Antibody-ALK5 inhibitor conjugates and uses thereof

Abstract

The present disclosure relates to antibody-drug conjugates comprising an ALK5 inhibitor and uses thereof.

Description

[0001] This application is a divisional application based on the Chinese invention patent application with application date of July 9, 2018, application number 201880096817.X, and invention name: "Antibody-ALK5 inhibitor conjugates and uses thereof". Technical Background

[0002] Members of the transforming growth factor-β (TGF-β) family of cytokines are multifunctional proteins that regulate a variety of biological processes during normal tissue development and in disease states. TGF-β family members are involved in inflammation, wound healing, extracellular matrix accumulation, bone formation, tissue development, cell differentiation, heart valve remodeling, tissue fibrosis and tumor progression, etc. (Barnard et al., 1990, Biochim Biophys Acta. 1032: 79-87; Sporn et al., 1992, J Cell Biol 119: 1017-1021; Yingling et al., 2004, Nature Reviews, 3: 1011-1022; Janssens et al., 2005, Endocr Rev., 26 (6): 743-74). To date, three mammalian isoforms have been identified: TGF-β1, TGF-β2 and TGF-β3. (Massague, 1990, Annu Rev Cell Biol 6:597-641). Other members of the transforming growth factor superfamily include activins, inhibins, bone morphogenetic proteins, growth and differentiation factors, and Muller inhibitors.

[0003] TGF-β1 transduces signals through two highly conserved single transmembrane serine / threonine kinase receptors, type I (ALK5) and type II TGF-β receptors. After ligand-induced binding and oligomerization, type II receptors phosphorylate serine / threonine residues in the GS region of ALK5, leading to ALK5 activation and the generation of new SMAD docking sites. SMADs are intracellular proteins that are specialized for transducing TGF-β signals from the extracellular environment into the cell nucleus. Upon activation, ALK5 phosphorylates Smad2 and Smad3 at their C-terminal SS×S-motif, leading to their dissociation from the receptor and formation of a complex with Smad4. The Smad complex then translocates into the nucleus and assembles with cell-specific DNA-binding cofactors to modify the expression of genes that regulate cell growth, differentiation, and development.

[0004] Activin transduces signals in a manner similar to TGF-β. Activin binds to the serine / threonine kinase, activin type II receptor (ActRIIB), and the activated type II receptor phosphorylates the serine / threonine residues in the GS region of ALK4. Activated ALK4 phosphorylates Smad2 and Smad3 in turn. Subsequent formation of heterologous Smad complexes with Smad4 leads to activin-induced gene transcription regulation.

[0005] TGF-β signaling is essential for maintaining immune homeostasis by regulating innate and adaptive immune cells, including T and B lymphocytes, NK cells, and antigen-presenting cells, such as dendritic cells. TGF-β is generally considered an immunosuppressive cytokine that plays a crucial role in thymic T cell development as well as in maintaining peripheral tolerance. TGF-β inhibits CD4 + and CD8 + T cell proliferation, cytokine production, cytotoxicity and differentiation into T helper cell subsets (Li et al., 2008, Cell 134: 392-404). TGF-β also plays a role in natural regulatory T cells (nT cells) produced by the thymus. regs ) and in the periphery in response to inflammation and various diseases (e.g., cancer). regs (iT regs ) plays an important role in the regulation of mitochondria (Tran et al., 2012, J Mol Cell Bio 4:29-37, 2012). reg It is CD4 + A small subset of T cells, usually CD25+Fo×P3+, actively inhibits T cell activation to help maintain peripheral T cell tolerance. reg It is essential for the survival and expansion of CD4 T cells in the periphery (Marie et al., 2005, J E x p Med 201:1061-67). Under appropriate inflammatory conditions, TGF-β converts naive CD4 + T cells transformed into Fo×P3 + iT regs , to suppress local tissue-resident T cells. iT cells are often found in the tumor itself. regs Levels increase, preventing T cell-mediated tumor clearance (Whiteside, 2014, E×pert Opin Biol Ther 14:1411-25).

[0006] In general, high levels of TGF-β expression are associated with poor clinical prognosis. Typically, tumors specify the TGF-β pathway and use it to avoid T cell-mediated tumor clearance (Yang et al., Trends Immunol 31:220-7, 2010; Tu et al., Cytokine Growth Factor Rev 25:423-35, 2014). This occurs in two ways. One is that TGF-β directly inhibits CD4+ and CD8 + T cell expansion, cytokine production and tumor cell killing. regs and iT regs TGF-β is essential for the survival and / or transformation of tumors, and they also inhibit immune-mediated tumor clearance. In multiple preclinical mouse models, neutralization of TGF-β has been shown to reduce tumor burden due to increased T cell-mediated tumor clearance. Importantly, inhibition of TGF-β signaling in T cells by expression of dominant-negative TGF-βRII or soluble TGF-β receptors is sufficient to restore effective immune-mediated tumor clearance in vivo. Gorelik et al., 2001, Nat Med 7:1118-22; Thomas et al., 2005, Cancer Cell 8:369-80.

[0007] In addition to its effect on the immune system, TGF-β signaling has a prominent but complex role in tumor development. Preclinical studies have shown that TGF-β has a paradoxical effect on the tumor itself and has a mixed effect on surrounding stromal cells. In the early stages of cancer progression, TGF-β inhibits tumor growth and amplification by regulating cell cycle mediators. However, in the late stage, TGF-β loses its growth inhibitory properties by inducing epithelial to mesenchymal transition (EMT) and by its effect on stromal fibroblasts, angiogenesis and extracellular matrix (ECM), and promotes tumor metastasis (Connolly et al., 2012, Int J Bio 8: 964-78). If delivered at the wrong stage, widespread inhibition of TGF-β signaling carries the risk of promoting tumor metastasis and / or inhibiting non-tumor stromal cell populations that indirectly aggravate tumor progression (Cui et al., 1996, Cell 86:531-; Siegel et al., 2003, PNAS 100:8430-35; Connolly et al., 2011, Cancer Res 71:2339-49; Achyut et al., 2013, PLOS Genetics 9:1-15). TGF-β inhibitors may cause tumors to become more invasive and metastatic, replacing the expected growth inhibitory effect.

[0008] Despite the paradoxical effect on the tumor itself and the widespread expression of TGF-β receptors, there has long been a focus on inhibiting the TGF-β pathway as a cancer therapy. Inhibitors have included neutralizing TGF-β antibodies, TGF-β2 antisense RNA, and small molecule ATP-competitive ALK5 kinase inhibitors. Some of the classic ALK5 inhibitors that have been developed are pyrazolyl, imidazole, and triazole (Bonafou× et al., 2009, E×pert Opin Ther Patents 19: 1759-69; Ling et al., 2011, CurrentPharma Biotech 12: 2190-2202). Many ALK5 inhibitors have been tested in in vitro cell-based assays and in vivo mouse xenograft and syngeneic tumor models, and significant efficacy has been demonstrated (Neuzillet et al., 2015, Pharm&Therapeutics 147: 22-31). However, most TGF-β inhibitors, especially ALK5 inhibitors, are still in the preclinical discovery stage due to concerns about host toxicity, as TGF-β receptors are ubiquitously expressed, and concerns about inadvertent promotion of tumor growth. For example, in preclinical toxicology studies in rats, two different series of ALK5 inhibitors showed heart valve lesions characterized by hemorrhage, inflammation, degeneration, and proliferation of valvular interstitial cells (Anderton et al., 2011, ToxPath 39:916-24).

[0009] Therefore, there is a need to target ALK5 inhibitors to cell types in which inhibition of TGF-β signaling is therapeutically useful while minimizing host tissue toxicity, such as that observed in cardiac tissue. Summary of the invention

[0010] To avoid on-target host toxicity and prevent unintentional worsening of tumor progression due to ALK5 inhibitor therapy, the inventors developed a new approach to direct compounds to only those cells that confer therapeutic benefit.

[0011] For the treatment of cancer, the method encompasses directing ALK5 inhibitors to the T cell compartment via antibodies to promote T cell-mediated tumor clearance and establish long-term remission without causing systemic toxicity. Without being bound by theory, it is believed that not only does inhibition of TGF-β signaling in T cells directly enhance T cell-mediated clearance, but it also inhibits T cell trafficking to induced T cells. regs transformation and decrease of natural T reg Therefore, inhibition of TGF-β signaling in T cells not only restores CD4 + and CD8 + T cell activity and removal of T reg"We found that inhibiting TGF-β signaling only in T cells would be safer than broad TGF-β inhibition, both from a tumor perspective and in terms of toxicity to host tissues."

[0012] Therefore, the present disclosure provides antibody-drug conjugates (ADCs), wherein the drug is an ALK5 inhibitor. The antibody component of the ADC can be an antibody or antigen-binding fragment that binds to a T cell surface molecule. Section 4.2 describes exemplary antibody components that can be used in the ADCs of the present disclosure. In some embodiments, the ALK5 inhibitor is an imidazole-benzodioxole compound, an imidazole-quinoxaline compound, a pyrazole-pyrrole compound, or a thiazole compound. Exemplary ALK5 inhibitors are described in Section 4.3 and Tables 1-3.

[0013] The ALK5 inhibitor can be coupled directly to the antibody component or connected to the antibody component through a linker. The linker can be a non-cleavable linker, or preferably, a cleavable linker. Exemplary non-cleavable linkers and cleavable linkers are described in Section 4.4. The average number of ALK5 inhibitor molecules attached per antibody or antigen-binding fragment can vary, and is typically 2 to 8 ALK5 inhibitor molecules per antibody or antigen-binding fragment. Drug loading is described in detail in Section 4.5.

[0014] The present disclosure also provides pharmaceutical compositions comprising the ADCs of the present disclosure. Exemplary pharmaceutical excipients that can be used to formulate pharmaceutical compositions comprising the ADCs of the present disclosure are described in Section 4.6.

[0015] The present disclosure also provides a method for treating cancer by administering an ADC of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need thereof. The ADC and pharmaceutical composition of the present disclosure can be administered as a monotherapy or as part of a combination therapy. Exemplary cancers that can be treated with the ADC and pharmaceutical composition of the present disclosure and exemplary combination therapies are described in Section 4.7. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of TGF-β on CD4 + and CD8 + During tumor progression, TGF-β, which can be derived from tumors and T cells themselves, inhibits CD4 + T cell functions, such as cytokine production, proliferation, and Th differentiation. At the same time, TGF-β also inhibits cytotoxic CD8 + The expression of granzymes and perforins in T cells inhibits tumor killing. + and CD8 +Both T cell populations can significantly inhibit T cell-mediated tumor clearance.

[0017] Figure 2 The role of TGF-β in T cells during tumor progression is shown. reg During tumor progression, nT cells are often found in tumors. reg and iT reg cells to control T cell-mediated functions in situ. TGF-β promotes nT reg Cell viability and iT reg T cells are transformed to inhibit T cell-mediated tumor clearance. reg The increase in cells ensures that T cells that infiltrate the tumor are also prevented from clearing it.

[0018] Figure 3 The ADC of the present disclosure is shown to be effective against CD4 + and CD8 + Mechanism of action of T cells. T cell targeted inhibition of TGF-β signaling restores CD4 + T cell activity and CD8 + T cell-mediated tumor killing.

[0019] Figure 4 The ADC of the present disclosure is shown to be reg Mechanism of action of cells. T cell-targeted inhibition of TGF-β signaling also blocks in situ T reg Mediated inhibition of immune-mediated tumor clearance.

[0020] Figures 5A-5D Compounds AD are shown to inhibit TGF-β-induced luciferase activity in HEK293T cells. Figure 5A : Compound A; Figure 5B : Compound B; Figure 5C : Compound C; Figure 5D : Compound D.

[0021] Figures 6A-6C MTS proliferation assay data for compounds AD are shown. Compound AC was expressed in CDC4 treated with TGF-β. + Restore proliferation in T cells. Fig. 6A : Data for compounds AD. Fig. 6A In the figures, the bars labeled "A", "B", "C", and "D" above "No TGF-β" show the results of experiments performed using the compounds at 100 nM and in the absence of TGF-β. Figure 6B : Data of compound B; Figure 6C : Data for compound C.

[0022] Figure 7A-7BLC-MS data for an exemplary ADC (ADC2) of the present disclosure is shown. Fig. 7A : LC-MS data of ADC heavy chain; Figure 7B : LC-MS data of ADC light chain.

[0023] Figure 8 is a chromatogram of ADC2 purified by SEC with a S-4FB / Ab ratio of 6. SEC analysis of purified ADC2 showed less than 5% aggregation.

[0024] Figures 9A-9F An exemplary antibody of the present disclosure (anti-transferrin receptor antibody R17217) is shown in primary mouse CD4 + Induces internalization of the antibody target transferrin receptor (TfR) on T cells. Fig. 9A : control without anti-transferrin receptor antibody; Fig. 9B : Incubate with anti-transferrin receptor antibody for 15 min; Fig. 9C : Incubate with anti-transferrin receptor antibody for 30 min; Fig.9D : Incubate with anti-transferrin receptor antibody for 60 min; Fig.9E : Incubate with anti-transferrin receptor antibody for 180 min; Fig.9F : Mean fluorescence intensity (MFI) during 3 hours.

[0025] Fig.10 Reversal of TGF-β-mediated proliferation inhibition in mouse CTLL2 cells by an exemplary ADC of the present disclosure (ADC1) is shown.

[0026] Fig.11 The results show that primary CD8 TGF-β activated by an exemplary ADC (ADC1) of the present disclosure + Inhibition of Granzyme B Expression in T Cells. ADC1 partially restored Granzyme B expression compared with free ALK5 inhibitor.

[0027] Fig.12 An exemplary ADC (ADC1) of the present disclosure is shown to reduce iT reg produced, similar to 100 mM of free ALK5 inhibitor.

[0028] Figures 13A-13D It shows that CD5 ( Fig.13A and Fig. 13C ) and CD2( Fig. 13B and Fig.13D ) internalized into primary activated mouse CD3 + in T cells.

[0029] Fig.14The results show that activated mouse CD3 + CD8 T cells expressing granzyme (GzmB) after 36 hours of incubation + T cell levels.

[0030] Fig.15 The results show that activated mouse CD3 + IL2 levels secreted by T cells after 36 h of incubation.

[0031] Fig.16 The results show that activated mouse CD3 + IFN-γ levels secreted by T cells after 36 h of incubation.

[0032] Fig.17 The results show that activated mouse CD3 + The amount of T cell proliferation after T cell incubation for 72 hours.

[0033] Fig.18 Internalization of CD7 into primary activated human CD3 + in T cells. DETAILED DESCRIPTION

[0034] The present disclosure provides antibody-drug conjugates (ADCs) that can be used to treat cancer, which include an antibody component covalently bound to an ALK5 inhibitor directly or through a linker. An overview of the ADCs of the present disclosure is given in Section 4.1. The antibody component of the ADC can be a complete antibody or a fragment thereof. The antibodies that can be used in the ADCs of the present disclosure are described in detail in Section 4.2. The ALK5 inhibitors that can be used in the ADCs of the present disclosure are described in Section 4.3. The ADCs of the present disclosure generally include a linker between the antibody and the ALK5 inhibitor. Exemplary linkers that can be used in the ADCs of the present disclosure are described in Section 4.4. Each antibody of the ADCs of the present disclosure can include a different number of ALK5 inhibitor portions. The drug loading will be discussed in detail in Section 4.5. The present disclosure also provides pharmaceutical preparations containing the ADCs of the present disclosure. Pharmaceutical preparations containing ADCs are described in Section 4.6. The present disclosure also provides methods for treating various cancers using the ADCs of the present disclosure. Methods for treating cancer using the ADCs of the present disclosure as a monotherapy or as part of a combination therapy are described in Section 4.7.

