroblasts were seeded at 50,000 cells / ml in 24 wells (500 μl per well) and incubated overnight. Cells were starved with serum for 18 hours to reduce the effect of serum on TGFβ-regulated genes, and then pretreated with SYN-301, SYN-302, isotype control ADC, anti-FAP antibody, or compound C at 1 μg / ml. TGF-β was added and the cells were incubated for 19 hours. Cells were then scraped into RLT buffer (Qiagen) and RNA was extracted using the Qiagen RNaeasy kit. RNA was reverse transcribed into cDNA, and qPCR was performed using TaqMan primers for COL4A1, FN1, and LRRC15. GAPDH was used as a normalizing agent.

[0406] The results are shown in Figures 7A-7B SYN-301 partially blocks TGF-β-induced gene responses, reducing COL4A1 expression by approximately 25-30%. Figure 7A This reduces FN1 expression by approximately 20-25%. Figure 7A and reduced LRRC15 expression by approximately 15-20%. Figure 7B SYN-302 has a milder response in blocking TGF-β signaling, while unconjugated anti-FAP antibodies and isotype control ADCs do not inhibit TGF-β signaling.

[0407] 7. Specific Implementation Plan

[0408] This disclosure is illustrated by the following specific implementation plan.

[0409] 1. A targeted drug conjugate comprising an ALK5 inhibitor operatively linked to a targeting moiety which binds to a cell surface molecule expressed on the surface of myofibroblasts, activated fibroblasts, fibroblasts transforming into myofibroblasts, or combinations thereof.

[0410] 2. The targeted drug conjugate according to embodiment 1, wherein the targeting portion binds to molecules on the surface of myofibroblast cells.

[0411] 3. A targeted drug conjugate according to embodiment 1 or embodiment 2, wherein the targeted portion binds to activated fibroblast cell surface molecules.

[0412] 4. A targeted drug conjugate according to any one of embodiments 1 to 3, wherein the targeting portion binds to a cell surface molecule of a fibroblast transforming into a myofibroblast.

[0413] 5. A targeted drug conjugate according to any one of embodiments 1 to 4, wherein the ALK5 inhibitor has an IC50 of at least 20 nM. 50 .

[0414] 6. A targeted drug conjugate according to any one of embodiments 1 to 5, wherein the ALK5 inhibitor is an imidazole compound, a pyrazole compound, or a thiazole compound.

[0415] 7. The targeted drug conjugate according to embodiment 6, wherein the ALK5 inhibitor is an imidazole compound.

[0416] 8. The targeted drug conjugate according to embodiment 6, wherein the ALK5 inhibitor is a pyrazole compound.

[0417] 9. The targeted drug conjugate according to embodiment 6, wherein the ALK5 inhibitor is a thiazole compound.

[0418] 10. The targeted drug conjugate according to embodiment 6, wherein the ALK5 inhibitor is an imidazole compound, which is an imidazole-benzodioxol compound or an imidazole-quinoxaline compound.

[0419] 11. The targeted drug conjugate according to embodiment 10, wherein the ALK5 inhibitor is an imidazole-benzodioxane compound.

[0420] 12. The targeted drug conjugate according to embodiment 10, wherein the ALK5 inhibitor is an imidazole-quinoxaline compound.

[0421] 13. The targeted drug conjugate according to embodiment 6, wherein the ALK5 inhibitor is a pyrazole compound, which is a pyrazole-pyrrolo compound.

[0422] 14. The targeted drug conjugate according to embodiment 6, wherein the ALK5 inhibitor is an imidazole-benzodioxane compound, an imidazole-quinoxaline compound, a pyrazole-pyrrolo compound, or a thiazole compound.

[0423] 15. A targeted drug conjugate according to any one of embodiments 1 to 4, wherein the ALK5 inhibitor is compound C.

[0424] 16. A targeted drug conjugate according to any one of embodiments 1 to 4, wherein the ALK5 inhibitor is compound N.

[0425] 17. A targeted drug conjugate according to any one of embodiments 1 to 16, wherein the ALK5 inhibitor is connected to the targeted portion via a connector.

[0426] 18. The targeted drug conjugate according to embodiment 17, wherein the linker is a PEG-containing linker.

[0427] 19. A targeted drug conjugate according to embodiment 17 or embodiment 18, wherein the adapter is a multivalent adapter.

[0428] 20. A targeted drug conjugate according to any one of embodiments 17 to 19, wherein the connector is an uncuttable connector.

[0429] 21. The targeted drug conjugate according to embodiment 20, wherein the indestructible linker is an N-maleimidomethylcyclohexane 1-carboxylate, maleimidocaproyl, or mercaptoacetamidocaproyl linker.

[0430] 22. The targeted drug conjugate according to embodiment 21, wherein the incisor is an N-maleimide methylcyclohexane 1-carboxylic acid ester conjugate.

[0431] 23. The targeted drug conjugate according to embodiment 21, wherein the incisor is a maleimide hexanoyl conjugate.

[0432] 24. The targeted drug conjugate according to embodiment 21, wherein the indestructible linker is a mercaptoacetaminohexanoyl linker.

