Antibody-ALK5 inhibitor conjugates and their uses

Abstract

To provide antibody-drug conjugates to target ALK5 inhibitors to cell types in which the inhibition of TGF-β signaling is therapeutically useful, while minimizing host tissue toxicity.SOLUTION: The present invention provides 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.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] 1. Background Members of the transforming growth factor-beta (TGF-β) family of cytokines Members of the IL-1 receptor are involved in a wide variety of biological processes, both during normal tissue development and in disease states. TGF-β family members are multifunctional proteins that regulate inflammation, Wound healing, extracellular matrix accumulation, bone formation, tissue development, cell differentiation, heart valve remodeling It is involved in inflammation, tissue fibrosis, and tumor progression (Barnard et al., 1990, Biochim. m Biophys Acta. 1032:79-87, Sporn et al., 1992, J Cell Biol 119:1017-1021, Yingl ing et al., 2004, Nature Reviews, 3:1011-1022, Janssens et al., 2005, Endocr Rev ., 26(6):743-74). Three mammalian isoforms have been identified so far: TGF-β 1, TGF-β2, and TGF-β3 (Massague, 1990, Annu Rev Cell Biol 6:597-6 41) Other members of the transforming growth factor superfamily include actinomycin A, actinomycin B, and actinomycin C. bin, inhibin, bone morphogenetic proteins, growth and differentiation factors, and Müllerian inhibitors Examples of such substances include:

[0002] TGF-βΙ binds two highly conserved single transmembrane serine / threonine kinase receptors. Transduces signals through type I (ALK5) and type II TGF-β receptors Upon ligand-induced binding and oligomerization, the type II receptor binds to the GS of ALK5. phosphorylates serine / threonine residues in the ALK5 domain, thereby activating ALK5. and the generation of new SMAD introduction sites. SMADs are transported from the extracellular environment to the nucleus of the cell. It is an intracellular protein that is specialized to transduce the signal of TGF-β. ALK5 phosphorylates Smad2 and Smad3 at the C-terminal SSXS motif. This leads to their dissociation from the receptors and the formation of a complex with Smad4. Smad complexes translocate into the nucleus, assemble with cell-specific DNA-binding cofactors, and regulate cell growth and development. It modulates the expression of genes that regulate differentiation and development.

[0003] Activin transduces signals in a manner similar to TGF-β. Activin / threonine kinase that binds to and activates the activin type II receptor (ActRIIB) The type II receptor hyperphosphorylates serine / threonine residues in the GS domain of ALK4. Activated ALK4 then hyperphosphorylates Smad2 and Sma d3, resulting in the formation of a hetero-Smad complex with Smad4. This results in activin-induced regulation of gene transcription.

[0004] TGF-β signaling mediates the expression of T and B lymphocytes, NK cells, and dendritic cells. By regulating both innate and adaptive immune cells, including antigen-presenting cells such as TGF-β is generally involved in the regulation of T cell proliferation in the thymus and is essential for maintaining immune homeostasis. Immunosuppressive cytokines play an essential role in cell development and maintaining peripheral tolerance. TGF-β is considered to be a CD4 + and CD8 + Both proliferation of T cells, inhibits T-helper subset differentiation, cytotoxicity, and T-kappa kinase production (Li et al. ., 2008, Cell 134:392-404). TGF-β also stimulates natural regulatory T cells ( The development of nTregs and their peripheral expression in response to various diseases such as inflammation and cancer. It plays an important role in the generation of inducible Treg (iTreg) (Tran et al., 2012, J Mol Cell Bio 4:29-37, 2012). nTregs are typically CD25+ FoxP3+ and actively suppresses T cell activation, helping to maintain peripheral T cell tolerance. TGF-β is a CD4+ T cell subset that accounts for a small proportion of peripheral reg of It is important for survival and expansion (Marie et al., 2005, J Exp Med 201:1061-67). Under inflammatory conditions, TGF-β inhibits naive CD4 + T cells, FoxP3 + iT reg to suppress local tissue-resident T cells. reg is often , and within the tumor itself have been found to prevent T cell-mediated tumor clearance (Whites ide, 2014, Expert Opin Biol Ther 14:1411-25).