[0035] 4.1 Antibody-drug conjugates

[0036] The ADCs of the present disclosure are generally composed of an ALK5 inhibitor covalently attached to an antibody, typically via a linker, such that the covalent attachment does not interfere with binding to the antibody target.

[0037] Techniques for coupling drugs to antibodies are well known in the art (see, e.g., Hellstrom et al., Controlled Drug Delivery, 2nd ed., 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). The ALK5 inhibitor is preferably attached to the antibody component of the ADC of the present disclosure by site-specific coupling. For example, the ALK5 inhibitor can be coupled to the antibody component 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, e.g., Zhou, 2017, Biomedicines 5(4):E64, the contents of which are incorporated herein by reference in their entirety.

[0038] In one embodiment, the antibody or its fragment is fused to the amino acid sequence of another protein (or part thereof, for example, at least 10, 20 or 50 amino acid parts of the protein) through the N-terminus or C-terminus of the antibody or internally by a covalent bond (e.g., a peptide bond). The antibody or its fragment can be connected to another protein at the N-terminus of the antibody constant domain. Recombinant DNA procedures can be used to produce such fusions, for example, as described in WO 86 / 01533 and EP0392745. In another embodiment, the effector molecule can increase the half-life in vivo, and / or enhance the delivery of antibodies across the epithelial barrier to the immune system. 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 No. WO 2005 / 117984.

[0039] The metabolic process or reaction can be an enzymatic process, such as proteolytic cleavage of a peptide linker of an ADC, or hydrolysis of a functional group (e.g., an acylhydrazone, ester, or amide). Intracellular metabolites include, but are not limited to, antibodies and free drugs that have undergone intracellular cleavage after entering, diffusing, taking up, or transporting into a cell.

[0040] The terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction on an antibody-drug conjugate (ADC) inside a cell whereby the covalent attachment, i.e., linker, between the drug moiety (D) and the antibody (Ab) is broken, resulting in the dissociation of the free drug from the antibody inside the cell. Thus, the cleaved portion of the ADC is an intracellular metabolite.

[0041] 4.2 Antibody components

[0042] The present disclosure provides antibody drug conjugates, wherein the antibody component is bound to a T cell surface molecule. Unless otherwise specified, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or immunoreacts with a specific antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heterologous coupling antibodies (e.g., bispecific antibodies, double antibodies, three antibodies and four antibodies) and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments. In addition, unless otherwise specified, the term "monoclonal antibody" (mAb) means including complete molecules 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 a complete antibody, are cleared faster from the circulation of animals or plants, and may have less nonspecific tissue binding (Wahl et al., 1983, J.Nucl.Med.24:316) compared to complete antibodies.

[0043] The term "scFv" refers to a single-chain Fv antibody in which the variable domains from the heavy and light chains of a traditional antibody have been linked to form one chain.

[0044] Reference to "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, including the heavy chain of Fv, scFv or Fab. Reference to "VL" refers to the variable region of the immunoglobulin light chain, including the light chain of Fv, scFv, dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with the same structural characteristics. Although antibodies exhibit binding specificity to specific targets, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Natural antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at the amino terminus, followed by a number of constant domains. Each light chain has a variable domain at the amino terminus (VL) and a constant domain at the carboxyl terminus.

[0045] In order to optimally deliver the ALK5 inhibitor within the cell, the antibody is preferably internalized. After the internalized antibody binds to the target molecule on the cell surface, it is internalized by the cell due to the binding. The result of this is that the ADC is taken up by the cell. Methods that allow the internalization of an antibody to be determined after binding to its antigen are known to the skilled person and are described, for example, on page 80 of PCT Publication No. WO 2007 / 070538 and in Section 5.11 below. Once internalized, if the ALK5 inhibitor is attached to the antibody using a cleavable linker (e.g., as described in Section 4.4), the ALK5 inhibitor can be released from the antibody by cleavage in the lysosome or by other cellular mechanisms.

[0046] The term "antibody fragment" refers to a portion of a full-length antibody, typically the target binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. The "Fv" fragment is the smallest antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain (VH-VL dimer) in tight non-covalent association. In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer binding specificity to the target to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three CDRs specific for a target) can have the ability to recognize and bind to a target. A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of an antibody in a single polypeptide chain. Typically, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. A "single domain antibody" is composed of a single VH or VL domain that exhibits sufficient affinity for TNF-α. In a specific embodiment, the single domain antibody is a camelid antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231: 25-38).

[0047] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that some residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. The F(ab') fragment is produced by cleavage of the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digest product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.

[0048] In certain embodiments, the antibodies of the present 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, including any eukaryotic, prokaryotic or phage clone, rather than its production method. Monoclonal antibodies that can be used for the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant and phage display technology or a combination thereof. Antibodies of the present disclosure include chimeric antibodies, primatized antibodies, humanized antibodies or human antibodies.

[0049] The antibodies of the present disclosure 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 generally selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, e.g., 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. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.

[0050] The antibodies of the present disclosure can be humanized. A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, an immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other target binding subdomains of an antibody) that contains a minimal sequence of a non-human immunoglobulin. Typically, a humanized antibody will contain at least one, and typically substantially all of two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al.; European Patent Publication No. EP239400; PCT Publication No. WO 91 / 09967; U.S. Patent No. 5,225,539; European Patent Publication No. EP592106; European Patent Publication No. EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et 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.

[0051] The antibodies of the present disclosure may be human antibodies. Completely "human" antibodies may be required for therapeutic treatment of human patients. As used herein, "human antibodies" include antibodies with human immunoglobulin amino acid sequences, and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and not expressing endogenous immunoglobulins. Human antibodies can be manufactured by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. 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 produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, e.g., PCT Publications WO 98 / 24893, WO 92 / 01047, WO 96 / 34096, WO 96 / 33735; U.S. Patent Nos. 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 and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, IL), Amgen (Thousand Oaks, CA), and Regeneron (Tarrytown, NY) can be hired to provide human antibodies against selected antigens using techniques similar to those described below. A technique called "guided selection" can be used to generate fully human antibodies that recognize a selected epitope. In this method, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1988, Biotechnology 12: 899-903).

[0052] The antibodies of the present disclosure may be primatized. The term "primatized antibody" refers to an antibody comprising a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and 5,693,780, which are incorporated herein by reference in their entirety.

[0053] Antibodies of the present disclosure include derivative antibodies. For example, but not limited to, derivative antibodies are usually modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to cellular ligands or other proteins (see Section 4.1 for discussion of antibody conjugates), etc. Many chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, derivatives can contain one or more non-natural amino acids, such as using ambr× technology (see, e.g., Wolfson, 2006, Chem. Biol. 13 (10): 1011-2).

[0054] In another embodiment of the present disclosure, the antibody or fragment thereof may be an antibody or antibody fragment whose sequence has been modified to change at least one constant region-mediated biological effector function relative to the corresponding wild-type sequence. For example, in some embodiments, the antibody of the present disclosure may be modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody, for example, to reduce binding to an Fc receptor (FcγR) or to C1q. FcγR and C1q binding may be reduced by mutating the immunoglobulin constant region fragment of the antibody in a specific region necessary for FcγR or C1q interaction (see, e.g., Canfield and Morrison, 1991, JE×p. 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).

[0055] Reduction in the FcγR binding ability of an antibody can also reduce other effector functions that rely on FcγR interactions, such as opsonization, phagocytosis, and antibody-dependent cellular cytotoxicity ("ADCC"), while reduction in C1q binding can reduce complement-dependent cytotoxicity ("CDCC"). Thus, reduction or elimination of effector function can prevent T cells targeted by the ADCs of the present disclosure from being destroyed by ADCC or CDC. Thus, in some embodiments, the effector function of an antibody is modified by selective mutation of the Fc portion of the antibody such that it retains antigen specificity and internalization ability, but eliminates ADCC / CDC function.

[0056] Many mutations for reducing FcγR and C1q binding have been described in the art, and such mutations may be included in the ADCs of the present disclosure. For example, U.S. Pat. No. 6,737,056 discloses that a single Fc region amino acid modification at position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 results in reduced binding to FcγRII and FcγRII. U.S. Pat. No. 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 Publication No. WO 2014 / 190441 describes a modified Fc domain with reduced FcγR binding having 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, L234D / L235E / E269K / D265S / K322E / E333K: E233K / L234R / L235R / E269K / D265S / K322E / E333K mutations, wherein the mutation before the semicolon is set in the first Fc polypeptide and the mutation after the semicolon is in the second Fc polypeptide of the Fc dimer. 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 Biol Chem 292:3900-08; Wang et al., 2018, Protein Cell 9:63-73).

[0057] As an alternative to mutating the constant region to reduce effector function, such as by mutating the Fc domain as described above, effector function can be eliminated through the use of antibody fragments (eg, Fab, Fab' or F(ab')2 fragments).

[0058] In other embodiments of the present disclosure, antibodies or fragments thereof may be modified to obtain or improve at least one constant region-mediated biological effector function relative to an unmodified antibody, such as to enhance FcγR interactions (see, e.g., US2006 / 0134709). For example, an antibody of the present disclosure may have a constant region that binds to FcγRIIA, FcγRIIB, and / or FcγRIIIA with greater affinity than the corresponding wild-type constant region.

[0059] Thus, the antibodies of the present disclosure may have alterations in biological activity resulting in reduced opsonization, phagocytosis or ADCC. Such alterations are known in the art. For example, antibody modifications that reduce ADCC activity are described in U.S. Pat. No. 5,834,597.

[0060] In another aspect, the antibody or fragment thereof may be an antibody or antibody fragment that has been modified to increase or decrease its binding affinity to the fetal Fc receptor FcRn, for example, by mutating the immunoglobulin constant region fragment in a specific region involved in the FcRn interaction (see, e.g., WO 2005 / 123780). Such mutations may increase the binding of the antibody to FcRn, which protects the antibody from degradation and increases its half-life.

[0061] In yet another aspect, the antibody has one or more amino acids inserted into one or more hypervariable regions thereof, e.g., 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 No. 2007 / 0280931.

[0062] The target of the antibody will depend on the desired therapeutic application of the ADC. Typically, the target is a molecule present on the surface of a cell to which the ALK5 inhibitor (e.g., T cell) and the antibody (preferably internalized when bound to the target) are delivered. Internalizing antibodies are described in, for example, 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.

[0063] It is desirable to generate antibodies that bind to T cell surface molecules for use in applications where ADCs are intended to stimulate the immune system by reducing TGF-β activity. Without being bound by theory, it is believed that delivery of ALK5 inhibitors to T cells can activate CD4 + and / or CD8 + T cell activity and inhibit regulatory T cell activity, both of which contribute to immune tolerance of tumors. Therefore, the use of antibodies that bind to T cell surface molecules in ADCs of the present disclosure can be used to treat various cancers, for example, as described in Section 4.7 below. In various embodiments, the antibodies bind to CD4 + T cells, CD8 + T cells, T REG In some embodiments, the antibody binds to a pan-T cell surface molecule. Examples of T cell surface molecules suitable for targeting include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD70, CD71, CD103, CD184, Tim3, LAG3, CTLA4, and PD1. Examples of antibodies that bind to T cell surface molecules and are considered to be internalizing include OKT6 (anti-CD1; ATCC deposit number CRL8020), OKT11 (anti-CD2; ATCC deposit number CRL8027), OKT3 (anti-CD3; ATCC deposit number CRL8001), OKT4 (anti-CD4; ATCC deposit number CRL8002), OKT8 (anti-CD8; ATCC deposit number CRL8014), 7D4 (anti-CD25; ATCC deposit number CRL1698), OKT9 (anti-CD71; ATCC deposit number CRL8021), CD28.2 (anti-CD28, BD Biosciences). Biosciences catalog number 556620), UCHT1 (anti-CD3, Bio×Cell catalog number BE0231), M290 (anti-CD103, Bio×Cell catalog number BE0026), and FR70 (anti-CD70, Bio×Cell catalog number BE0022).

[0064] In some embodiments, the targeted T cell surface molecule is a T cell surface molecule that can be recycled back to the cell surface through the endosome after internalization (see, Goldenring, 2015 Curr. Opin. Cell Biol., 35: 117-22). It is believed that exemplary T cell surface molecules that can be recycled through the endosome include CD5 and CD7. Without being bound by theory, it is believed that targeting T cell surface molecules that can be recycled through the endosome can promote the delivery of ALK5 inhibitors to ALK5, because ALK5 can also be recycled through the endosome. Therefore, targeting T cell surface molecules that can be recycled through the endosome may help bring ALK5 inhibitors closer to ALK5.

[0065] 4.3 ALK5 inhibitors

[0066] The ALK5 inhibitors of the present disclosure are preferably small molecules that competitively and reversibly bind to the ATP binding site in the cytoplasmic kinase domain of the ALK5 receptor, preventing downstream R-Smad phosphorylation.

[0067] However, an ALK5 inhibitor may not need to be specific or selective for ALK5 compared to other TGF-β family receptors, such as ALK4 and / or ALK7 and / or TGF-β receptor II. In some embodiments, an ALK5 inhibitor has activity against both ALK5 and TGF-β receptor II. Although it is preferred that the ALK5 inhibitor has limited inhibitory activity against the BMP II receptor, this is not required because the ADCs of the present disclosure target T cells in which BMP II activity is minimal or absent.

[0068] The IC of the ALK5 inhibitors of the present disclosure when measured in an in vitro cell assay using T cells from at least 3 subjects, at least 5 subjects, or at least 10 subjects. 50 Preferably 100 nM or less, more preferably 50 nM or less, most preferably 20 nM or less. Exemplary cell assays are described in Section 5.6 below. When the ADC targets human rather than mouse T cell surface molecules, human cells can be used instead of mouse cells and antibodies that recognize humans can be used instead of antibodies that recognize mouse CD28 and CD3.

[0069] Illustrative examples of ALK5 inhibitors suitable for use in the antibody-drug conjugates of the present disclosure include imidazole-benzodioxole compounds, imidazole-quinoxaline compounds, pyrazole-pyrrole compounds, or thiazole compounds.

[0070] According to one aspect of the present disclosure, the imidazole-benzodioxole ALK5 inhibitor has the following formula:

[0071]

[0072] Where R 1 is hydrogen or a lower alkyl radical having 1 to about 5 carbon atoms, R 2 is hydrogen or a lower alkyl radical having 1 to about 5 carbon atoms, R 3 is an amide, a nitrile, an alkynyl group having 1 to about 3 carbon atoms, a carboxyl group having 1 to about 5 carbon atoms, or an alkanol group, A is a direct bond or an alkyl group having 1 to about 5 carbon atoms, and B is a direct bond or an alkyl group having 1 to about 5 carbon atoms. In some individual preferred embodiments of the present disclosure, R 2 is hydrogen or methyl, A has 1 carbon atom, and B is a direct bond to the benzyl group, and R 3 In a preferred embodiment of a combination of the present disclosure, R 2 is hydrogen or methyl, A has 1 carbon atom, and B is a direct bond to the benzyl group.