[0433] 25. A targeted drug conjugate according to any one of embodiments 17 to 19, wherein the connector is a cuttable connector.

[0434] 26. The targeted drug conjugate according to embodiment 25, wherein the cleavable linker is a peptide linker.

[0435] 27. The targeted drug conjugate according to embodiment 25, wherein the cleavable linker is a dipeptide linker, a disulfide linker, or a hydrazone linker.

[0436] 28. The targeted drug conjugate according to embodiment 27, wherein the cleavable linker is a dipeptide linker.

[0437] 29. The targeted drug conjugate according to embodiment 26, wherein the cleavable linker is a tripeptide linker.

[0438] 30. The targeted drug conjugate according to embodiment 26, wherein the cleavable linker is a tetrapeptide linker.

[0439] 31. The targeted drug conjugate according to embodiment 30, wherein the peptide linker is a gly-gly-phenylalanine-gly (gly-gly-phe-gly) linker.

[0440] 32. The targeted drug conjugate according to embodiment 27, wherein the cuttable connector is a disulfide connector.

[0441] 33. The targeted drug conjugate according to embodiment 27, wherein the cuttable connector is a hydrazone connector.

[0442] 34. The targeted drug conjugate according to embodiment 27, wherein the linker is a protease-sensitive valine-citrulline dipeptide linker.

[0443] 35. The targeted drug conjugate according to embodiment 27, wherein the linker is a protease-sensitive phenylalanine-lysine dipeptide linker.

[0444] 36. The targeted drug conjugate according to embodiment 27, wherein the linker is a glutathione-sensitive disulfide linker.

[0445] 37. The targeted drug conjugate according to embodiment 27, wherein the connector is an acid-sensitive disulfide connector.

[0446] 38. A targeted drug conjugate according to any one of embodiments 1 to 37, wherein the ALK5 inhibitor is conjugated to the target moiety via site-specific conjugation.

[0447] 39. A targeted drug conjugate according to embodiment 38, wherein the ALK5 inhibitor is conjugated via one or more cysteine, lysine, or glutamine residues on the targeting moiety.

[0448] 40. A targeted drug conjugate according to embodiment 39, wherein the ALK5 inhibitor is conjugated via one or more cysteine ​​residues on the targeting moiety.

[0449] 41. A targeted drug conjugate according to embodiment 39, wherein the ALK5 inhibitor is conjugated via one or more lysine residues on the targeting moiety.

[0450] 42. A targeted drug conjugate according to embodiment 39, wherein the ALK5 inhibitor is conjugated via one or more glutamine residues on the targeting moiety.

[0451] 43. A targeted drug conjugate according to embodiment 38, wherein the ALK5 inhibitor is conjugated via one or more non-natural amino acid residues on the targeting moiety.

[0452] 44. The targeted drug conjugate according to embodiment 43, wherein one or more non-natural amino acid residues comprise p-acetylphenylalanine (pAcF).

[0453] 45. The targeted drug conjugate according to embodiment 43, wherein one or more non-natural amino acid residues comprise p-azidomethyl-L-phenylalanine (pAMF).

[0454] 46. ​​The targeted drug conjugate according to embodiment 43, wherein one or more non-natural amino acid residues comprise selenocysteine ​​(Sec).

[0455] 47. A targeted drug conjugate according to embodiment 38, wherein the ALK5 inhibitor is conjugated via one or more glycans on the targeting moiety.

[0456] 48. The targeted drug conjugate according to embodiment 47, wherein one or more polysaccharides comprise fucose.

[0457] 49. The targeted drug conjugate according to embodiment 47, wherein one or more polysaccharides comprise 6-thiofucose.

[0458] 50. The targeted drug conjugate according to embodiment 47, wherein the one or more polysaccharides comprise galactose.

[0459] 51. The targeted drug conjugate according to embodiment 47, wherein one or more polysaccharides comprise N-acetylgalactosamine (GalNAc).

[0460] 52. The targeted drug conjugate according to embodiment 47, wherein the one or more polysaccharides comprise N-acetylglucosamine (GlcNAc).

[0461] 53. The targeted drug conjugate according to embodiment 47, wherein the one or more polysaccharides comprise sialic acid (SA).

[0462] 54. A targeted drug conjugate according to any one of embodiments 38 to 53, wherein the ALK5 inhibitor is conjugated via a linker.

[0463] 55. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 1 to 30.

[0464] 56. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 1 to 20.

[0465] 57. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 1 to 15.

[0466] 58. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 2 to 12.

[0467] 59. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 4 to 15.

[0468] 60. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 6 to 12.

[0469] 61. A targeted drug conjugate according to any one of embodiments 1 to 54, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 2 to 8.

[0470] 62. The targeted drug conjugate according to embodiments 1 to 61, wherein the targeted portion is internalized.

[0471] 63. A targeted drug conjugate according to any one of embodiments 1 to 62, wherein the targeting portion comprises an antibody or an antibody fragment.