[0005] In general, high levels of TGF-β expression are associated with poor clinical prognosis. In many cases, tumors have co-opted the TGF-β pathway and are using it to mediate 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. First, TGF- β promotes CD4+ and CD8+ T cell expansion, cytokine production, and tumor cell death The second is that TGF-β directly inhibits nT reg and iT reg are important for the survival and / or transformation of tumors, respectively, which also play a role in immune-mediated tumor clearance. In multiple preclinical mouse models, neutralization of TGF-β inhibits T cell proliferation and It has been demonstrated that tumor burden is reduced due to increased IL-1 mediated tumor clearance. Importantly, this is achieved through expression of dominant-negative TGF-βRII or through the expression of soluble TGF-β. Inhibition of TGF-β signaling in T cells using β receptors in vivo This is sufficient to restore effective immune-mediated tumor clearance. Gorelik et al., 20 01, Nat Med 7:1118-22, Thomas et al., 2005, Cancer Cell 8:369-80.

[0006] Besides its effects on the immune system, TGF-β signaling is also important in tumor development. Preclinical studies have demonstrated that TGF-β acts in a paradoxical manner against the tumor itself. It has been shown that IL-1 has a potent inhibitory effect on the IL-1 receptor, and a confounding effect on surrounding stromal cells. In the early stages, TGF-β promotes tumor growth and proliferation through the regulation of cell cycle mediators. However, at later stages, TGF-β loses its growth-inhibitory properties and Through induction of epithelial-mesenchymal transition (EMT), as well as through the regulation of stromal fibroblasts, angiogenesis, and and promotes tumor metastasis through its effects on the extracellular matrix (ECM) (Connolly et al., 2013). t al., 2012, Int J Bio 8:964-78). If delivered at the wrong stage, TGF-β signaling Broad-spectrum inhibition of cellular signaling carries the risk of promoting tumor metastasis and / or inhibiting non-tumor There is a risk that these tumors may inhibit tumor-promoting stromal cell populations and indirectly exacerbate 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 Instead of the intended growth-blocking effect, the inhibitors caused tumors to become more aggressive and metastatic. There is a possibility that this will happen.

[0007] Despite the paradoxical effects on the tumor itself, and widespread expression of TGF-β receptors Inhibition of the TGF-β pathway as a cancer treatment has long been of interest. -β neutralizing antibodies, TGF-β2 antisense RNA, and small molecule ATP-competitive ALK5 kinase Some of the classical ALK5 inhibitors being developed are pyrazole-based , imidazoles, and triazoles (Bonafoux et al., 2009, Expert Opin Ther Patents 19:1759-69, Ling et al., 2011, Current Pharma Biotech 12:2190-2202 Many ALK5 inhibitors have been shown to be effective in in vitro cell-based assays as well as in vivo Tested in both ivo mouse xenograft and syngeneic tumor models, demonstrating significant efficacy (Neuzillet et al., 2015, Pharm & Therapeutics 147:22-31). The ubiquitous expression of GF-β receptors reduces the risk of host toxicity and tumor growth. Many TGF-β inhibitors, especially ALK5 inhibitors, have not yet been approved for preclinical development due to concerns that they may promote For example, in preclinical toxicology studies in rats, A series of ALK5 inhibitors has been developed that prevents bleeding, inflammation, degeneration, and proliferation of interstitial cells in the valve. Heart valve lesions were identified (Anderton et al., 2011, Tox Path 39:916-24). Summary of the Invention

[0008] Thus, while minimizing host tissue toxicity such as that observed in cardiac tissue. Targeting ALK5 inhibitors to cell types where inhibition of TGF-β signaling is therapeutically beneficial There is a need to target this.