[0073] According to another aspect of the present disclosure, the imidazole-quinoxaline ALK5 inhibitor has the following formula:

[0074]

[0075] Where R 1 is hydrogen or a lower alkyl radical having 1 to about 5 carbon atoms, R 2 is hydrogen, halogen or lower alkyl having 1 to about 5 carbon atoms, R 3 is an amide, a nitrile, an alkynyl group having 1 to about 3 carbon atoms, a carboxyl group having 1 to about 5 carbon atoms, or an alkanol group, A is a direct bond or an alkyl group having 1 to about 5 carbon atoms, and B is a direct bond or an alkyl group having 1 to about 5 carbon atoms. In some individual preferred embodiments of the present disclosure, R 2 is hydrogen or methyl, halogen (including fluorine or chlorine), A has 1 carbon atom, and B is a direct bond to the benzyl group, and R 3 In a preferred embodiment of a combination of the present disclosure, R 2 is hydrogen or methyl, A has 1 carbon atom, and B is a direct bond to the benzyl group.

[0076] According to another aspect of the present disclosure, the pyrazole ALK5 inhibitor has the following formula:

[0077]

[0078] Where R 2 is hydrogen, halogen or lower alkyl having 1 to about 5 carbon atoms, R 4is hydrogen, halogen, lower alkyl having 1 to about 5 carbon atoms, alkoxy having 1 to about 5 carbon atoms, haloalkyl, carboxyl, carboxyalkyl ester, nitrile, alkylamine or a group having the formula:

[0079]

[0080] Where R 5 is lower alkyl having 1 to about 5 carbon atoms, halogen or morpholino, and R 6 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.

[0081] According to another aspect of the present disclosure, the pyrazole-pyrrole ALK5 inhibitor has the following formula:

[0082]

[0083] Where R 7 is hydrogen, halogen, lower alkyl having 1 to about 5 carbon atoms, alkanol, morpholino or alkylamine, R 2 is hydrogen, halogen or lower alkyl having 1 to about 5 carbon atoms, and R 8 is hydrogen, hydroxy, amino, halogen or a group having the formula:

[0084]

[0085] Where R 5 is piperazinyl, R 6 is morpholino, piperidinyl, piperazinyl, alkoxy, hydroxy, oxane, halogen, thioalkyl or alkylamine, and A is a lower alkyl having 1 to about 5 carbon atoms.

[0086] According to another aspect of the present disclosure, the thiazole ALK5 inhibitor has the following formula:

[0087]

[0088] Where R 9 is hydrogen, halogen or lower alkyl having 1 to about 5 carbon atoms, and R 10 is hydrogen or a lower alkyl group having 1 to about 5 carbon atoms.

[0089] In certain embodiments, the ALK5 inhibitor is selected from any of the compounds designated A to N in Table 1 below:

[0090]

[0091]

[0092]

[0093]

[0094] In some additional specific embodiments, the ALK5 inhibitor is selected from any of the compounds named 1 to 283 in Table 2 below:

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] The preparation and use of ALK5 inhibitors are well known and well documented in the scientific and patent literature. PCT Publication No. WO 2000 / 61576 and U.S. Patent Publication No. US2003 / 0149277 disclose triaryl imidazole derivatives and their use as ALK5 inhibitors. PCT Publication No. WO 2001 / 62756 discloses pyridyl imidazole derivatives and their use as ALK5 inhibitors. PCT Publication No. WO 2002 / 055077 discloses the use of imidazolyl cyclic acetal derivatives as ALK5 inhibitors. PCT Publication No. WO 2003 / 087304 discloses trisubstituted heteroaryls and their use as ALK5 and / or ALK4 inhibitors. WO 2005 / 103028, U.S. Patent Publication No. US2008 / 0319012, and U.S. Patent No. 7,407,958 disclose 2-pyridyl-substituted imidazoles as ALK5 and / or ALK4 inhibitors. One of the representative compounds, IN-1130, showed ALK5 and / or ALK4 inhibitor activity in several animal models. The following patents and patent publications provide additional examples of ALK5 inhibitors and provide illustrative synthetic schemes and methods for using ALK5 inhibitors: U.S. Patent Nos. 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, and PCT Publication Nos. WO 2004 / 065392, WO 2009 / 050183, WO 2009 / 133070, WO 2011 / 146287, and WO 2013 / 009140. The foregoing patents and patent publications are incorporated herein by reference in their entirety.

[0108] 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

[0109] 396129-53-6), IN-1130, GW-788388, and D4476 (EMD4 Biosciences Merck KGaA, Darmstadt, Germany).

[0110] The structures and names of the ALK5 inhibitors described herein refer to the molecule prior to attachment to an antibody and / or linker.

[0111] Preferred ALK5 inhibitors are those that can be attached to the linker via a free NH or NH2 group (preferably attached to an NH or NH2 group) or a moiety of 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 as shown in Table 2). The ALK5 inhibitor can be derivatized to add a free NH or NH2 group. The design of derivatized ALK5 inhibitors should preferably consider the structure activity relationship (SAR) of the inhibitor to reduce the possibility of eliminating inhibitory activity when adding NH or NH2 groups, although this activity can also be determined empirically. Exemplary derivatized counterparts of several compounds shown in Table 1 are shown in Table 3.

[0112]

[0113]

[0114] 4.4 Connectors

[0115] Typically, the ADC comprises a linker between the ALK5 inhibitor and the antibody. The linker is a moiety comprising a covalent bond or a chain of atoms that covalently attaches the antibody to the drug moiety. In various embodiments, the linker comprises a divalent group, such as an alkyl diyl, an aryl diyl, a heteroaryl diyl, such as -(CR2) n O(CR2) n - moieties, repeating units of alkoxy (e.g., polyvinyloxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyvinylamino, Jeffamine TM ); and diacid esters and amides, including succinates, succinamides, diglycolates, malonates, and caproamides.

[0116] The linker can comprise one or more linker components, such as extensions and spacers. For example, a peptidyl linker can comprise two or more amino acids and optionally one or more extensions and / or spacer components. Various linker components are known in the art, some of which are described below.

[0117] The linker can be a "cleavable linker" to facilitate release of the drug in the cell. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., 1992, Cancer Research 52: 127-131; U.S. Pat. No. 5,208,020) can be used.

[0118] Examples of linkers and linker components known in the art include maleimidocaproyl (mc); maleimidocaproyl-p-aminobenzyl carbamate; maleimidocaproyl-peptide-aminobenzyl carbamate linkers, for example, maleimidocaproyl-L-phenylalanine-L-lysine-p-aminobenzyl carbamate and maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl carbamate (vc); N-succinimidyl 3-(2-pyridyldithio) propionate (also known as N-succinimidyl 4-(2-pyridyldithio) pentanoate or SPP); 4-succinimidyl-oxycarbonyl-2-methyl-2-(2-pyridyldithio)-toluene (SMPT) ; N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP); N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB); 2-iminothiolane; S-acetyl succinic anhydride; disulfide benzyl carbamate; carbonate; hydrazone linker; N-(α-maleimidoacetoxy) succinimide ester; N-[4-(p-azidosalicyloylamino)butyl]-3'-(2'-pyridyldithio) propionamide (AMAS); N[β-maleimidopropoxy] succinimide ester (BMPS); [N-ε-maleimidocaproyloxy] succinimide ester (EMCS); N-[γ-maleimidobutyryloxy] succinimide ester (GM BS); succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylic-[6-amidohexanoate] (LC-SMCC); succinimidyl-6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPDP); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB); succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC); N-succinimidyl-3-[2-pyridyldithio]-propionamido (SPDP); [N-ε-maleimidocaproyloxy]sulfosuccinimide ester (sulfonic acid- EMCS); N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS); 4-sulfosuccinimidyl-6-methyl-α-(-pyridyldithio)anilinohexanoate (sulfo-LC-SMPT); sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate (sulfo-LC-SPDP); m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS); N-sulfosuccinimidyl [4-iodoacetyl] aminobenzoate (sulfo-SIAB); sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC);Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (sulfo-SMPB); ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS); disuccinimidyl tartrate (DST); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); diethylenetriamine-pentaacetic acid (DTPA); thiourea linker; and oxime-containing linker. ;

[0119] In some embodiments, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker releases the ALK5 inhibitor from the antibody in a suitable environment. In yet other embodiments, the linker is non-cleavable, and the drug is released, for example, by degradation of the antibody in lysosomes (see, U.S. Patent Publication 2005 / 0238649, which is incorporated herein by reference in its entirety for all purposes).

[0120] Examples of non-cleavable linkers that can be used in the ADCs of the present disclosure include linkers of N-maleimidomethylcyclohexane 1-carboxylate, maleimidocaproyl, or mercaptoamidoacetamidocaproyl.

[0121] In some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease, including but not limited to a lysosomal or endosomal protease. In some embodiments, the peptidyl linker comprises a peptidyl component of at least two amino acids in length or at least three amino acids in length or more.

[0122] Cleavage agents may 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 active drugs within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptidyl linker (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker) that can be cleaved by the thiol-dependent protease cathepsin-B. Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, which is incorporated herein by reference in its entirety for all purposes.

[0123] In some embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin using a val-cit linker).

[0124] In other embodiments, the cleavable linker is sensitive to pH, that is, sensitive to hydrolysis at certain pH values. Generally, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, e.g., U.S. Patent Nos. 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 linkers are relatively stable under neutral pH conditions (e.g., those in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0125] In yet other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). Various disulfide bonds are known in the art, including, for example, those that can be 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-pyridyldithio) toluene)-, SPDB, and SMPT (see, e.g., 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 U.S. Pat. No. 4,880,935).

[0126] In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (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).

[0127] Typically, the linker is substantially insensitive to the extracellular environment. As used herein, in the context of a linker, "substantially insensitive to the extracellular environment" means that no more than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of the linker is cleaved in an ADC sample when the ADC is present in an extracellular environment (e.g., plasma).

[0128] For example, whether the linker is substantially insensitive to the extracellular environment can be determined by incubating the ADC 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.

[0129] In other non-mutually exclusive embodiments, the linker can promote cellular internalization. In certain embodiments, when coupled to a therapeutic agent (i.e., in the context of a linker-therapeutic agent portion of an ADC as described herein), the linker promotes cellular internalization. In yet other embodiments, when coupled to both an ALK5 inhibitor and an antibody, the linker promotes cellular internalization.

[0130] In many embodiments, the joint is suicide-type. As used herein, the term "suicide-type" refers to a difunctional chemical part that can covalently link two spaced chemical parts together to form a stable trimolecular part. If the key between it and the first part is cleaved, the joint will spontaneously separate from the second chemical part. See, for example, PCT Publication Nos. 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 relate to drug-cleavable substrate conjugates, wherein the drug and cleavable substrate are optionally connected by a suicide-type joint, and are all clearly incorporated by reference. An example of a suicide Spacer unit that can be used to generate a suicide linker is depicted in Formula I below.

[0131] Various exemplary linkers that can be used with the compositions and methods of the invention are described in PCT Publication No. WO 2004 / 010957, U.S. Patent Publication No. US2006 / 0074008, U.S. Patent Publication No. 2005 / 0238649, and U.S. Patent Publication No. US2006 / 0024317 (each of which is incorporated herein by reference in its entirety for all purposes).

[0132] The ADC of the present disclosure may have the following Formula I, wherein an antibody (Ab) is coupled to one or more ALK5 inhibitor drug moieties (D) via an optional linker (L).

[0133] Ab-(LD)p I

[0134] Thus, the antibody can be coupled to the drug directly or through a linker. In Formula I, p is the average number of drug (i.e., ALK5 inhibitor) moieties per antibody, which can range, for example, from about 1 to about 20 drug moieties per antibody, and in certain embodiments, from 2 to about 8 drug moieties per antibody. Additional details of drug loading are described in Section 4.5 below.

[0135] In some embodiments, the linker component may include an "extension" that connects the antibody to another linker component or a drug moiety, for example, via a cysteine ​​residue. Exemplary extensions are shown below (where the left wavy line indicates the site of covalent attachment to the antibody, and the right wavy line indicates the site of covalent attachment to another linker component or a drug moiety):

[0136]

[0137] See, US Patent No. 9,109,035; Ducry et al., 2010, Bioconjugate Chem. 21:5-13.

[0138] In some embodiments, the linker component may include an amino acid unit. In one such embodiment, the amino acid unit allows protease cleavage of the linker, thereby promoting the release of the drug from the ADC when exposed 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-glycine (gly-gly-gly). The amino acid unit may include naturally occurring amino acid residues as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid units can be designed and optimized for selectivity for enzymatic cleavage by specific enzymes, for example, cathepsins B, C and D or plasmin proteases.

[0139] In some embodiments, the linker component may include a "spacer" unit that connects the antibody to the drug portion directly or through a stretcher and / or an amino acid unit. The spacer unit may be "suicide" or "non-suicide". A "non-suicide" spacer unit is a spacer unit in which some or all of the spacer units remain bound to the drug portion after enzymatic (e.g., proteolytic) cleavage of the ADC. Examples of non-suicide spacers include, but are not limited to, glycine spacers and glycine-glycine spacers. A "suicide" spacer unit allows the release of the drug portion without a separate hydrolysis step. In certain embodiments, the spacer unit of the linker includes a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is formed between the benzyl alcohol and the cytotoxic agent. See, e.g., Hamann et al., 2005, E×pert Opin. Ther. Patents 15: 1087-1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments, the phenylene portion of the p-aminobenzyl unit is replaced by Q m Substituted, wherein Q is --C1-C8 alkyl, --O--(C1-C8 alkyl), -halogen, -nitro or -cyano; and m is an integer from 0 to 4. Examples of suicide spacer units also include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, e.g., U.S. Patent Publication No. US2005 / 0256030), such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetal. Spacers that undergo cyclization after amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, Amer. Chem. Soc. 94:5815); and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a suicide spacer useful in ADCs.

[0140] In one embodiment, the Spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit as described below, which can be used to incorporate and release a variety of drugs.

[0141]

[0142] wherein Ab and D are as defined above for Formula I; A is a stretch, a is an integer from 0 to 1; W is an amino acid unit, and 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; and p ranges from 1 to about 20.

[0143] The linker can comprise any one or more of the above-mentioned linker components. In certain embodiments, the linker is as shown in the brackets of the following ADC formula:

[0144] Ab–(–[Aa-Ww-Yy]-D) p II

[0145] wherein Ab, A, a, W, w, D and p are as defined in the preceding paragraph; Y is a spacer unit, y is 0, 1 or 2; and Exemplary embodiments of such linkers are described in U.S. Patent Publication No. 2005 / 0238649A1, which is incorporated herein by reference.

[0146] Exemplary linker components and combinations thereof are shown below in the context of an ADC of Formula II:

[0147]

[0148] Linker components, including stretchers, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US Patent Publication No. 2005 / 0238649.

[0149] 4.5 Drug loading

[0150] The drug loading is represented by p and is the average number of ALK5 inhibitor moieties per antibody in the molecule. The 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 moieties (D) per antibody, although typically the average number is a fraction or decimal. Typically, the loading of the ALK5 inhibitor averages 2 to 8 drug moieties per antibody, more preferably 2 to 4 drug moieties per antibody or 5 to 7 drug moieties per antibody.

[0151] As will be appreciated by those skilled in the art, in many cases, reference to an ADC is shorthand for a population or collection of ADC molecules (sometimes in the context of a pharmaceutical composition), each molecule being comprised of an antibody covalently attached to one or more ALK5 inhibitor moieties, and the drug loading ratio represents the average drug loading in the population or collection, although the ratio based on individual molecules in the population may differ from one ADC molecule to another. In some embodiments, the population or collection comprises ADC molecules comprising antibodies covalently attached to anywhere from 1 to 30 drug moieties, in some embodiments, anywhere between 1 to 20, between 1 to 15, between 2 to 12, or between 2 to 8 drug moieties. Preferably, the average value in the population is as described in the preceding paragraph, e.g., 2 to 8 drug moieties per antibody, more preferably 4 to 8 drug moieties per antibody or 5 to 7 drug moieties per antibody.