[0472] 64. The targeted drug conjugate according to embodiment 63, wherein the targeted portion comprises an antibody.

[0473] 65. A targeted drug conjugate according to embodiment 64, wherein the antibody is a monoclonal antibody.

[0474] 66. A targeted drug conjugate according to embodiment 65, wherein the antibody is human or humanized.

[0475] 67. The targeted drug conjugate according to embodiment 66, wherein the antibody is human.

[0476] 68. A targeted drug conjugate according to embodiment 66, wherein the antibody is humanized.

[0477] 69. A targeted drug conjugate according to embodiment 63, wherein the targeting portion comprises an antibody fragment.

[0478] 70. The targeted drug conjugate according to embodiment 69, wherein the antibody fragment is a fragment of a monoclonal antibody.

[0479] 71. A targeted drug conjugate according to embodiment 70, wherein the antibody fragment is a fragment of a human or humanized antibody.

[0480] 72. The targeted drug conjugate according to embodiment 71, wherein the antibody fragment is a fragment of a human antibody.

[0481] 73. The targeted drug conjugate according to embodiment 71, wherein the antibody fragment is a fragment of a humanized antibody.

[0482] 74. A targeted drug conjugate according to any one of embodiments 69 to 73, wherein the antibody fragment is Fab, Fab', F(ab')2, Fv, scFv, dsFv or a single-domain antibody.

[0483] 75. A targeted drug conjugate according to embodiment 74, wherein the antibody fragment is Fab.

[0484] 76. A targeted drug conjugate according to embodiment 74, wherein the antibody fragment is Fab'.

[0485] 77. The targeted drug conjugate according to embodiment 74, wherein the antibody fragment is F(ab')2.

[0486] 78. A targeted drug conjugate according to embodiment 74, wherein the antibody fragment is Fv.

[0487] 79. A targeted drug conjugate according to embodiment 74, wherein the antibody fragment is scFv.

[0488] 80. The targeted drug conjugate according to embodiment 79, wherein the scFv comprises a polypeptide linker between the VH and VL domains of the scFv.

[0489] 81. A targeted drug conjugate according to embodiment 74, wherein the antibody fragment is dsFv.

[0490] 82. The targeted drug conjugate according to embodiment 74, wherein the antibody fragment is a single-domain antibody.

[0491] 83. The targeted drug conjugate according to embodiment 82, wherein the single-domain antibody is camel V. H H antibody fragments or humanized camel V H H antibody fragment.

[0492] 84. A targeted drug conjugate according to any one of embodiments 1 to 62, wherein the targeting portion is not based on immunoglobulins.

[0493] 85. A targeted drug conjugate according to any one of embodiments 1 to 84, wherein the cell surface molecule is a human cell surface molecule.

[0494] 86. A targeted drug conjugate according to any one of embodiments 1 to 85, wherein the cell surface molecule is FAP, PDGFR-β, FGFR1, PPAR-γ, FSP1, GFAP, fascin, CD147, CXCR4, αvβ6, AXL, or MERTK.

[0495] 87. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is FAP.

[0496] 88. A targeted drug conjugate according to embodiment 87, wherein the targeting portion preferentially binds to a membrane-bound FAP relative to a soluble FAP.

[0497] 89. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is PDGFR-β.

[0498] 90. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is FGFR1.

[0499] 91. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is PPAR-γ.

[0500] 92. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is FSP1.

[0501] 93. The targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is GFAP.

[0502] 94. The targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is myofascitis.

[0503] 95. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is CD147.

[0504] 96. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is CXCR4.

[0505] 97. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is αvβ6.

[0506] 98. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is AXL.

[0507] 99. A targeted drug conjugate according to embodiment 86, wherein the cell surface molecule is MERTK.

[0508] 100. A targeted drug conjugate according to any one of embodiments 1 to 85, wherein the cell surface molecule is LRRC15.

[0509] 101. A targeted drug conjugate according to any one of embodiments 1 to 100, which promotes apoptosis of myofibroblasts in contact with the targeted drug conjugate.

[0510] 102. A targeted drug conjugate according to any one of embodiments 1 to 100, which promotes the dedifferentiation of myofibroblasts in contact with the targeted drug conjugate.

[0511] 103. A targeted drug conjugate according to embodiment 102, wherein dedifferentiation is measured by a reduction in smooth muscle actin expression.

[0512] 104. A targeted drug conjugate according to any one of embodiments 1 to 103, comprising an Fc domain substituted with one or more amino acid-substituted amino acids having a reduced effector function.

[0513] 105. The targeted drug conjugate according to embodiment 104, wherein one or more substitutions comprise N297A, N297Q, N297G, D265A / N297A, D265A / N297G, L235E, L234A / L235A, L234A / L235A / P329A, L234D / L235E:L234R / L235R / E233K, L234D / L235E / D265S:E233K / L234R / L235R / D265S, L234D / L235E / E269K:E233K / L234R / L235R / E269K, L234D / L235E / K322A: E233K / L234R / L235R / K322A, L234D / L235E / P329W: E233K / L234R / L235R / P329W, L234D / L235E / E269K / D265S / K322A: E233K / L234R / L235R / E269K / D265S / K322A or L234D / L235E / E269K / D265S / K322E / E333K: E233K / L234R / L235R / E269K / D265S / K322E / E333K.