[0009] 2. Overview Avoid targeted host toxicity and unintended tumor progression with ALK5 inhibitor therapy To prevent deterioration, the inventors target compounds only to those cells that provide therapeutic benefit. We developed a novel approach to

[0010] For the treatment of cancer, this approach involves antibody-directing ALK5 inhibitors to the T cell compartment. This promotes T cell-mediated tumor clearance and provides long-term protection without systemic toxicity. Without being bound by theory, the present invention includes the following: Inhibition of TGF-β signaling not only directly enhances T cell-mediated clearance, T cells are transformed into inducible T reg This inhibits the conversion of natural T reg Survival rate Therefore, inhibition of TGF-β signaling in T cells is thought to reduce the CD4 + and CD8 + Not only does it restore T cell activity, but it also stimulates T reg "brake" This effectively reconnects the immune system by removing TG only in T cells. Inhibition of F-β signaling has broad spectrum effects, both from the tumor perspective as well as from the host tissue toxicity perspective. It is safer than conventional TGF-β inhibition.

[0011] Thus, the present disclosure provides antibody-drug conjugates (ADCs) in which the drug is an ALK5 inhibitor. The antibody component of the ADC is an antibody or antigen-binding domain that binds to a T cell surface molecule. Section 4.2 provides exemplary antibodies that can be used in the ADCs of the disclosure. In some embodiments, the ALK5 inhibitor is an imidazole-based Benzodioxole compounds, imidazole-quinoxaline compounds, pyrazole-pyrrolo compounds Exemplary ALK5 inhibitors are those described in Section 4.3, and As described in Tables 1 to 3.

[0012] The ALK5 inhibitor can be directly conjugated to an antibody component or can be phospho-conjugated. The linker can be a non-cleavable linker or a , 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 combined can vary and generally is determined by the number of antibodies or antigen-binding fragments. The drug loading ranges from 2 to 8 ALK5 inhibitor molecules per piece. Drug loading is described in detail in Section 4.5. It has been done.

[0013] The present disclosure further provides pharmaceutical compositions comprising the ADCs of the present disclosure. Exemplary pharmaceutical additives that can be used to formulate a pharmaceutical composition include those listed in Section 4. It is stated in verse 6.

[0014] The present disclosure relates to a method for administering an ADC of the present disclosure or a pharmaceutical composition of the present disclosure to a subject in need thereof. The present disclosure further provides a method of treating cancer by administering to a subject a therapeutically effective amount of the ADC or pharmaceutical composition of the present disclosure. The compounds of the present disclosure can be administered as monotherapy or as part of a combination therapy. Exemplary Cancers That Can Be Treated with DCs and Pharmaceutical Compositions, and Exemplary Combination Therapies is described in Section 4.7. [Brief description of the drawings]

[0015] [Figure 1] The effect of TGF-β on CD4+ and CD8+ T cells is shown. During tumor progression, TGF-β, which can originate from both tumor 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 tumor death by inhibiting the expression of granzymes and perforin in cytotoxic CD8+ T cells. Inhibiting both CD4+ and CD8+ T cell populations completely suppresses T cell-mediated tumor clearance. [Diagram 2] 1 shows the effect of TGF-β on Treg cells during tumor progression. During tumor progression, nTreg and iTreg cells are typically found in tumors and control T cell-mediated functions in situ. TGF-β promotes nTreg cell survival and iTreg cell conversion to suppress T cell-mediated tumor clearance. Increased Treg cells at tumor sites ensures that tumor-infiltrating T cells also prevent tumors from clearing tumors. [Diagram 3]

[0023] Figure 1 shows the mechanism of action of the ADCs of the present disclosure in CD4+ and CD8+ T cells. T cell-targeted inhibition of TGF-β signaling restores CD4+ T cell activity and CD8+ T cell-mediated tumor death. [Figure 4]