[0152] Some ADC populations may be in the form of compositions comprising an ADC described herein and an antibody molecule lacking a drug moiety, such as an antibody to which attachment of an ALK5 antibody was unsuccessful.

[0153] The average amount of ALK5 inhibitor moiety per antibody in the ADC formulation from the conjugation reaction can be characterized by conventional methods such as mass spectrometry and ELISA assays.

[0154] The quantitative distribution of the ADC, represented by p, can also be determined. In some cases, the separation, purification and characterization of homogeneous ADC can be obtained, for example, by electrophoresis, where p is a certain value of the ADC loaded with other ALK5 inhibitors.

[0155] For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, in the case where the attachment is cysteine ​​thiol, as in the above exemplary embodiments, the antibody may have only one or several cysteine ​​thiol groups, or may have only one or several sufficiently reactive thiol groups, which can be attached to the joint by them. In certain embodiments, a higher drug loading, such as p>5, may cause the aggregation, insolubility, toxicity or cell permeability loss of certain antibody-drug conjugates. In certain embodiments, the drug loading of the ADC of the present disclosure is 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 3.7. In fact, it has been shown that for some ADCs, the optimal ratio of the drug portion of each antibody can be less than 8, and can be about 2 to about 5. See US Patent Publication No. US2005 / 0238649 (incorporated herein by reference in its entirety).

[0156] In certain embodiments, during the coupling reaction, less than the theoretical maximum drug moiety is coupled to the antibody. The antibody may include, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Typically, antibodies do not include many free and reactive cysteine ​​thiol groups that may be connected to the drug moiety; in fact, most cysteine ​​thiol residues in antibodies exist in the form of disulfide bridges. In certain embodiments, antibodies may be reduced under partial or total reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0157] The loading (drug / antibody ratio) of the ADC can be controlled in various ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the coupling reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine ​​thiol modification, (iv) genetically engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug attachments (e.g., thioMab or thioFab prepared as disclosed in PCT Publication No. WO 2006 / 034488 (incorporated herein by reference in its entirety)).

[0158] It will be appreciated that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent and then reacts with a drug moiety reagent, then the resulting product is a mixture of ADC compounds having one or more distributions of drug moieties attached to the antibody. The average amount of drug per antibody can be calculated from the mixture by a dual ELISA antibody assay that is specific for the antibody and specific for the drug. Individual ADC molecules in the mixture can be identified by mass spectrometry and separated by HPLC (e.g., hydrophobic interaction chromatography).

[0159] In some embodiments, a homogeneous ADC having a single loading value can be separated from the conjugation mixture by electrophoresis or chromatography.

[0160] 4.6 Formulation and Administration

[0161] Suitable routes of administration for ADC include, but are not limited to, oral, parenteral, rectal, transmucosal, enteral administration, 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 a solid tumor or a hematological tumor.

[0162] The immunoconjugate can be formulated according to known methods to prepare a pharmaceutically useful composition, whereby the ADC is combined with a pharmaceutically suitable excipient in a mixture. Sterile phosphate buffered saline is an example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those skilled in the art. See, for example, Ansel et al., Pharmaceutical Dosage Forms And Drug Delivery Systems, 5th Edition (Lea & Febiger 1990) and Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990) and revised editions.

[0163] In a preferred embodiment, the ADC is formulated in Good's biological buffer (pH 6-7) using a buffer 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) [Pipes]. A more preferred buffer is MES or MOPS, preferably at a concentration of 20 to 100 mM, more preferably about 25 mM. Most preferably, it is 25 mM MES, pH 6.5. The formulation may also contain 25 mM trehalose and 0.01% v / v polysorbate 80 as excipients, with the final buffer concentration being changed to 22.25 mM due to the added excipients. The preferred storage method is as a lyophilized formulation of the conjugate, stored at a temperature of -20°C to 2°C, most preferably stored at 2°C to 8°C.

[0164] ADC can be formulated for intravenous administration by, for example, bolus injection, slow perfusion or continuous perfusion. Preferably, ADC is perfused in less than about 4 hours, and more preferably in less than about 3 hours. For example, the initial 25-50 mg can be perfused in 30 min, preferably even in 15 min, and the remaining in the next 2-3 hours. Injectable preparations can exist in unit dosage form, for example in ampoules or multi-dose containers, with preservatives added thereto. The composition can take the form of a suspension, solution or emulsion, for example, in an oily or aqueous vehicle, and can include a preparaton, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the active ingredient can be in powder form, to be constituted with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0165] Other pharmaceutical methods can be used to control the duration of ADC action. Controlled release formulations can be prepared by using polymer complexes or adsorption of ADC. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of polyanhydride copolymers of stearic acid dimer and sebacic acid Sherwood et al., 1992, Bio / Technology 10:1446. The rate of ADC release from this matrix depends on the molecular weight of ADC, the amount of ADC in the matrix, and the size of the dispersed particles. Saltzman et al., 1989, Biophys. J. 55:163; Sherwood et al., supra. Other solid dosage forms are described in Ansel et al., Pharmaceutical Dosage Forms And Drug Delivery Systems, 5th edition (Lea &

[0166] Febiger 1990) and Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company 1990), and revised editions.

[0167] In general, the dosage of ADC administered to humans will vary depending on factors such as the patient's age, weight, height, sex, general medical condition, and prior medical history. It may be desirable to provide the recipient with a dosage of ADC ranging from about 0.3 mg / kg to 5 mg / kg as a single intravenous infusion, although lower or higher doses may be administered as the circumstances require. For a 70 kg patient, for example, a dosage of 0.3-5 mg / kg would be 21-350 mg, or for a 1.7 m patient would be 12-20 6The dosage can be repeated as needed, for example, once a week for 2-10 weeks, once a week for 8 weeks, or once a week for 4 weeks. According to the needs of maintenance therapy, the frequency can also be reduced, for example, once every other week for several months, or once a month or once a quarter for multiple months. The preferred dosage can include but is not limited to 0.3mg / kg, 0.5mg / kg, 0.7mg / kg, 1.0mg / kg, 1.2mg / kg, 1.5mg / kg, 2.0mg / kg, 2.5mg / kg, 3.0mg / kg, 3.5mg / kg, 4.0mg / kg, 4.5mg / kg and 5.0mg / kg. For weekly use, a more preferred dosage is 0.6mg / kg, and for less frequent administration, it is 1.2mg / kg. Any amount of 0.3 to 5mg / kg can be used. The dosage is preferably administered multiple times once a week. A minimum dosage regimen of 4 weeks, more preferably 8 weeks, more preferably 16 weeks or longer may be used, with the dosage frequency depending on toxic side effects and recovery therefrom, which is primarily related to hematotoxicity. The administration regimen may include administration once or twice a week, with a cycle selected from: (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) monthly. The cycle may be repeated 2, 4, 6, 8, 10 or 12 times or more.

[0168] Alternatively, the ADC can be administered in a dosage form every 2 or 3 weeks, repeated for a total of at least 3 doses. Alternatively, twice a week for 4-6 weeks. The dosage can be once every other week, or even less frequently, so that the patient can recover from any drug-related toxicity. Alternatively, the dosage regimen can be reduced, i.e., every 2 or 3 weeks for 2-3 months. The dosage regimen can optionally be repeated at other intervals, and the dosage can be given by various parenteral routes, with appropriate adjustments to the dosage and regimen.

[0169] 4.7 Treatment

[0170] The ADC of the present disclosure can be used to treat various cancers. The ADC can be used as a monotherapy or as part of a combination therapy regimen, for example, with a standard of care agent or regimen. Suitable antibodies included in the ADC for treating cancer are those that target T cell surface antigens. Exemplary antibodies are described in Section 4.2.

[0171] Examples of cancers that can be treated using the ADCs of the present disclosure include, but are not limited to, pancreatic cancer, glioblastoma, myelodysplastic syndrome, prostate cancer, liver cancer (e.g., hepatocellular carcinoma), melanoma, breast cancer, and urothelial cancer (e.g., bladder cancer, urethral cancer, and ureteral cancer).

[0172] For the treatment of melanoma harboring BRAF mutations, the ADCs of the present disclosure can be used in combination with drugs that specifically target BRAF mutations (e.g., vemurafenib, dapafenib, and trametinib).

[0173] For the treatment of malignant melanoma, the ADC of the present disclosure may be used in combination with a checkpoint inhibitor (e.g., ipilimumab or nivolumab or pembrolizumab).

[0174] For the treatment of non-small cell lung cancer (NSCLC), the ADC of the present disclosure can be used in combination with standard care chemotherapy (e.g., cisplatin, carboplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, etoposide or vinblastine included). In addition, ADC can be used in combination with targeted therapy (e.g., bevacizumab or erbitux).

[0175] For the treatment of bladder cancer, the ADCs of the present disclosure may be used in combination with standard of care treatments, including but not limited to cisplatin, mitomycin-C, carboplatin, docetaxel, paclitaxel, doxorubicin, 5-FU, methotrexate, vinblastine, ifosfamide, and pemetrexed.

[0176] For the treatment of renal cancer, the ADCs of the present disclosure can be used in combination with standard of care treatments, such as agents that block angiogenesis and / or specific tyrosine kinases, such as sorafenib, sunitinib, temsirolimus, everolimus, pazopanib, and axitinib.

[0177] For the treatment of breast cancer, the ADC of the present disclosure can be used in combination with standard care chemotherapeutics, such as anthracyclines (doxorubicin or epirubicin) and taxanes (paclitaxel or docetaxel) as well as fluorouracil, cyclophosphamide and carboplatin. In addition, the ADC of the present disclosure 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.

[0178] For pancreatic cancer, the ADC of the present disclosure can be used in combination with standard care chemotherapeutic agents (e.g., gemcitabine, 5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin or docetaxel). In addition, ADC can be used in combination with targeted therapies that inhibit EGFR (e.g., erlotinib).

[0179] For glioblastoma, the ADCs of the present disclosure may be used in combination with standard of care chemotherapeutic agents such as carboplatin, cyclophosphamide, etoposide, lomustine, methotrexate, or procarbazine.

[0180] For prostate cancer, the ADCs of the present disclosure may be used in combination with standard of care chemotherapeutics including docetaxel, optionally with the steroids prednisone or cabazitaxel.

[0181] 5. Examples

[0182] The following abbreviations are used throughout the examples:

[0183] Boc – tert-Butyloxycarbonyl

[0184] DCM – dichloromethane

[0185] DMA-Dimethylamine

[0186] DMF – Dimethylformamide

[0187] DIPEA – N,N-Diisopropylethylamine

[0188] EtOAc – Ethyl acetate

[0189] EtOH – ethanol

[0190] Fmoc – fluorenylmethoxycarbonyl

[0191] HOBt – Hydroxybenzotriazole

[0192] MeOH – methanol

[0193] NaHMDS – Sodium hexamethyldisilazide

[0194] RT – Room temperature, about 21°C

[0195] TBTU – O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

[0196] TEA – Triethylamine

[0197] THF – Tetrahydrofuran

[0198] TFA – trifluoroacetic acid

[0199] TMS – imidazole-1-(trimethylsilyl)imidazole

[0200] 5.1 Example 1: Synthesis of 4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-amine (Compound A)

[0201] Compound A was prepared according to the general method in Scheme 1 below:

[0202]

[0203] Solution 1

[0204] 5.1.1 2-Methyl-1,5-naphthyridine (A1)

[0205] A mixture of concentrated sulfuric acid (2.5ml), sodium m-nitrobenzenesulfonate (2.08g, 9.24mmol), boric acid (445mg, 7.21mmol) and ferrous sulfate heptahydrate (167mg, 0.60mmol) was stirred at room temperature. Glycerol (1.5ml) was added to the reaction mixture, followed by 5-amino-2-methylpyridine (A-SM) (500mg, 4.62mmol) and water (2.5ml), and heated at 135°C for 18h. After the reaction was completed by TLC measurement, the reaction mixture was cooled to about 21°C, alkalized with 4N NaOH, and extracted with EtOAc (2×100ml). The organic extracts were combined, washed with water (200ml), dried over Na2SO4 and evaporated under reduced pressure to obtain crude compound A1. The crude product was purified by silica gel column chromatography using (2% MeOH / CH2Cl2) to obtain compound A1 (200mg, 30%) as a light brown crystalline solid.

[0206] 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)

[0207] LC-MS (ESI): m / z 145 [M+H] +

[0208] 5.1.2 1-(6-methylpyridin-2-yl)-2-(1,5-naphthyridin-2-yl)ethan-1-one (A2)

[0209] A solution of A1 (200 mg, 1.38 mmol) and 6-methylpicolinic acid methyl ester (209 mg, 1.38 mmol) in anhydrous THF (10 ml) is placed under N2 atmosphere and cooled to -78 ° C. Potassium bis (trimethylsilyl) amide (0.5 M in toluene, 6.9 ml, 3.47 mmol) is added dropwise in 5 min. The reaction mixture is stirred for 1 h at -78 ° C, and then warmed to about 21 ° C and kept for 20 h. After the reaction is completed (measured by TLC), the reaction mixture is quenched with saturated ammonium chloride solution (20 ml). The aqueous layer is extracted with EtOAc (2 × 20 ml). The combined organic extracts are washed with water (100 ml), dried over Na2SO4 and evaporated to obtain crude compound A2. The crude material is purified by column chromatography (1% MeOH / CH2Cl2) to obtain compound A2 (110 mg, 30.5%) as an orange-yellow solid.

[0210] 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)

[0211] LC-MS (ESI): m / z 264 [M+H] +

[0212] 5.1.3 4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1,3-thiazol-2-amine (Compound A)

[0213] 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, and then concentrated under reduced pressure to obtain crude A3 (120 mg), which was used in 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 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 (2×20 ml) and washed with water (50.0 ml). The separated organic layer was then washed with 1N HCl (30 ml×2). The combined aqueous layers were basified with 35% aqueous sodium hydroxide solution (20 ml) and extracted with CH2Cl2 (2××20 ml). The organic layer was dried over sodium sulfate and evaporated to give crude compound A. Crude compound A was recrystallized from acetonitrile (2 ml) to obtain purified compound A (35 mg, 49% yield over 2 steps) as a yellow crystalline solid.

[0214] 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)

[0215] LC-MS (ESI): m / z 320 [M+H] +

[0216] UPLC purity: 97.6%

[0217] 5.2 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)

[0218] Compound B was prepared according to the general method in Scheme 2 below:

[0219]

[0220] Solution 2

[0221] 5.2.1 tert-Butyl (2-chloroethyl) (methyl) carbamate (B7)

[0222] In the time of 1h, a solution of B6 (1g, 7.69mmol) in water (4ml) and a solution of TEA (1ml, 7.69mmol) in THF (4ml) were added simultaneously to a stirred solution of Boc anhydride (1.7ml, 7.30mmol) in THF (4ml). The resulting mixture was stirred for 16h at about 21°C. The reaction mixture was diluted with a saturated NaCl solution (20ml) and extracted with DCM (3×50ml). The combined organic extracts were dried over Na2SO4 and concentrated in vacuo to obtain a crude compound, which was purified by silica gel column chromatography using 10% EtOAc / hexane to obtain compound B7 (1g, 5.18mmol, 71%) in the form of a light yellow liquid.