[0514] 106. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise N297A.

[0515] 107. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise N297Q.

[0516] 108. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise N297G.

[0517] 109. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise D265A / N297A.

[0518] 110. A targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise D265A / N297G.

[0519] 111. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L235E.

[0520] 112. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234A / L235A.

[0521] 113. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234A / L235A / P329A.

[0522] 114. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E:L234R / L235R / E233K.

[0523] 115. A targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E / D265S:E233K / L234R / L235R / D265S.

[0524] 116. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E / E269K:E233K / L234R / L235R / E269K.

[0525] 117. The targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E / K322A:E233K / L234R / L235R / K322A.

[0526] 118. A targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E / P329W:E233K / L234R / L235R / P329W.

[0527] 119. A targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E / E269K / D265S / K322A:E233K / L234R / L235R / E269K / D265S / K322A.

[0528] 120. A targeted drug conjugate according to embodiment 105, wherein one or more substitutions comprise L234D / L235E / E269K / D265S / K322E / E333K:E233K / L234R / L235R / E269K / D265S / K322E / E333K.

[0529] 121. A pharmaceutical composition comprising a targeted pharmaceutical conjugate according to any one of embodiments 1 to 120 and a pharmaceutically acceptable carrier.

[0530] 122. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 1 and 30.

[0531] 123. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:targeting portion ratio between 1 and 20.

[0532] 124. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 1 and 15.

[0533] 125. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:targeting portion ratio between 2 and 12.

[0534] 126. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 4 and 15.

[0535] 127. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 6 and 12.

[0536] 128. A pharmaceutical composition according to embodiment 121, wherein at least 30% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 2 and 8.

[0537] 129. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 1 and 30.

[0538] 130. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 1 and 20.

[0539] 131. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 1 and 15.

[0540] 132. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:targeting portion ratio between 2 and 12.

[0541] 133. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 4 and 15.

[0542] 134. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 6 and 12.

[0543] 135. A pharmaceutical composition according to embodiment 121, wherein at least 40% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 2 and 8.

[0544] 136. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 1 and 30.

[0545] 137. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 1 and 20.

[0546] 138. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 1 and 15.

[0547] 139. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 2 and 12.

[0548] 140. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:target portion ratio between 4 and 15.

[0549] 141. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 6 and 12.

[0550] 142. A pharmaceutical composition according to embodiment 121, wherein at least 50% of the targeted pharmaceutical conjugate molecule in the pharmaceutical composition has an ALK5 inhibitor:targeting portion ratio between 2 and 8.

[0551] 143. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 1 and 30.

[0552] 144. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 1 and 20.

[0553] 145. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 1 and 15.

[0554] 146. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:targeting portion ratio between 2 and 12.

[0555] 147. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 4 and 15.

[0556] 148. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:target portion ratio between 6 and 12.

[0557] 149. A pharmaceutical composition according to embodiment 121, wherein at least 60% of the targeted pharmaceutical conjugate molecules in the pharmaceutical composition have an ALK5 inhibitor:targeting portion ratio between 2 and 8.

[0558] 150. A method of treating fibrosis in a subject in need, comprising administering to the subject a targeted pharmaceutical conjugate according to any one of embodiments 1 to 120 or a pharmaceutical composition according to any one of embodiments 121 to 149.

[0559] 151. The method according to embodiment 150, wherein the fibrosis is pulmonary fibrosis.

[0560] 152. The method according to embodiment 151, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF).

[0561] 153. The method according to embodiment 150, wherein the fibrosis is liver fibrosis.

[0562] 154. The method according to embodiment 150, wherein the fibrosis is renal fibrosis.

[0563] 155. The method according to embodiment 150, wherein the fibrosis is cardiac fibrosis.

[0564] 156. The method according to embodiment 150, wherein the fibrosis is skin fibrosis.

[0565] 157. The method according to embodiment 150, wherein the fibrosis is esophageal fibrosis.

[0566] 158. The method according to embodiment 153, wherein the subject suffers from NASH, for example, has been diagnosed with NASH.

[0567] 159. The method according to any one of embodiments 150 to 157, wherein the subject suffers from systemic sclerosis, for example, has been diagnosed with systemic sclerosis.

[0568] 160. A method of treating a subject suffering from, for example, a person diagnosed with systemic sclerosis, comprising administering to the subject a targeted drug conjugate according to any one of embodiments 1 to 120 or a pharmaceutical composition according to any one of embodiments 121 to 149.

[0569] 161. A method of treating a subject with NASH, such as a subject who has been diagnosed with NASH, comprising administering to the subject a targeted drug conjugate according to any one of embodiments 1 to 120 or a pharmaceutical composition according to any one of embodiments 121 to 149.

[0570] 162. The method of claim 160 or embodiment 161, wherein the subject exhibits signs and / or symptoms of fibrosis.