[0223] 1 H NMR (400MHz, CDCl3): δ3.58-3.52(m,4H),2.93(s,3H),1.46(s,9H)

[0224] 5.2.2 Tert-butyl methyl (2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)carbamate (Intermediate-B)

[0225] To a stirred solution of 4-hydroxyphenylboronic acid pinacol ester (789 mg, 3.58 mmol) in DMF (13 ml) under argon atmosphere was added B7 (900 mg, 4.66 mmol), KI (18 mg, 0.10 mmol) and CsCO (2.57 g, 7.88 mmol). The reaction mixture was heated to 65 ° C and stirred for 16 h. 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 / hexanes to obtain intermediate-B (580 mg, 1.53 mmol, 43%) as a light yellow solid.

[0226] 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)

[0227] 5.2.3 2-(2-Bromopyridin-4-yl)-1-(pyridin-2-yl)ethan-1-one (B2)

[0228] To a stirred solution of 2-bromo-4-methylpyridine (B1) (2 g, 11.62 mmol) in THF (30 ml) was added a solution of NaHMDS (2 M in THF, 12.7 ml, 25.58 mmol) dropwise under argon at -78 ° C. The yellow solution was stirred for 30 min at -78 ° C. A solution of ethyl picolinate (1.72 ml, 12.79 mmol) in THF (10 ml) was then added, the reaction mixture was heated to about 21 ° C and stirred for 16 h. The solvent was evaporated under reduced pressure, and the solid residue was ground with diethyl ether, filtered and washed with diethyl ether. The solid was then diluted with saturated NH4Cl solution (30 ml), and the aqueous phase was extracted with EtOAc (2×200 ml). The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography using 10% EtOAc / hexanes to obtain Compound B2 (2.06 g, 7.46 mmol, 64.3%) as a yellow solid.

[0229] 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)

[0230] LC-MS(ESI): m / z 277[M] +

[0231] 5.2.4 2-Bromo-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine (B3)

[0232] 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. DMA (0.6 ml, 4.61 mmol) was added dropwise, and the mixture was stirred for 2 h at about 21 ° C under an argon atmosphere. Hydrazine monohydrate (1.15 ml, 23.09 mmol) was added dropwise, and the resulting mixture was heated at 50 ° C for 3 h, and at about 21 ° C for 16 h. 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 obtain a crude compound. The crude product was purified by silica gel column chromatography using 30% EtOAc / hexane to obtain compound B3 (560 mg, 1.86 mmol, 60.6%) as a yellow solid.

[0233] 1 H NMR (500MHz, 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)

[0234] LC-MS(ESI): m / z 301[M] +

[0235] 5.2.5 2-Bromo-4-(3-(pyridin-2-yl)-1-trityl-1H-pyrazol-4-yl)pyridine (B4)

[0236] To a stirred solution of B3 (500 mg, 1.66 mmol) in acetone (10 ml) was added KCO (1.37 g, 9.99 mmol) and trityl chloride (464 mg, 2.49 mmol). The reaction mixture was then heated to reflux and stirred for 24 h. The reaction mixture was filtered and the filtrate was concentrated, then distributed between CHCl (20 ml) and water (10 ml). The organic phase was dried over NaSO and concentrated. The crude solid was purified by silica gel column chromatography using 2% MeOH / CHCl to obtain compound B4 (402 mg, 0.74 mmol, 44%) as a light yellow solid.

[0237] 1H 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)

[0238] 5.2.6 tert-Butyl methyl (2-(4-(4-(3-(pyridin-2-yl)-1-trityl-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)carbamate (B5)

[0239] To a stirred solution of B4 (100 mg, 0.18 mmol) in toluene (2 ml) under an argon atmosphere was added a solution of intermediate-B (185 mg, 0.49 mmol) in EtOH (0.75 ml), followed by 2M 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 complete consumption of the starting material (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 obtain a crude product, which was purified by silica gel column chromatography using 30% EtOAc / hexane to obtain compound B5 (70 mg, 0.09 mmol, 53%) as a colorless solid.

[0240] 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)

[0241] 5.2.7 N-methyl-2-(4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethan-1-amine hydrochloride (Compound B)

[0242] To a stirred solution of B5 (70 mg, 0.09 mmol) in CH2Cl2 (6 ml) was added 4N HCl in 1,4-dioxane (0.5 ml) at 0°C. The reaction mixture was stirred under argon atmosphere for 1 h. After complete consumption of the starting material (monitored by TLC), the solvent was evaporated under reduced pressure to obtain the crude compound, which was triturated with n-pentane (2×1 ml) and dried to obtain compound B HCl salt (25 mg, 0.06 mmol, 69%) as a colorless solid.

[0243] 1 H 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)

[0244] LC-MS (ESI): m / z 372 [M+H] +

[0245] 5.3 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)

[0246] Compound C was prepared according to the general method in Scheme 3 below:

[0247]

[0248] Solution 3

[0249] 5.3.1 2-(2-Bromopyridin-4-yl)-1-(6-methylpyridin-2-yl)ethan-1-one (C2)

[0250] To a stirred solution of 2-bromo-4-methylpyridine (B1) (1 g, 5.81 mmol) in THF (15 ml) was added a solution of NaHMDS (2 M in THF, 6.39 ml, 12.8 mmol) dropwise under argon at -78 °C. The yellow solution was stirred for 30 min at -78 °C. A solution of methyl 6-methylpicolinate (1.19 ml, 8.72 mmol) in THF (7 ml) was then added, and the reaction mixture was warmed to about 21 °C and stirred for 16 h. The solvent was evaporated under reduced pressure, and the solid residue was ground with diethyl ether, filtered and washed with diethyl ether. The solid was then diluted with a saturated NH4Cl solution (20 ml), 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 / hexanes to obtain Compound C2 (1.1 g, 3.79 mmol, 65.4%) as a yellow solid.

[0251] 1 H 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)

[0252] LC-MS(ESI): m / z 291[M] +

[0253] 5.3.2 2-Bromo-4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]pyridine (C3)

[0254] A solution of C2 (300 mg, 1.03 mmol) in dry DMF (1 ml) was treated with glacial acetic acid (0.14 ml, 2.48 mmol) in DMF under argon. DMA (0.2 ml, 1.55 mmol) was added dropwise, and the mixture was stirred for 1 h at about 21 ° C under an argon atmosphere. Hydrazine monohydrate (0.37 ml, 7.75 mmol) was added dropwise, and the resulting mixture was heated at 50 ° C for 3 h and at about 21 ° C for 16 h. 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 obtain crude C3. Crude C3 was purified by silica gel column chromatography using 2% MeOH / DCM to obtain purified C3 (172 mg, 0.54 mmol, 53%) as a yellow solid.

[0255] 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)

[0256] LC-MS (ESI): m / z 315 [M+H] +

[0257] 5.3.3 2-Bromo-4-(3-(6-methylpyridin-2-yl)-1-trityl-1H-pyrazol-4-yl)pyridine (C4)

[0258] To a stirred solution of C3 (40 mg, 0.12 mmol) in acetone (2 ml) was added KCO (53 mg, 0.38 mmol) and trityl chloride (53 mg, 0.19 mmol). The reaction mixture was then heated to reflux and stirred for 24 h. The reaction mixture was filtered and the filtrate was concentrated and then partitioned between CHCl (5 ml) and water (5 ml). The organic phase was dried over NaSO and concentrated. The crude solid was purified by silica gel column chromatography using 2% MeOH / CHCl to obtain compound C4 (30 mg, 0.05 mmol, 41%) as a light yellow solid.

[0259] 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,

[0260] 1H),2.53(s,3H)

[0261] LC-MS (ESI): m / z 558 [M+H] +

[0262] 5.3.4 tert-Butyl methyl (2-(4-(4-(3-(6-methylpyridin-2-yl)-1-trityl-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)carbamate (C5)

[0263] To a stirred solution of C4 (150 mg, 0.26 mmol) in toluene (5 ml) under argon atmosphere was added a solution of intermediate-B (152 mg, 0.40 mmol) in EtOH (1 ml), followed by 2M 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 complete consumption of the starting material (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 obtain crude C5, which was purified by silica gel column chromatography using 30% EtOAc / hexane to obtain purified C5 (51 mg, 0.07 mmol, 26%) as a brown solid.

[0264] 1 H 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)

[0265] 5.3.5 N-methyl-2-(4-4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}phenoxy)ethan-1-amine (Compound C)

[0266] To a stirred solution of C5 (51 mg, 0.07 mmol) in CH2Cl2 (5 ml) was added 4N HCl in 1,4-dioxane (0.3 ml) at 0°C. The reaction mixture was then stirred under an argon atmosphere for 1 h. After complete consumption of the starting material (monitored by TLC), the solvent was evaporated under reduced pressure to obtain crude compound C. The crude compound C was then triturated with n-pentane (2×1 ml) and dried to obtain the HCl salt of compound C (20 mg, 0.05 mmol, 74%) as a brown solid.

[0267] 1 H NMR (400MHz, DMSO-d6): δ8.93 (brs, 2H), 8.61 (d, J = 5.6Hz,

[0268] 1H),8.56(brs,1H),8.33(brs,1H),8.03(d,J=8.8Hz,2H),7.88(t,J=7.6Hz,

[0269] 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)

[0270] LC-MS (ESI): m / z 386 [M+H] +

[0271] 5.4 Example 4: Synthesis of (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonylamino)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (Compound D)

[0272] Compound D was prepared according to the general method in Scheme 4 below:

[0273]

[0274] Solution 4

[0275] 5.4.1 Methyl 1-acetyl-2-oxoindoline-6-carboxylate (D2)

[0276] A stirred solution of methyl 2-oxoindoline-6-carboxylate (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 complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to about 21 ° C. The precipitate was filtered, washed with n-hexane (2×50 ml), and dried in vacuo to obtain compound D2 (1.5 g, 61.5%) as a yellow solid.

[0277] 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)

[0278] 5.4.2(Z)-1-Acetyl-3-(hydroxy(phenyl)methylene)-2-oxoindoline-6-carboxylic acid methyl ester (D3)

[0279] Under an inert atmosphere at 0 ° C, TBTU (2.69g, 8.36mmol), benzoic acid (903mg, 7.40mmol) and triethylamine (2.2ml) were added to a stirred solution of compound D2 (1.5g, 6.43mmol) in DMF (10ml). The reaction mixture was warmed to about 21 ° C and stirred for 16h. After the starting material was completely consumed (monitored by TLC), the reaction mixture was quenched with ice-cold water (30ml) and extracted with EtOAc (2×40ml). The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to obtain a crude product D3, which was purified by silica gel column chromatography using 80% EtOAc / hexanes to obtain compound D3 (900mg, 42%) as a yellow solid.

[0280] 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)

[0281] LC-MS (ESI): m / z 338.3 [M+H] +

[0282] 5.4.3 (Z)-3-(Hydroxy(phenyl)methylene)-2-oxoindoline-6-carboxylic acid (D4)

[0283] At about 21 ° C, 1N NaOH solution (15ml) was added to a stirred solution of compound D3 (900mg, 2.67mmol) in MeOH (15ml). The mixture was heated to 100 ° C and stirred for 6h. After the complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to about 21 ° C, quenched with 1N HCl aqueous solution (13ml) and stirred for 30min. The precipitated solid was filtered and washed with 20% EtOAc / hexanes to obtain compound D4 (580mg, 77%) as an off-white solid, which can be used in the next step without further purification.

[0284] 1 H NMR (400MHz, DMSO-d6): δ12.76(brs,1H),11.61(brs,1H),7.77-7.50(m,8H),7.13(brs,1H)

[0285] 5.4.4 (Z)-N-ethyl-3-(hydroxy(phenyl)methylene)-2-oxoindoline-6-carboxamide (fragment A)

[0286] Under an inert atmosphere at about 21 ° C, 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) under an inert atmosphere. After 30 min, 2N ethylamine in THF (2.1 ml, 4.12 mmol) was added at 0 ° C and stirred for 1 h. The reaction mixture was then warmed to about 21 ° C and stirred for another 16 h. After complete consumption of the starting material (monitored by TLC), the volatiles were removed in vacuo. The residue was diluted with water (15 ml), filtered and washed with 20% EtOAc / hexane (2×10 ml) to obtain a crude product, which was purified by silica gel column chromatography using 10% MeOH / CH2Cl2 to obtain fragment A (410 mg, 64.5%) as an off-white solid.

[0287] 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)

[0288] LC-MS (ESI): m / z 307.1 (MH + )

[0289] 5.4.5 N-(2-(Dimethylamino)ethyl)-N-(4-nitrophenyl)methanesulfonamide (D8)

[0290] To a stirred solution of compound D7 (800 mg, 3.70 mmol) in acetone (15 ml) was added potassium carbonate (1.32 g, 9.62 mmol), sodium iodide (110 mg, 0.74 mmol) and compound B6 (799 mg, 5.55 mmol) at 0 ° C under an inert atmosphere. The reaction mixture was heated to 50 ° C and stirred for 20 h. After complete consumption of the starting material (monitored by TLC), the volatiles were removed in vacuo. 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 in vacuo to obtain a crude product, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 to obtain compound D8 (460 mg, 43%) as a light yellow solid.

[0291] 1H 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)

[0292] LC-MS (ESI): m / z 288.3 [M+H] +

[0293] 5.4.6 N-(4-aminophenyl)-N-(2-(dimethylamino)ethyl)methanesulfonamide (fragment B)

[0294] To a stirred solution of compound D8 (460 mg, 1.60 mmol) in MeOH (10 ml) was added 10% Pd / C (40 mg) and stirred at about 21° C. for 3 h under hydrogen atmosphere (balloon pressure). After complete consumption of the starting material (monitored by TLC), the reaction mixture was washed with water. The mixture was filtered through a pad of Celite® and washed with MeOH (10 ml). The filtrate was concentrated in vacuo to afford the crude product, which was purified by silica gel column chromatography using 10% MeOH / CH 2 Cl 2 to afford Fragment B (300 mg, 73%) as a light yellow solid.

[0295] 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)

[0296] LC-MS (ESI): m / z 258.2 [M+H] +

[0297] 5.4.7 (Z)-3-(((4-(N-(2-(dimethylamino)ethyl)methylsulfonylamino)phenyl)amino)(phenyl)methylene)-N-ethyl-2-oxoindoline-6-carboxamide (D5)

[0298] 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 h under microwave. After the starting material was consumed (monitored by TLC and LC-MS), the volatiles were removed in vacuo. The residue was diluted with water (10 ml) and extracted with EtOAc (3 × 25 ml) to obtain a crude product, which was purified by preparative HPLC to obtain compound D5 (150 mg, 42%) as a light yellow solid.

[0299] 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)

[0300] LC-MS (ESI): m / z 548.6 [M+H] +

[0301] 5.4.8 (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonylamino)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (Compound D)

[0302] 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 dry toluene (3 ml) at about 21 ° C under an inert atmosphere. The reaction mixture was heated to reflux temperature (120 ° C) and maintained for 16 h. After the starting material was consumed (monitored by TLC), the reaction mixture was cooled to about 21 ° C, diluted with EtOAc (30 ml), and washed with 1N HCl aqueous solution (15 ml). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to monodemethylate the compound (40 mg) protected with di-troc.

[0303] The crude product from the above reaction was dissolved in acetic acid (3ml), and zinc powder (9mg, 0.13mmol) was added under an inert atmosphere at about 21°C. The reaction mixture was heated to 50°C and stirred for 8h. After the complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to about 21°C, and volatiles were removed in vacuo. The residue was diluted with water (20ml) and extracted with EtOAc (2×25ml). The combined organic extracts were washed with saturated NaHCO solution (20ml), dried over NaSO, filtered and concentrated under reduced pressure to obtain crude compound D, which was purified by silica gel column chromatography using 5-6% MeOH / CHCl to obtain 12mg compound D, with HPLC purity of 83%.

[0304] The reaction was repeated on a 60 mg scale and the resulting crude product was combined with the above batch and purified by preparative HPLC to afford Compound D (8.0 mg, 6.3%) as a light yellow solid.

[0305] 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)

[0306] LC-MS (ESI): m / z 534.6 [M+H] +

[0307] UPLC purity: 99.18%

[0308] 5.5 Example 5: Alternative Synthesis of (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonylamino)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide (Compound D)

[0309] Compound D was also prepared according to the general method in Scheme 5 below:

[0310]

[0311] Solution 5

[0312] 5.5.1 N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfonamide (D9)

[0313] Under an inert atmosphere, 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) and stirred for 30 min at about 21 ° C. To this mixture, 1,2-dibromoethane (2.18 g, 11.60 mmol) was added at about 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 about 21 ° C, 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 in vacuo to obtain a crude product, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 to obtain 1.2 g of D9 mixture, which was a mixture containing 40% unreacted starting material. The resulting mixture was used directly in the next step without further purification.

[0314] 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)

[0315] 5.5.2 N-(2-(Methylamino)ethyl)-N-(4-nitrophenyl)methanesulfonamide (D10)

[0316] Under inert atmosphere at about 21 ° C, triethylamine (1.6 ml) and methylamine (2M in THF; 9.3 ml, 18.63 mmol) were added to a stirred solution of compound D9 (1.2 g, impure) in THF (10 ml) in a sealed tube. The reaction mixture was heated to 80 ° C and kept for 16 h. After the starting material was completely consumed (monitored by TLC), the reaction mixture was cooled to about 21 ° C and concentrated under reduced pressure to obtain crude D10. Crude D10 was purified by using silica gel column chromatography 15% MeOH / CH2Cl2 to obtain compound D10 (500 mg, two-step total yield 39%) as a yellow solid.

[0317] 1H NMR (500MHz, DMSO-d6): δ8.94(brs,1H),8.31(d,J=9.0Hz,2H),7.80(d,J=8.5 Hz,2H),4.06(t,J=6.0Hz,2H),3.15(s,3H),3.00(t,J=6.0Hz,2H),2.55(s,3H)

[0318] 5.5.3 tert-Butyl methyl (2-(N-(4-nitrophenyl)methylsulfonylamino)ethyl) carbamate (D11)

[0319] To a stirred solution of D10 (500 mg, 1.83 mmol) in CH2Cl2 (10 ml) was added triethylamine (0.4 ml, 2.61 mmol) and Boc-anhydride (659 mg, 3.02 mmol) under inert conditions at about 21° C. and maintained for 5 h. After complete consumption of the starting material (monitored by TLC), the volatiles were removed in vacuo to obtain the crude product, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 to obtain D11 (320 mg, 47%) as a colorless thick syrup.

[0320] 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)

[0321] LC-MS (ESI): m / z 274.2 (M + -B℃)

[0322] 5.5.4 tert-Butyl (2-(N-(4-aminophenyl)methylsulfonylamino)ethyl)(methyl)carbamate (Boc variant of fragment B)

[0323] To a solution of compound D11 (250 mg, 0.67 mmol) in EtOH (10 ml) was added Raney-Ni (40 mg) under hydrogen atmosphere (balloon pressure) at about 21°C and stirred for 1 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was washed with water and stirred for 1 h. The combined filtrates were concentrated in vacuo to afford the crude product, which was purified by silica gel column chromatography using 10% MeOH / CH 2 Cl 2 to afford the Boc-variant of fragment B (180 mg, 77%) as a light yellow solid.

[0324] 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)

[0325] LC-MS (ESI): m / z 244.2 (M + -B℃)

[0326] 5.5.5 tert-Butyl (Z)-(2-(N-(4-(((6-(ethylcarbamoyl)-2-oxoindolin-3-ylidene)(phenyl)methyl)amino)phenyl)methylsulfonylamino)ethyl)(methyl)carbamate (D10)

[0327] A solution of fragment A (70 mg, 0.22 mmol), 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 under microwave. After consumption of the starting material (monitored by TLC and LC-MS), the volatiles were removed in vacuo to obtain a residue, which was purified by preparative HPLC to obtain compound D10 (50 mg, 36%) as a light yellow solid.

[0328] 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)

[0329] LC-MS (ESI): m / z 634.6 [M+H] +

[0330] 5.5.6 (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonylamino)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxamide hydrochloride (HCl salt of Compound D)

[0331] To a stirred solution of compound D10 (20 mg, 0.03 mmol) in diethyl ether (3 ml) was added 4N HCl in 1,4-dioxane (0.3 ml) at 0°C under an inert atmosphere. The reaction mixture was stirred at about 21°C for 1 h. After complete consumption of the starting material (monitored by TLC), the volatiles were removed in vacuo to obtain a crude product, which was triturated with n-pentane (2×4 ml) to obtain compound D (12 mg, 71%) as a light yellow solid HCl salt.

[0332] 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).

[0333] LC-MS (ESI): m / z 534.7 [M+H] +

[0334] UPL purity: 96.26%

[0335] 5.6 Example 6: In vitro determination of compound AD test activity

[0336] 5.6.1 N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfonamide (2)

[0337] Compounds AD were tested to determine whether they could inhibit TGF-β-induced luciferase activity in HEK293T cells in vitro.

[0338] 30,000 HEK293T cells were seeded in a 96-well white flat-bottom plate overnight. The next day, 100 ng of SMAD luciferase reporter plasmid was transfected into the cells per well using lipofectamine for 24 h. The next day, cells were treated with compound AD and 100 pM TGFβ for 24 h. Luciferase activity was measured using a luciferase assay kit (Promega). The assay was performed twice for compounds A, B and D and three times for compound C. The results are shown in Table 4.

[0339]

[0340]

[0341] The activity data for Experiment 1 are shown in FIG5 .

[0342] Compounds AC showed the greatest inhibitory activity.

[0343] 5.6.2 MTS proliferation assay

[0344] Compounds AD were tested to determine whether they could be activated in primary mouse CD4 + Inhibits TGF-β signaling in T cells.

[0345] Using RoboSep TM Primary mouse CD4 T cells were isolated from the spleen of C57 / B6 mice using the Stemcell Technologies + T cells. 0.5 μg / ml hamster anti-mouse CD3e antibody (145-2C11; eBioscience) was coated on a 96-well flat-bottom plate overnight. 5 Purified CD4 + T cells were incubated with 1 μg / ml soluble hamster anti-mouse CD28 antibody (37.51, BD Biosciences), 1 nM TGF-β1 and 8-fold serial dilutions of compound AD. After 72 hours, cell proliferation was measured using MTS assay (Promega) according to the manufacturer's instructions. The results are shown in Table 5.

[0346]

[0347] The data from Experiment 1 are shown in FIG6 .

[0348] In two different experiments, no IC values ​​were obtained for compound D. 50 Compound A in mouse CD4 + No consistent effects were shown in T cells either. However, both compounds B and C reversed TGFβ-mediated inhibition of T cell proliferation.

[0349] Based on both assays, Compound C was selected for conjugation into ADC.

[0350] 5.7 Example 7: Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2H-pyrrol-1(5H)-yl)hexanoylamino)-3-methylbutanoylamino)-5-ureidopentanamido)benzylmethyl(2-(4-(4-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)carbamate

[0351] Compound C was linked to a valine-citrulline linker according to the general method in Scheme 6 below:

[0352]

[0353] Solution 6

[0354] At 0° C., L1 (122 mg, 0.165 mmol, 1.1 eq.) and TEA (52 μl, 0.375 mmol, 2.5 eq.) were added to a solution of compound C (58 mg, 0.150 mmol, 1.0 eq.) in DMF (2 ml), and the reaction mixture was stirred at about 21° C. for 2 h to obtain crude ADC-1. The crude ADC-1 was purified by preparative HPLC to obtain purified ADC-1 (34 mg, 24% yield) as a white solid.

[0355] 5.8 Example 8: Generation of Antibody Drug Conjugate 1 (ADC1)

[0356] Anti-mouse transferrin receptor antibody R17217 and rat anti-mouse IgG2A isotype control antibody (Bio×Cell) were dialyzed overnight in coupling buffer (25mM sodium borate / 25mM NaCl and 0.3mM EDTA, final pH 7.4). The antibodies were reduced for 2h using tris(2-carboxyethyl)phosphine (TCEP) at a reduction ratio of 10-30. ADC-1 was dissolved in DMSO to a final concentration of 10mM and then coupled to the antibody at a coupling 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 during the coupling step. The final ADC was dialyzed overnight in PBS, filtered using a 0.22μm filter, and analyzed by HPLC-HIC to determine the DAR and by HPLC-SEC to determine the aggregation level. For HPLC-HIC, samples were analyzed at The butyl-NPR column was run at a flow rate of 0.5 ml / min. Phase A was 25 mM sodium phosphate and 1.5 M ammonium sulfate at pH 6.95, while phase B was 75% 25 mM sodium phosphate and 25% isopropanol at pH 6.95. For HPLC-SEC analysis, a G3000SW column (Tosoh Bioscience), flow rate 0.25 ml / min at 280 nM, 25 min.

[0357] 5.9 Example 9: Synthesis of Compound C linked to a disulfide linker (ADC-2) Compound C was linked to a disulfide linker according to the general method in Schemes 7A-B below:

[0358]

[0359] Scheme 7A

[0360]

[0361] Option 7B

[0362]

[0363] Scheme 7B (continued)

[0364] 5.9.1 Synthesis of Intermediate A

[0365] 2-Chlorotrityl chloride resin (L2) (4 g, 4 mmol) was washed with DCM (2 x 40 ml), swelled in 50 ml DCM for 10 min, and then drained. Fmoc-Cys(Trt)-OH (L3) (7.03 g, 12 mmol) was dissolved in 40 ml DCM and added to the vessel containing 2-chlorotrityl chloride resin. 8.7 ml DIPEA (6.8 ml, 40 mmol) was added to the vessel, and the mixture was vortexed at about 21°C for 2 h. 10 ml methanol was then added to the mixture and vortexed for 30 min. The resulting resin (L4) was then drained and washed five times with DMF. Resin L4 was then deprotected by adding about 40 ml of a solution of 20% piperidine in DMF to resin L4 to provide resin L5, shaking the mixture, and then draining the liquid from the resin. An additional 40 ml of 20% piperidine in DMF was added to the resin and shaken for 15 min. Resin L5 was then drained and washed with DMF (6 x 40 ml).

[0366] The Fmoc-amino acid solution was prepared by separately 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).

[0367] Fmoc-Asp(OtBu)-OH solution was added to resin L5 and shaken for 60 min to provide 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.

[0368] In an exemplary synthesis, dried resin L10 (8 g) was added to a flask, and 80 ml of cleavage solution (TFA:TES:EDT:HO=90:5:3:2, v / v / v / v) was added. The reaction was allowed to proceed for 1.5 h. The resin was then separated from the reaction mixture by filter press. 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 provide 1.1 g of intermediate A (yield: 37%) as a white solid. LC-MS (ESI) m / z: 752 [M+H] +.

[0369] 5.9.2 2-(Pyridin-2-yldisulfanyl)ethylmethyl(2-(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)ethyl)carbamate (L12)

[0370] To a solution of compound C (40 mg, 0.1038 mmol) and 4-nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate (L11) (80 mg, 0.2272 mmol) in DMF (5 ml) was added DIPEA (0.5 ml) and HOBt (14 mg, 0.1038 mmol). The mixture was stirred at about 21 ° C. under N2 for 16 h to provide L12. The crude L12 was purified by preparative HPLC to give 35 mg of purified L12 (yield 56%) as a white solid.

[0371] 5.9.3 (2R,5S,8S,11S,14S,19S)-19-amino-5,8,14-tris(carboxymethyl)-11-(3-guanidinopropyl)-2-(((2-(methyl(2-(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)ethyl)carbamoyloxy)ethyl)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaeicosane-1,20-dioic acid (L13)

[0372] To a solution of L12 (35 mg, 0.058 mmol) in THF / H2O (5 ml / 5 ml) was added intermediate A (80 mg, 0.106 mmol) under N2. The mixture was stirred at about 21 °C for 16 h to provide L13. The crude L13 was purified by preparative HPLC to provide 23 mg of purified L13 (yield 31%) as a white solid.

[0373] 5.9.4 (2R,5S,8S,11S,14S,19S)-19-(2-(tert-butoxycarbonylaminooxy)acetylamino)-5,8,14-tris(carboxymethyl)-11-(3-guanidinopropyl)-2-(((2-(methyl(2-(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridine-2-1)phenoxy)ethyl)carbamoyloxy)ethyl)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaeicosane-1,20-dioic acid (L15)

[0374] To a solution of L13 (32 mg, 0.025 mmol) in DMF (3 ml) was added 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylaminooxy)acetate (L14) (28 mg, 0.097 mmol), followed by TEA (0.5 ml). The reaction mixture was stirred at about 21 ° C for 16 h under N2 atmosphere to provide L15. Crude L15 was purified by preparative HPLC to provide 12 mg of purified L15 (yield 33%) as a white solid.

[0375] 5.9.5 (2R, 5S, 8S, 11S, 14S, 19S)-19-(2-(aminooxy)amidoacetamido)-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)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaeicosane-1,20-dioic acid (ADC-2)

[0376] To a mixture of L15 (12 mg, 0.0085 mmol) in DCM (5 ml) was added TFA (1 ml). The mixture was stirred at about 21 °C for 30 min to provide ADC-2. The crude ADC-2 was concentrated and purified with preparative HPLC to provide 3.5 mg of purified ADC-2 (yield 31%) as a white solid.

[0377] 5.10 Example 10: Generation of Antibody Drug Conjugate 2 (ADC2)

[0378] ADC-2 was attached to the anti-TfR antibody via antibody lysine residues according to the general method in Scheme 8 below:

[0379]

[0380] Solution 8

[0381] The heterobifunctional linker S-4FB was purchased from Solulink. Rat anti-mouse IgG2a and anti-mouse transferrin receptor antibody R17217 were 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 about 21°C for 3h. The antibody solution modified with S-4FB was combined with a 2-hydrazinopyridine solution (0.5mM in 100mM MES buffer, pH 5.0) and incubated at 37°C for 30min at various coupling ratios of 5-50. The S4FB / Ab molar substitution ratio was determined by UV-Vis at A354. Zeba TMThe modified antibody was purified by spin desalting column, the buffer was exchanged to 50mM phosphate buffer (pH 6.5, 150mM NaCl), and then mixed with linker-SS-drug ADC-2 (10mM in DMSO) at different molar ratios at 37°C for 24h to provide ADC2. The next day, the ADC2 sample was dialyzed against PBS overnight. The sample was filtered and then tested by HPLC-SEC, SDS-PAGE and LC-MS. The exemplary LC-MS data of ADC2 prepared with S-4FB / Ab ratio of 6 and ADC-2 / Ab ratio of 20 are shown in Figure 7. Figure 7 shows that the average DAR of the tested ADC2 sample is 4.99, the DAR of the heavy chain is 1.97, and the DAR of the light chain is 0.53.

[0382] If aggregation of ADC2 was detected to be more than 5% by HPLC-SEC, the aggregated components were separated by AKTA with SEC column (GE Healthcare Life Sciences, Superde × 200 increase 10 / 300 GL) and analyzed again by HPLC-SEC. The chromatogram of ADC2 purified by SEC to remove aggregates is shown in Figure 8 middle.