[0571] 163. The method according to implementation scheme 160 or implementation scheme 161, wherein the subject does not exhibit signs and / or symptoms of fibrosis.

[0572] 164. The method according to any one of embodiments 150 to 163, wherein the targeted drug conjugate or pharmaceutical composition is administered as part of a combination therapy regimen comprising the administration of one or more second therapeutic agents, optionally wherein the one or more agents are not targeted drug conjugates (each a “second therapeutic agent”) according to any one of embodiments 1 to 120.

[0573] 165. The method according to embodiment 164, wherein the targeted drug conjugate or drug composition is administered in combination with a standard care therapy or treatment regimen.

[0574] 166. The method according to embodiment 164 or 165, wherein the combination therapy includes administering at least one second therapeutic agent to the subject.

[0575] 167. The method according to any one of embodiments 164 to 166, wherein the second therapeutic agent comprises pirfenidone, nintedanib, pentraxin-2, pamrevlumab, prednisone, cortisone, cyclophosphamide, or azathioprine.

[0576] 168. The method according to embodiment 167, wherein the second therapeutic agent comprises pirfenidone.

[0577] 169. The method according to implementation scheme 167 or implementation scheme 168, wherein the second therapeutic agent comprises nintedanib.

[0578] 170. The method according to any one of embodiments 167 to 169, wherein the second therapeutic agent comprises pentraxin-2.

[0579] 171. The method according to any one of embodiments 167 to 170, wherein the second therapeutic agent comprises pamrevlumab.

[0580] 172. The method according to any one of embodiments 167 to 171, wherein the second therapeutic agent comprises prednisone.

[0581] 173. The method according to any one of embodiments 167 to 172, wherein the second therapeutic agent comprises cortisone.

[0582] 174. The method according to any one of embodiments 167 to 173, wherein the second therapeutic agent comprises cyclophosphamide.

[0583] 175. The method according to any one of embodiments 167 to 174, wherein the second therapeutic agent comprises azathioprine.

[0584] 176. The method according to any one of embodiments 164 to 175, comprising treating the subject with the combination therapy.

[0585] 177. The method according to any one of embodiments 164 to 176, comprising administering the second therapeutic agent to the subject.

[0586] 178. A method of treating a subject with cancer, comprising administering to the subject in need a targeted drug conjugate according to any one of embodiments 1 to 120 or a pharmaceutical composition according to any one of embodiments 121 to 149.

[0587] 179. The method according to embodiment 178, wherein the cancer is urothelial carcinoma.

[0588] 180. The method according to embodiment 179, wherein the cancer is bladder cancer.

[0589] 181. The method according to embodiment 179, wherein the cancer is urethral cancer.

[0590] 182. The method according to embodiment 179, wherein the cancer is ureteral cancer.

[0591] 183. The method according to embodiment 178, wherein the cancer is lung cancer.

[0592] 184. The method according to embodiment 183, wherein the cancer is NSCLC.

[0593] 185. The method according to embodiment 184, wherein the NSCLC is adenocarcinoma.

[0594] 186. The method according to embodiment 184, wherein the NSCLC is squamous cell carcinoma.

[0595] 187. The method according to embodiment 184, wherein the NSCLC is large cell carcinoma.

[0596] 188. The method according to embodiment 183, wherein the cancer is small cell lung cancer.

[0597] 189. The method according to embodiment 178, wherein the cancer is breast cancer.

[0598] 190. The method according to embodiment 178, wherein the cancer is pancreatic cancer.

[0599] 191. The method according to embodiment 178, wherein the cancer is prostate cancer.

[0600] 192. The method according to embodiment 178, wherein the cancer is esophageal cancer.

[0601] 193. The method according to implementation scheme 178, wherein the cancer is colorectal cancer.

[0602] 194. The method according to implementation scheme 193, wherein the colorectal cancer is adenocarcinoma.

[0603] 195. The method according to implementation scheme 193, wherein the colorectal cancer is a carcinoid tumor.

[0604] 196. The method according to embodiment 193, wherein the colorectal cancer is a gastrointestinal stromal tumor.

[0605] 197. The method according to implementation scheme 193, wherein the colorectal cancer is colorectal lymphoma.

[0606] 198. The method according to embodiment 178, wherein the cancer is head and neck cancer.

[0607] 199. The method according to implementation scheme 178, wherein the cancer is ovarian cancer.

[0608] 200. The method according to embodiment 178, wherein the cancer is kidney cancer.

[0609] 201. The method according to embodiment 178, wherein the cancer is gastric adenocarcinoma.

[0610] 202. The method according to any one of embodiments 178 to 201, wherein the targeted drug conjugate or pharmaceutical composition is administered as part of a combination therapy regimen comprising administration of one or more second therapeutic agents, optionally wherein the one or more agents are not targeted drug conjugates (each a “second therapeutic agent”) according to any one of embodiments 1 to 120.

[0611] 203. The method according to embodiment 202, wherein the targeted drug conjugate or drug composition is administered in combination with a standard care therapy or treatment regimen.

[0612] 204. The method according to embodiment 202 or 203, wherein the combination therapy includes administering at least one second therapeutic agent to the subject.

[0613] 205. The method according to any one of embodiments 202 to 204, wherein the combination therapy comprises immunotherapy, optionally wherein the immunotherapy is a checkpoint inhibitor therapy, chimeric antigen receptor (CAR) therapy, adoptive T-cell therapy, oncolytic virus therapy, dendritic cell vaccine therapy, STING agonist therapy, TLR agonist therapy, intratumoral CpG therapy, or cytokine therapy.

[0614] 206. The method according to any one of embodiments 202 to 205, wherein the combination therapy comprises checkpoint inhibitor therapy.

[0615] 207. The method according to embodiment 206, wherein the checkpoint inhibitor therapy comprises T-cell checkpoint inhibitor therapy.

[0616] 208. The method according to embodiment 207, wherein the T-cell checkpoint inhibitor therapy comprises an antibody or an antigen-binding fragment thereof.

[0617] 209. The method according to any one of embodiments 206 to 208, wherein the checkpoint inhibitor therapy targets PD1, PDL1, CTLA4, TIGIT, LAG3, OX40, CD40 VISTA, or a combination thereof.

[0618] 210. The method according to embodiment 209, wherein the checkpoint inhibitor therapy targets PD1.

[0619] 211. The method according to implementation plan 210, wherein the second therapeutic agent is pembrolizumab.

[0620] 212. The method according to implementation plan 210, wherein the second therapeutic agent is nivolumab.

[0621] 213. The method according to implementation plan 210, wherein the second therapeutic agent is cimipril.

[0622] 214. The method according to implementation plan 210, wherein the second therapeutic agent is dotalipramab.

[0623] 215. The method according to any one of embodiments 209 to 214, wherein the checkpoint inhibitor therapy targets PDL1.

[0624] 216. The method according to implementation plan 215, wherein the second treatment is atezolizumab.

[0625] 217. The method according to implementation plan 215, wherein the second therapeutic agent is acitumab.

[0626] 218. The method according to implementation plan 215, wherein the second therapeutic agent is durvalumab.

[0627] 219. The method according to any one of embodiments 209 to 218, wherein the checkpoint inhibitor therapy targets CTLA4.

[0628] 220. The method according to implementation plan 219, wherein the second therapeutic agent is ipilimumab.

[0629] 221. The method according to any one of embodiments 209 to 220, wherein the checkpoint inhibitor therapy targets TIGIT.

[0630] 222. The method according to implementation scheme 221, wherein the second therapeutic agent is etigilimab.

[0631] 223. The method according to implementation plan 221, wherein the second therapeutic agent is tiragolumab.

[0632] 224. The method according to implementation plan 221, wherein the second therapeutic agent is AB154.

[0633] 225. The method according to any one of embodiments 209 to 224, wherein the checkpoint inhibitor therapy targets LAG3.

[0634] 226. The method according to implementation plan 225, wherein the second therapeutic agent is LAG525.

[0635] 227. The method according to implementation plan 225, wherein the second therapeutic agent is Sym022.

[0636] 228. The method according to implementation plan 225, wherein the second therapeutic agent is relatlimab.

[0637] 229. The method according to implementation plan 225, wherein the second therapeutic agent is TSR-033.

[0638] 230. The method according to any one of embodiments 209 to 229, wherein the checkpoint inhibitor therapy targets OX40.

[0639] 231. The method according to implementation plan 230, wherein the second therapeutic agent is MEDI6469.

[0640] 232. The method according to implementation plan 230, wherein the second therapeutic agent is PF-04518600.

[0641] 233. The method according to implementation plan 230, wherein the second therapeutic agent is BMS 986178.

[0642] 234. The method according to any one of embodiments 209 to 233, wherein the checkpoint inhibitor therapy targets CD40.

[0643] 235. The method according to implementation plan 234, wherein the second therapeutic agent is selicrelumab.

[0644] 236. The method according to implementation plan 234, wherein the second therapeutic agent is CP-870,893.

[0645] 237. The method according to implementation plan 234, wherein the second therapeutic agent is APX005M.

[0646] 238. The method according to any one of embodiments 209 to 237, wherein the checkpoint inhibitor therapy targets VISTA.

[0647] 239. The method according to implementation plan 238, wherein the second therapeutic agent is HMBD-002.

[0648] 240. The method according to any one of embodiments 202 to 239, wherein the second therapeutic agent is a chimeric antigen receptor (CAR).

[0649] 241. The method according to any one of embodiments 202 to 240, wherein the combination therapy comprises adoptive T-cell therapy.

[0650] 242. The method according to embodiment 241, wherein the adoptive T-cell therapy is autologous T-cell therapy.

[0651] 243. The method according to any one of embodiments 202 to 242, wherein the combination therapy comprises oncolytic virus therapy.

[0652] 244. The method according to any one of embodiments 202 to 243, wherein the combination therapy comprises dendritic cell vaccine therapy.

[0653] 245. The method according to any one of embodiments 202 to 244, wherein the combination therapy comprises STING agonist therapy.