[0383] 5.11 Example 11: Antibody-induced receptor internalization assay

[0384] 96-well flat-bottom plates were coated with anti-mouse CD3e antibody overnight at 4°C. TM CD4 T cells were isolated from mouse spleen using the Stemcell Technologies cell separation system. + T cells. At 37 degrees, about 2 × 10 per well 5 The cells were plated with soluble anti-CD28 antibody for 24-48 hours. After activation, CD4 + T cells were washed and re-plated with 5 μg / ml (anti-transferrin receptor) primary antibody at 37 degrees at the indicated time points to induce internalization. The reaction was terminated with ice-cold staining buffer and kept on ice to terminate internalization. At the end of the assay, the cells were washed twice in ice-cold staining buffer to remove unbound antibodies. The cells were pelleted and then stained with goat anti-rat secondary antibody conjugated with PE and incubated on ice for 30 minutes. The cells were washed with staining buffer and then analyzed for expression by FACS. As shown in Figure 9, TfR expression began to increase within 1 hour and within 3 hours in primary CD4 + Internalization in T cells has been shown to result in more than 70% internalization of TfR by the anti-transferrin receptor antibody R17217.

[0385] 5.12 Example 12: In vitro assay

[0386] 5.12.1 Proliferation assay

[0387] Mouse CTLL2 cells were cultured in 0.2 ng / ml IL2 at a rate of 1 × 10 5 Cells / well culture. 1 nM TGF-β, 1 μg / ml ADC and / or 100 nM ALK5 inhibitor were added to each well as indicated. Compound C was added to these wells for 24 h. Proliferation was quantified by adding BrdU reagent (Abcam) to each well for another 12 h and then analyzed by ELISA.

[0388] like Fig.10 As shown, treatment of CTLL2 cells with TGF-β inhibited proliferation by approximately 60%. However, addition of ADC1 (DAR 2-4, 4-6, or 6-8) resulted in almost complete reversal of TGF-β inhibition and restoration of CTLL2 proliferation, similar to cells treated with ALK5 inhibitor alone. Cells treated with rat anti-mouse IgG2A isotype control ALK5 ADC did not restore CTLL2 proliferation. There was no inhibition of proliferation in cells treated with ADC1 in the absence of TGF-β or in cells treated with naked Tfr antibody alone, indicating that ADC1 does not affect proliferation unless TGF-β is present (data not shown).

[0389] 5.12.2 Granzyme B expression assay

[0390] Using EasySep TM Mouse T Cell Isolation Kit (Negative Selection) (Stemcell Technologies) was used to purify mouse CD3 + CD3 T cells were activated using plate-bound anti-CD3e and soluble anti-CD28 as described previously. + T cells were washed and incubated with 5% serum plus 1 nM TGF-β- / + The ADC culture medium was replated.

[0391] The Golg stop reagent was added in the last 4 h, and the cells were then immunostained for surface CD8 (BD) and intracellular GzmB (eBioscience) and analyzed by flow cytometry. Granzyme B (GzmB) is a + Serine protease released by T cells to kill tumor cells. Therefore, increased expression of GzmB indicates that CD8 + Cytotoxic T cell activation.

[0392] like Fig.11 As shown, although TGF-β inhibits the expression of+ However, treatment with ADC1 on all three DARS2-4, 4-6, and 6-8 also restored GzmB expression, comparable to the ALK5 compound. In addition, the rat anti-mouse IgG2A isotype control ALK5 ADC was unable to restore GzmB expression.

[0393] 5.12.3 iTreg conversion assay

[0394] Isolate naive CD4 T cells from isolated mouse spleen cells using a negative selection kit. Adjust the cell density to 0.4 × 10 6 cells / ml, and 10 ng / ml of mouse IL-2, 20 ng / ml of TGF-β and 1 μg / ml of soluble anti-CD28 were added to the cell suspension.

[0395] Anti-mouse CD3 antibody was coated on a 24-well plate at 10 μg / ml and incubated overnight at 4°C. The antibody was then aspirated from the plate. 1 ml of cell suspension was added to each well of the 24-well plate. 3 μg / ml and 5 μg / ml of ADC1 (DAR 4-6), anti-transferrin receptor antibody, rat anti-mouse IgG2A isotype control ALK5 ADC, and 100 nM and 1 μM of ALK5 inhibitor compound C were added to separate wells of the 24-well plate. The cells were then cultured for 72 h. TfR expression was tested at 48 h (data not shown). The cells were stained for Fo×P3 (eBioscience Fo×P3 staining buffer) and sorted by FACS at 72 h.

[0396] like Fig.12 As shown, 5 μg / ml of ADC1 (+CD71-ALK5 ADC) moderately reduced the amount of iTreg generation, similar to 100nM free ALK5 inhibitor alone (+ALK5 inh 100nM). In contrast, control ALK5 ADC (+Iso-ALK5 ADC) and naked anti-TfR antibody (+anti-CD71) had no effect on iTreg Fo×P3 expression.

[0397] 5.13 Example 13: Synthesis and Characterization of Compound N

[0398] Compound N was synthesized according to the general method in Scheme 9 below:

[0399]

[0400] Solution 9

[0401] Compound N was compared to Compound C in a number of in vitro assays. A summary of their IC50 activities in recombinant kinase assays and their Ki values ​​are shown in Table 6. Table 6 also shows the activity of Compound C in inhibiting TGF-β signaling in human HEK cells and mouse T cells. In the recombinant assay, Compound C was found to be 10 times more potent than Compound N.

[0402]

[0403] 5.14 Example 14: Internalization of CD2 and CD5 into T cells

[0404] Two different internalization studies were performed to measure CD2 and CD5 internalization after incubation of T cells with anti-CD2 and anti-CD5 antibodies, respectively.

[0405] 5.14.1 Study 1: No Antibody Elution

[0406] Mouse CD3 was activated with plate-bound anti-CD3 antibody (1 μg / ml) plus soluble anti-CD28 antibody (2 μg / ml). + T cells 36h. Wash cells and incubate with 1 μg / ml rat anti-mouse CD2 antibody (clone 12-15, Southern Biotech, catalog number 1525), rat anti-mouse CD5 antibody (clone 53-7.3, Southern Biotech, catalog number 1547) or rat isotype control antibody at specified time points (0, 15min or 0.5, 1, 3 or 6h) at 37 degrees. At each time point, the assay was terminated by placing the cells on ice. CD2 and CD5 expression were detected using fluorescently coupled secondary antibodies.

[0407] At 6 h, more than 60% of CD5 and more than 50% of CD2 were internalized into mouse CD3 + T cells (respectively Fig.13A and Fig. 13B ).

[0408] 5.14.2 Study 2: Antibody Elution

[0409] Study 1 was repeated except that free antibody was incubated with cells for 30 min at 4°C to saturate all receptors on the cell surface. Excess antibody in the supernatant was washed away prior to the start of the time course.

[0410] At 6 h, nearly 90% of CD5 and more than 50% of CD2 were internalized into mouse CD3 + T cells (respectively Fig. 13C and Fig.13D ).

[0411] 5.14.3 Discussion

[0412] In Study 1, new and recycled receptors, if present, could have continued to reach the cell surface throughout the time course and could bind to free antibodies in the medium. In Study 2, unbound antibodies were washed away before the start of the time course, so only the internalization of those receptors present at the start of the time course could be monitored. For CD2, the results of Studies 1 and 2 were similar, indicating that CD2 cannot turn over rapidly. For CD5, internalization increased by approximately 20% in the washout study (Study 2), indicating that new receptors were recycled, or increased by de novo synthesis, over the 6h time course. Recycling is believed to be a possible option, as a large amount of de novo synthesis is not expected over a 6-hour time course. Therefore, the results of Studies 1 and 2 suggest that CD5 may be recycled back to the cell surface more than CD2.

[0413] 5.15 Example 15: Generation and Characterization of ADCs Targeting CD2 and CD5

[0414] 5.15.1 Example 15: Generation of ADC

[0415] Four ALK5-ADCs, referred to in this example as TGF-β antagonists (T3A) targeting T cells, were prepared using rat anti-mouse CD2 antibodies (clone 12-15, Southern Biotech, catalog number 1525) and rat anti-mouse CD5 antibodies (clone 53-7.3, Southern Biotech, catalog number 1547). T3A was prepared using two linker-ALK5 inhibitor payloads, one of which contained a cleavable Val-Cit (VC) linker attached to ALK5-Compound C and the other contained a non-cleavable maleimidocaproyl (MC) linker attached to Compound N.

[0416] The antibody, linker and ALK5 payload combinations of four T3A are shown in Table 7:

[0417]

[0418] T3A#2-#5 were purified by size exclusion chromatography (SEC) and the drug-antibody ratio was calculated by hydrophobic interaction chromatography (HIC). The aggregation percentage, percentage of unbound antibody and DAR value of each T3A#2-#5 are shown in Table 8.

[0419]

[0420] 5.15.2 ADC Characterization

[0421] To determine the efficacy of T3A#2-5 in reversing TGF-β-mediated immunosuppression, mouse CD3 + T cells were activated with anti-CD3 plus anti-CD28 antibodies for 36-72 h in the presence of 1 nM TGF-β plus small molecule ALK5 inhibitor compound C (positive control), T3A#2-5, or isotype control T3A (negative control). After 36 h, CD8 + T cell levels as a marker of cytotoxicity ( Fig.14 ), and secreted cytokine IL2 ( Fig.15 ) and IFN-γ levels ( Fig.16 Finally, after 72 h, the amount of T cell proliferation was measured by Cell Titer Glo (Promega) ( Fig.17 ). All of these assays correlated with tumor clearance in vivo.

[0422] The functional capacity observed relative to activated T cells (set as 100%) was Figure 14-17 T3A#5 restored GzmB expression and T cell proliferation, but only partially restored IFN-γ expression. No effect on IL2 expression was observed.

[0423] 5.15.3 Discussion

[0424] The data of the above examples show that the level of target expression on T cells is important for efficacy in primary T cell assays. Both CD2 and CD5 are highly expressed in more than 85% of naive T cells and activated T cells, unlike CD71, which is highly expressed only in 20%-50% of activated T cells. However, although both CD2 and CD5 are highly expressed on T cells, it is observed that ADCs targeting CD5 have higher efficacy than ADCs targeting CD2. Based on the receptor internalization patterns observed with CD2 and CD5 in Example 14, at 6h, about 85% of CD5 is internalized, but only 53% of CD2 is internalized into primary mouse T cells. In addition, CD5 seems to begin to internalize faster than CD2. The data show that the amount of internalization also affects efficacy.

[0425] The data also suggest that both the linker and the release mechanism of the ALK5 inhibitor attached to the antibody are important for efficacy. The cathepsin B-cleavable VC linker in combination with an anti-CD5 antibody (T3A#5) was the most effective T3A. However, the non-cleavable MC linker in combination with an anti-CD5 antibody (T3A#4) also had some activity when attached to an anti-CD5 antibody.

[0426] Based on testing in primary mouse T cells, the efficacy of T3A can be ranked as follows: 1) T3A#5, 2) T3A#4, 3) T3A#3, and 4) T3A#2.

[0427] Without being bound by theory, it is believed that for high ADC activity, the ADC should target a T cell target that is widely expressed in naive and activated T cells (e.g., expressed in ≥70% of cells) and rapidly internalized and has an established intracellular release mechanism (e.g., proteolytic processing).

[0428] 5.16 Example 16: Internalization of CD7 into T cells

[0429] Internalization studies were performed to measure CD7 internalization after T cells were incubated with two different anti-CD7 antibodies.

[0430] Human CD3 was activated with plate-bound anti-CD3 antibody (1 μg / ml) plus soluble anti-CD28 antibody (2 μg / ml). + T cells 40h. Wash cells and incubate with 1 μg / ml anti-human CD7 antibody (clone 124-D1 and 4H9, Caprico Biotech) or rat isotype control antibody at 4 degrees for 30min to saturate all receptors on the cell surface. Wash away the remaining antibody in the supernatant, and then incubate the cells at 37 degrees for 0 to 6h. At each time point (5, 15, 30, 60, 180 and 360min), the assay is terminated by placing the cells on ice. CD7 expression is detected using a fluorescently coupled secondary antibody.

[0431] At 6 h, nearly 70%-80% of CD7 was internalized ( Fig.18 The amount of internalization was comparable to that of CD2 and CD5, indicating the suitability of CD7 as an ADC target.

[0432] 6. Specific implementation plan

[0433] The present disclosure is illustrated by the following specific embodiments.

[0434] 1. An antibody-ALK5 inhibitor conjugate (ADC) comprising an ALK5 inhibitor operably linked to an antibody or antigen-binding fragment that binds to a T cell surface molecule.

[0435] 2. The ADC according to embodiment 1, wherein the IC of the ALK5 inhibitor is 50 is at least 20 nM.

[0436] 3. The ADC according to embodiment 1 or embodiment 2, wherein the ALK5 inhibitor is an imidazole compound, a pyrazole compound or a thiazole compound.

[0437] 4. The ADC according to embodiment 3, wherein the ALK5 inhibitor is an imidazole compound.

[0438] 5. The ADC according to embodiment 3, wherein the ALK5 inhibitor is a pyrazole compound.

[0439] 6. The ADC according to embodiment 3, wherein the ALK5 inhibitor is a thiazole compound.

[0440] 7. The ADC according to embodiment 3, wherein the ALK5 inhibitor is an imidazole compound, which is an imidazole-benzodioxole compound or an imidazole-quinoxaline compound.

[0441] 8. The ADC according to embodiment 7, wherein the ALK5 inhibitor is an imidazole-benzodioxole compound.

[0442] 9. The ADC according to embodiment 7, wherein the ALK5 inhibitor is an imidazole-quinoxaline compound.

[0443] 10. The ADC according to embodiment 3, wherein the ALK5 inhibitor is a pyrazole compound, which is a pyrazole-pyrrole compound.

[0444] 11. The ADC according to embodiment 3, wherein the ALK5 inhibitor is an imidazole-benzodioxole compound, an imidazole-quinoxaline compound, a pyrazole-pyrrole compound or a thiazole compound.

[0445] 12. The ADC according to any one of embodiments 1 to 11, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a linker.

[0446] 13. The ADC of embodiment 12, wherein the linker is a non-cleavable linker.

[0447] 14. The ADC according to embodiment 13, wherein the non-cleavable linker is a linker of N-maleimidomethylcyclohexane 1-carboxylate, maleimidocaproyl or mercaptoacetamidocaproyl.

[0448] 15. The ADC according to embodiment 14, wherein the non-cleavable linker is an N-maleimidomethylcyclohexane 1-carboxylate linker.

[0449] 16. The ADC of embodiment 14, wherein the non-cleavable linker is a maleimidocaproyl linker.

[0450] 17. The ADC according to embodiment 14, wherein the non-cleavable linker is a thioacetamidohexanoyl linker.

[0451] 18. The ADC of embodiment 12, wherein the linker is a cleavable linker.

[0452] 19. The ADC according to embodiment 18, wherein the cleavable linker is a dipeptide linker, a disulfide linker or a hydrazone linker.

[0453] 20. The ADC according to embodiment 19, wherein the cleavable linker is a dipeptide linker.

[0454] 21. The ADC of embodiment 19, wherein the cleavable linker is a disulfide linker.

[0455] 22. The ADC of embodiment 19, wherein the cleavable linker is a hydrazone linker.

[0456] 23. The ADC according to embodiment 19, wherein the linker is a valine-citrulline dipeptide linker that is sensitive to proteases.