[0654] 246. The method according to any one of embodiments 202 to 245, wherein the combination therapy comprises TLR agonist therapy.

[0655] 247. The method according to any one of embodiments 202 to 246, wherein the combination therapy comprises chemotherapy.

[0656] 248. The method according to implementation plan 247, wherein the second therapeutic agent is an antimetabolite, alkylating agent, an anthracycline, antimicrotubule agent, platinum compound, taxane, topoisomerase inhibitor, or vinca alkaloid.

[0657] 249. The method according to implementation plan 248, wherein the second therapeutic agent is an antimetabolite.

[0658] 250. The method according to embodiment 249, wherein the antimetabolite is 5-fluorouracil.

[0659] 251. The method according to embodiment 249, wherein the antimetabolite is gemcitabine.

[0660] 252. The method according to embodiment 249, wherein the antimetabolite is methotrexate.

[0661] 253. The method according to implementation scheme 248, wherein the second therapeutic agent is an alkylating agent.

[0662] 254. The method according to embodiment 253, wherein the alkylating agent is cyclophosphamide.

[0663] 255. The method according to embodiment 253, wherein the alkylating agent is dacarbazine.

[0664] 256. The method according to embodiment 253, wherein the alkylating agent is nitrogen mustard (mechlorethamine).

[0665] 257. The method according to embodiment 253, wherein the alkylating agent is diziquone.

[0666] 258. The method according to embodiment 253, wherein the alkylating agent is temozolomide.

[0667] 259. The method according to implementation plan 248, wherein the second therapeutic agent is an anthracycline.

[0668] 260. The method according to embodiment 259, wherein the anthracycline is doxorubicin.

[0669] 261. The method according to embodiment 259, wherein the anthracycline is epirubicin.

[0670] 262. The method according to implementation scheme 248, wherein the second therapeutic agent is an antimicrotubule agent.

[0671] 263. The method according to embodiment 262, wherein the antimicrotubule agent is vinblastine.

[0672] 264. The method according to embodiment 248, wherein the second therapeutic agent is a platinum compound.

[0673] 265. The method according to embodiment 264, wherein the platinum compound is cisplatin.

[0674] 266. The method according to embodiment 264, wherein the platinum compound is oxaliplatin.

[0675] 267. The method according to implementation plan 248, wherein the second therapeutic agent is taxane.

[0676] 268. The method according to embodiment 267, wherein the taxane is paclitaxel.

[0677] 269. The method according to embodiment 267, wherein the taxane is docetaxel.

[0678] 270. The method according to embodiment 248, wherein the second therapeutic agent is a topoisomerase inhibitor.

[0679] 271. The method according to embodiment 270, wherein the topoisomerase inhibitor is etoposide.

[0680] 272. The method according to embodiment 270, wherein the topoisomerase inhibitor is mitoxantrone.

[0681] 273. The method according to implementation plan 248, wherein the second therapeutic agent is vinca alkaloid.

[0682] 274. The method according to embodiment 273, wherein the vinca alkaloid is vincristine.

[0683] 275. The method according to any one of embodiments 202 to 274, wherein the combination therapy comprises intratumoral CpG therapy.

[0684] 276. The method according to any one of embodiments 202 to 275, wherein the second therapeutic agent is an ADC having a cytotoxic payload.

[0685] 277. The method according to embodiment 276, wherein the ADC having a cytotoxic payload targets the FAP.

[0686] 278. The method according to implementation plan 277, wherein the second therapeutic agent is OMTX705.

[0687] 279. The method according to any one of embodiments 202 to 278, wherein the second therapeutic agent is a cytokine.

[0688] 280. The method according to embodiment 279, wherein the cytokine is IL2.

[0689] 281. The method according to implementation scheme 279, wherein the cytokine is IL12.

[0690] 282. The method according to embodiment 279, wherein the cytokine is IFN-α.

[0691] 283. The method according to embodiment 279, wherein the cytokine is IFN-γ.

[0692] 284. The method according to any one of embodiments 202 to 283, comprising treating the subject with the combination therapy.

[0693] 285. The method according to any one of embodiments 202 to 284, comprising administering the second therapeutic agent to the subject.

[0694] 286. A method for promoting the dedifferentiation of myofibroblasts into quiescent fibroblasts, comprising contacting the myofibroblasts with a targeted drug conjugate according to any one of embodiments 1 to 120 or a drug composition according to any one of embodiments 121 to 149.

[0695] 287. A method for promoting the dedifferentiation of activated fibroblasts into resting fibroblasts, comprising contacting the activated fibroblasts with a targeted drug conjugate according to any one of embodiments 1 to 120 or a drug composition according to any one of embodiments 121 to 149.

[0696] 288. A method for promoting the dedifferentiation of fibroblasts that have transformed into myofibroblasts into quiescent fibroblasts, comprising contacting the myofibroblasts that have transformed into myofibroblasts with a targeted drug conjugate according to any one of embodiments 1 to 120 or a drug composition according to any one of embodiments 121 to 149.