[0457] 24. The ADC according to embodiment 19, wherein the linker is a glutathione-sensitive disulfide linker.

[0458] 25. The ADC according to embodiment 19, wherein the linker is an acid-labile disulfide linker.

[0459] 26. The ADC according to any one of embodiments 1 to 25, wherein the ALK5 inhibitor is conjugated to the antigen or antigen-binding fragment by site-specific conjugation.

[0460] 27. The ADC according to embodiment 26, wherein the ALK5 inhibitor is conjugated via one or more cysteine, lysine or glutamine residues on the antibody or antigen-binding fragment.

[0461] 28. The ADC according to embodiment 27, wherein the ALK5 inhibitor is conjugated via one or more cysteine ​​residues on the antibody or antigen-binding fragment.

[0462] 29. The ADC of embodiment 27, wherein the ALK5 inhibitor is conjugated via one or more lysine residues on the antibody or antigen-binding fragment.

[0463] 30. The ADC according to embodiment 27, wherein the ALK5 inhibitor is conjugated via one or more glutamine residues on the antibody or antigen-binding fragment.

[0464] 31. The ADC according to embodiment 26, wherein the ALK5 inhibitor is conjugated via one or more non-natural amino acid residues on the antibody or antigen-binding fragment.

[0465] 32. The ADC of embodiment 31, wherein the one or more non-natural amino acid residues comprise p-acetylphenylalanine (pAcF).

[0466] 33. The ADC according to embodiment 31, wherein the one or more non-natural amino acid residues comprise p-azidomethyl-L-phenylalanine (pAMF)

[0467] 34. The ADC of embodiment 31, wherein the one or more non-natural amino acid residues comprise selenocysteine ​​(Sec).

[0468] 35. The ADC of embodiment 26, wherein the ALK5 inhibitor is conjugated via one or more glycans on the antibody or antigen-binding fragment.

[0469] 36. The ADC of embodiment 35, wherein the one or more glycans comprise fucose.

[0470] 37. The ADC according to embodiment 35, wherein the one or more glycans comprise 6-thiofucose.

[0471] 38. The ADC of embodiment 35, wherein the one or more glycans comprises galactose.

[0472] 39. The ADC of embodiment 35, wherein one or more glycans comprises N-acetylgalactosamine (GalNAc).

[0473] 40. The ADC of embodiment 35, wherein the one or more glycans comprise N-acetylglucosamine (GlcNAc).

[0474] 41. The ADC of embodiment 35, wherein the one or more glycans comprise sialic acid (SA).

[0475] 42. The ADC of any one of embodiments 26 to 41, wherein the ALK5 inhibitor is coupled via a linker.

[0476] 43. The ADC of any one of embodiments 1 to 42, wherein the average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 2 to 8.

[0477] 44. The ADC according to any one of embodiments 1 to 43, wherein the antibody is a monoclonal antibody.

[0478] 45. The ADC of embodiment 44, wherein the antibody is human or humanized.

[0479] 46. ​​The ADC of embodiment 45, wherein the antibody is human.

[0480] 47. The ADC of embodiment 45, wherein the antibody is humanized.

[0481] 48. The ADC according to any one of embodiments 1 to 47, wherein the antigen binding fragment is a Fab, Fab', F(ab')2 or Fv fragment.

[0482] 49. The ADC of embodiment 48, wherein the antigen binding fragment is a Fab.

[0483] 50. The ADC of embodiment 48, wherein the antigen binding fragment is Fab'.

[0484] 51. The ADC of embodiment 48, wherein the antigen binding fragment is F(ab')2.

[0485] 52. The ADC of embodiment 48, wherein the antigen binding fragment is a Fv fragment.

[0486] 53. The ADC according to any one of embodiments 48 to 52, wherein the antigen binding fragment is an antigen binding fragment of a human or humanized antibody.

[0487] 54. The ADC of embodiment 53, wherein the antigen binding fragment is an antigen binding fragment of a human antibody.

[0488] 55. The ADC of embodiment 53, wherein the antigen binding fragment is an antigen binding fragment of a humanized antibody.

[0489] 56. The ADC according to any one of embodiments 1 to 47, comprising an antibody.

[0490] 57. The ADC according to any one of embodiments 1 to 55, comprising an antigen binding fragment.

[0491] 58. The ADC of any one of embodiments 1 to 57, wherein the T cell surface molecule is CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD70, CD71, CD103, CD184, Tim3, LAG3, CTLA4 or PD1.

[0492] 59. The ADC of embodiment 58, wherein the T cell surface molecule is CD1.

[0493] 60. The ADC of embodiment 58, wherein the T cell surface molecule is CD2.

[0494] 61. The ADC of embodiment 58, wherein the T cell surface molecule is CD3.

[0495] 62. The ADC of embodiment 58, wherein the T cell surface molecule is CD4.

[0496] 63. The ADC of embodiment 58, wherein the T cell surface molecule is CD5.

[0497] 64. The ADC of embodiment 58, wherein the T cell surface molecule is CD6.

[0498] 65. The ADC of embodiment 58, wherein the T cell surface molecule is CD7.

[0499] 66. The ADC of embodiment 58, wherein the T cell surface molecule is CD8.

[0500] 67. The ADC of embodiment 58, wherein the T cell surface molecule is CD25.

[0501] 68. The ADC of embodiment 58, wherein the T cell surface molecule is CD28.

[0502] 69. The ADC of embodiment 58, wherein the T cell surface molecule is CD70.

[0503] 70. The ADC of embodiment 58, wherein the T cell surface molecule is CD71.

[0504] 71. The ADC of embodiment 58, wherein the T cell surface molecule is CD103.

[0505] 72. The ADC of embodiment 58, wherein the T cell surface molecule is CD184.

[0506] 73. The ADC of embodiment 58, wherein the T cell surface molecule is Tim3.

[0507] 74. The ADC of embodiment 58, wherein the T cell surface molecule is LAG3.

[0508] 75. The ADC of embodiment 58, wherein the T cell surface molecule is CTLA4.

[0509] 76. The ADC of embodiment 58, wherein the T cell surface molecule is PD1.

[0510] 77. The ADC according to any one of embodiments 1 to 57, wherein the T cell surface molecule is a T cell surface molecule capable of recycling through endosomes.

[0511] 78. The ADC of embodiment 77, wherein the T cell surface molecule is CD5 or CD7.

[0512] 79. The ADC of embodiment 78, wherein the T cell surface molecule is CD5.

[0513] 80. The ADC of embodiment 78, wherein the T cell surface molecule is CD7.

[0514] 81. The ADC according to any one of embodiments 1 to 80, comprising an Fc domain having one or more amino acid substitutions that reduce effector function.

[0515] 82. The ADC of embodiment 81, 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, L235E / L234A / L235A / P329A, 4D / L235E / K322A:E233K / L234R / L235R / K322A, L234D / L235E / P329W:E233K / L234R / L235R / P329W, L234D / L235E / E269K / D265S / K322A:E 233K / L234R / L235R / E269K / D265S / K322A or L234D / L235E / E269K / D265S / K322E / E333K:E233K / L234R / L235R / E269K / D265S / K322E / E333K.

[0516] 83. The ADC of embodiment 82, wherein the one or more substitutions comprise N297A.

[0517] 84. The ADC of embodiment 82, wherein the one or more substitutions comprise N297Q.

[0518] 85. The ADC of embodiment 82, wherein the one or more substitutions comprise N297G.

[0519] 86. The ADC of embodiment 82, wherein the one or more substitutions comprise D265A / N297A.

[0520] 87. The ADC of embodiment 82, wherein the one or more substitutions comprise D265A / N297G.

[0521] 88. The ADC of embodiment 82, wherein the one or more substitutions comprise L235E.

[0522] 89. The ADC of embodiment 82, wherein the one or more substitutions comprise L234A / L235A.

[0523] 90. The ADC of embodiment 82, wherein the one or more substitutions comprise L234A / L235A / P329A.

[0524] 91. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E:L234R / L235R / E233K.

[0525] 92. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E / D265S:E233K / L234R / L235R / D265S.

[0526] 93. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E / E269K:E233K / L234R / L235R / E269K.

[0527] 94. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E / K322A:E233K / L234R / L235R / K322A.

[0528] 95. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E / P329W:E233K / L234R / L235R / P329W.

[0529] 96. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E / E269K / D265S / K322A:E233K / L234R / L235R / E269K / D265S / K322A.

[0530] 97. The ADC of embodiment 82, wherein the one or more substitutions comprise L234D / L235E / E269K / D265S / K322E / E333K:E233K / L234R / L235R / E269K / D265S / K322E / E333K.

[0531] 98. A pharmaceutical composition comprising the ADC according to any one of embodiments 1 to 97 and a pharmaceutically acceptable carrier.

[0532] 99. A method of treating cancer, comprising administering the ADC according to any one of Embodiments 1 to 97 or the pharmaceutical composition according to Embodiment 98 to a subject in need thereof.

[0533] 100. The method of embodiment 99, wherein the cancer is an immunogenic cancer.

[0534] 101. The method of embodiment 100, wherein the cancer is a solid tumor expressing a tumor antigen.

[0535] 102. The method of embodiment 101, wherein the tumor antigen is gp100, melanA or MAGEA1.

[0536] 103. The method of embodiment 102, wherein the tumor antigen is gp100.

[0537] 104. The method of embodiment 102, wherein the tumor antigen is melanA.

[0538] 105. The method of embodiment 102, wherein the tumor antigen is MAGE A1.

[0539] 106. The method of embodiment 99, wherein the cancer is a solid tumor comprising immune infiltration.

[0540] 107. The method of any one of embodiments 99 to 106, wherein the cancer is treatable by immunotherapy.

[0541] 108. A method according to embodiment 107, wherein the immunotherapy is cytokine therapy, adoptive T cell therapy, chimeric antigen receptor (CAR) therapy, or T cell checkpoint inhibitor therapy.

[0542] 109. The method of embodiment 108, wherein the immunotherapy is cytokine therapy.

[0543] 110. A method according to embodiment 108, wherein the immunotherapy is adoptive T cell therapy.

[0544] 111. A method according to embodiment 108, wherein the immunotherapy is chimeric antigen receptor (CAR) therapy.

[0545] 112. A method according to embodiment 108, wherein the immunotherapy is T cell checkpoint inhibitor therapy.

[0546] 113. A method according to embodiment 108 or embodiment 112, wherein the T cell checkpoint inhibitor is an inhibitor of PD1, PDL1 or CTLA4.

[0547] 114. A method according to embodiment 113, wherein the T cell checkpoint inhibitor is an inhibitor of PD1.

[0548] 115. A method according to embodiment 113, wherein the T cell checkpoint inhibitor is an inhibitor of PDL1.

[0549] 116. A method according to embodiment 113, wherein the T cell checkpoint inhibitor is an inhibitor of CTLA4.

[0550] 117. A method according to any one of embodiments 99 to 116, wherein the cancer is non-small cell lung cancer (NSCLC), liver cancer, urothelial carcinoma, renal cancer, breast cancer, or melanoma.

[0551] 118. The method of embodiment 117, wherein the cancer is NSCLC.

[0552] 119. The method of embodiment 117, wherein the cancer is liver cancer.

[0553] 120. The method of embodiment 120, wherein the liver cancer is hepatocellular carcinoma.

[0554] 121. The method of embodiment 117, wherein the cancer is urothelial carcinoma.

[0555] 122. The method of embodiment 121, wherein the cancer is bladder cancer.

[0556] 123. The method of embodiment 117, wherein the cancer is renal cancer.

[0557] 124. The method of embodiment 117, wherein the cancer is breast cancer.

[0558] 125. The method of embodiment 117, wherein the cancer is melanoma.

[0559] 126. A method according to any one of embodiments 99 to 125, wherein the cancer is treatable by an ALK5 inhibitor.

[0560] 127. The method of any one of Embodiments 99 to 126, wherein the ADC or pharmaceutical composition is administered as a monotherapy.

[0561] 128. The method of any one of Embodiments 99 to 126, wherein the ADC or pharmaceutical composition is administered as part of a combination therapy regimen.

[0562] 129. The method of embodiment 128, wherein the ADC or pharmaceutical composition is administered in combination with a standard of care therapy or treatment regimen.

[0563] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure.

[0564] 7. References

[0565] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated as being incorporated by reference for all purposes. In the event of an inconsistency between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present specification are intended to prevail.

Claims

1. An antibody-ALK5 inhibitor conjugate (ADC) comprising an ALK5 inhibitor operably linked to an antibody or antigen-binding fragment that binds to a T cell surface molecule.

2. The ADC according to claim 1, wherein the IC of the ALK5 inhibitor is 50 is at least 20 nM.

3. The ADC according to claim 1, wherein the ALK5 inhibitor is an imidazole compound, a pyrazole compound or a thiazole compound.

4. The ADC according to claim 3, wherein the ALK5 inhibitor is an imidazole-benzodioxole compound, an imidazole-quinoxaline compound, a pyrazole-pyrrole compound or a thiazole compound.

5. The ADC of claim 1, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a non-cleavable linker or a cleavable linker.

6. The ADC of claim 5, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a non-cleavable linker, the non-cleavable linker being a linker of N-maleimidomethylcyclohexane 1-carboxylate, maleimidocaproyl or mercaptoacetamidocaproyl.

7. The ADC of claim 5, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a cleavable linker, which is a dipeptide linker, a disulfide linker, or a hydrazone linker.

8. The ADC according to claim 7, wherein the linker is a protease-sensitive valine-citrulline dipeptide linker, a glutathione-sensitive disulfide linker or an acid-sensitive disulfide linker.

9. The ADC of claim 1, wherein the ALK5 inhibitor is coupled via one or more cysteine ​​residues on the antibody or antigen-binding fragment or one or more lysine residues on the antibody or antigen-binding fragment, optionally wherein the ALK5 inhibitor is coupled via a linker.

10. The ADC of claim 1, wherein the average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule ranges from 2 to 8.

11. The ADC of claim 1, wherein the antibody is a monoclonal antibody.

12. The ADC of claim 11, wherein the antibody is human or humanized.

13. The ADC of claim 1, wherein the antigen binding fragment is a Fab, Fab', F(ab')2 or Fv fragment.

14. The ADC of claim 13, wherein the antigen-binding fragment is an antigen-binding fragment of a human or humanized antibody.

15. The ADC of claim 1, wherein the T cell surface molecule is CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD70, CD71, CD103, CD184, Tim3, LAG3, CTLA4 or PD1.

16. The ADC according to claim 1, wherein the T cell surface molecule is a T cell surface molecule capable of recycling through endosomes.

17. The ADC of claim 16, wherein the T cell surface molecule is CD5 or CD7.

18. A pharmaceutical composition comprising the ADC according to claim 1 and a pharmaceutically acceptable carrier.

19. A method of treating cancer comprising administering the ADC according to claim 1 to a subject in need thereof.

20. The method of claim 19, wherein the cancer is: (a) Immunogenic cancers; (b) solid tumors containing immune infiltrates; (c) solid tumors treatable by immunotherapy; or (d) Treatable with ALK5 inhibitors.

21. The method of claim 20, wherein the cancer is a solid tumor that expresses a tumor antigen.

22. The method of claim 20, wherein the cancer is treatable by immunotherapy and the immunotherapy is cytokine therapy, adoptive T cell therapy, chimeric antigen receptor (CAR) therapy, or T cell checkpoint inhibitor therapy.

23. The method of claim 19, wherein the ADC is administered as a monotherapy or as part of a combination therapy regimen.

Images