[0697] 289. The method according to any one of embodiments 286 to 288, wherein dedifferentiation includes a reduction in smooth muscle actin expression.

[0698] 290. A method for promoting apoptosis of myofibroblasts, comprising contacting the myofibroblasts with a targeted drug conjugate according to any one of embodiments 1 to 120 or a drug composition according to any one of embodiments 121 to 149.

[0699] 291. A method for promoting apoptosis of activated fibroblasts, comprising contacting the activated fibroblasts with a targeted drug conjugate according to any one of embodiments 1 to 120 or a drug composition according to any one of embodiments 121 to 149.

[0700] 292. A method for promoting apoptosis of fibroblasts transforming into myofibroblasts, comprising contacting the myofibroblasts transforming into myofibroblasts with a targeted drug conjugate according to any one of embodiments 1 to 120 or a drug composition according to any one of embodiments 121 to 149.

[0701] 293. The method according to any one of embodiments 286 to 292, wherein the contact is performed inside the subject.

[0702] 294. The method of claim 293, comprising administering the targeted drug conjugate or drug composition to the subject.

[0703] 295. Compound C or its salt.

[0704] 296. Compound N or its salt.

[0705] 297. A targeted drug conjugate comprising compound C operatively linked to a targeting moiety.

[0706] 298. A targeted drug conjugate comprising a compound N operatively linked to a targeting moiety.

[0707] 299. Compound C that is coupled with a connector.

[0708] 300. Compound N that is coupled with a connector.

[0709] Although various specific implementation schemes have been explained and described, it should be understood that various changes may be implemented without departing from the spirit and scope of this disclosure.

[0710] 8. References

[0711] All publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety for all purposes, just as each individual publication, patent, patent application or other document is individually indicated to be incorporated by reference for all purposes. In the event of any inconsistency between the teachings of one or more references incorporated herein and this disclosure, the teachings of this specification shall prevail.

Claims

1. A targeted drug conjugate comprising an ALK5 inhibitor operatively linked to a targeting moiety via a cleavable linker, the targeting moiety comprising an antibody or antibody fragment binding to fibroblast activation protein (FAP), wherein the ALK5 inhibitor is N-methyl-2-(4-(4-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethane-1-amine.

2. The targeted drug conjugate according to claim 1, wherein the linker is a PEG-containing linker.

3. The targeted drug conjugate according to claim 1 or claim 2, wherein the ALK5 inhibitor is connected to the target portion via a cleavable connector, the cleavable connector being a dipeptide connector, a disulfide connector, or a hydrazone connector.

4. The targeted drug conjugate according to claim 3, wherein the linker is a protease-sensitive valine-citrulline dipeptide linker, a glutathione-sensitive disulfide linker, or an acid-sensitive disulfide linker.

5. The targeted drug conjugate according to claim 4, wherein the linker is a valine-citrulline dipeptide linker.

6. The targeted drug conjugate according to claim 3, wherein the linker is a disulfide linker.

7. The targeted drug conjugate according to claim 1 or claim 2, wherein the ALK5 inhibitor is conjugated via one or more cysteine ​​residues on the targeting moiety or one or more lysine residues on the targeting moiety.

8. The targeted drug conjugate according to claim 1 or claim 2, wherein the average number of ALK5 inhibitor molecules per targeting moiety is in the range of 2 to 8.

9. The targeted drug conjugate according to claim 1 or claim 2, wherein the targeting portion comprises an antibody.

10. The targeted drug conjugate of claim 9, wherein the antibody is a monoclonal antibody.

11. The targeted drug conjugate of claim 10, wherein the antibody is human or humanized.

12. The targeted drug conjugate according to claim 1 or claim 2, wherein the targeting portion comprises an antibody fragment.

13. The targeted drug conjugate of claim 12, wherein the antibody fragment is Fab, Fab', F(ab')2, Fv, scFv, dsFv or a single-domain antibody.

14. The targeted drug conjugate of claim 12, wherein the antibody fragment is a fragment of a human antibody or a humanized antibody.

15. A pharmaceutical composition comprising a targeted pharmaceutical conjugate according to any one of claims 1-14 and a pharmaceutically acceptable carrier.

16. Use of the targeted drug conjugate according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, skin fibrosis or esophageal fibrosis.

17. The use according to claim 16, wherein the fibrosis is idiopathic pulmonary fibrosis (IPF).

18. The use according to claim 16 or claim 17, wherein the drug formulation is intended for administration as a single therapy.

19. The use according to claim 16 or claim 17, wherein the drug formulation is intended for administration as part of a combination therapy regimen.

20. The use according to claim 19, wherein the combination therapy regimen comprises pirfenidone or nintedanib.

21. Use of the targeted drug conjugate according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 in the preparation of a medicament for the treatment of systemic sclerosis.

22. Use of the targeted drug conjugate according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 15 in the preparation of a medicament for the treatment of urothelial carcinoma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, esophageal cancer, colorectal cancer, head and neck cancer, ovarian cancer, kidney cancer, or gastric adenocarcinoma.

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