[0023] Figure 1 shows the mechanism of action of the ADCs of the present disclosure on Treg cells. T cell-targeted inhibition of TGF-β signaling also blocks Treg-mediated suppression of immune-mediated tumor clearance in situ. [Figure 5A-B] Figure 5 shows the inhibition of TGF-β-induced luciferase activity in HEK293T cells by compounds A and B. Figure 5A: Compound A, Figure 5B: Compound B. [Figure 5C-D] Figure 5C shows the inhibition of TGF-β-induced luciferase activity in HEK293T cells by compounds C to D. Figure 5C: Compound C, Figure 5D: Compound D [Figure 6A] MTS proliferation assay data for compounds A-D are shown. Compounds A-C restore proliferation in TGF-β treated CDC4+ T cells. Figure 6A: Data for compounds A-D. In Figure 6A, the bars labeled "A", "B", "C", and "D" above "No TGF-β" show the results of experiments performed with compounds at 100 nM without TGF-β. Figure 6B: Data for compound B, Figure 6C: Data for compound C. [Figure 6B-C] MTS proliferation assay data for compounds A-D are shown. Compounds A-C restore proliferation in TGF-β treated CDC4+ T cells. Figure 6B: Data for compound B, Figure 6C: Data for compound C. [Figure 7] 7A-7B show LC-MS data for an exemplary ADC of the disclosure (ADC2). Figure 7A: LC-MS data for the ADC heavy chain, Figure 7B: LC-MS data for the ADC light chain. [Figure 8] Chromatogram of SEC purified ADC2 prepared with an S-4FB / Ab ratio of 6. SEC analysis of purified ADC2 shows less than 5% aggregation. [Figure 9A-B]Figure 9 shows that an exemplary antibody of the disclosure (anti-transferrin receptor antibody R17217) induces internalization of the antibody's target, transferrin receptor (TfR), in primary mouse CD4+ T cells. Figure 9A: Control without anti-transferrin receptor antibody, Figure 9B: 15 minute incubation with anti-transferrin receptor antibody. [Figure 9C-D] Figure 9C shows that an exemplary antibody of the disclosure (anti-transferrin receptor antibody R17217) induces internalization of the antibody's target, transferrin receptor (TfR), in primary mouse CD4+ T cells. Figure 9C: 30 minute incubation with anti-transferrin receptor antibody, Figure 9D: 60 minute incubation with anti-transferrin receptor antibody. [Figure 9E-F] Figure 9E shows that an exemplary antibody of the disclosure (anti-transferrin receptor antibody R17217) induces internalization of the antibody's target, the transferrin receptor (TfR), in primary mouse CD4+ T cells. Figure 9E: 180 minutes of incubation with anti-transferrin receptor antibody, Figure 9F: Mean fluorescence intensity (MFI) over a 3 hour time course. [Figure 10] 1 shows reversal of TGF-β-mediated inhibition of proliferation in mouse CTLL2 cells by an exemplary ADC of the disclosure (ADC1). [Figure 11] 1 shows derepression of granzyme B expression in primary CD8+ T cells activated with TGF-β by an exemplary ADC of the disclosure (ADC1). ADC1 partially restores granzyme B expression comparable to free ALK5 inhibitors. [Figure 12] FIG. 1 shows that an exemplary ADC of the disclosure (ADC1) reduces iTreg generation similarly to 100 mM free ALK5 inhibitor. [Figure 13A-B] Internalization of CD5 (FIG. 13A) as well as CD2 (FIG. 13B) into activated primary murine CD3+ T cells is shown. [Figure 13C-D] Internalization of CD5 (FIG. 13C) as well as CD2 (FIG. 13D) into activated primary murine CD3+ T cells is shown. [Figure 14]Levels of CD8+ T cells expressing granzyme (GzmB) are shown following a 36 hour incubation of activated mouse CD3+ T cells in the presence of T3A #2-#5. [Figure 15] Shown are levels of secreted IL2 following 36 hour incubation of activated mouse CD3+ T cells in the presence of T3A #2-#5. [Figure 16] Shown are levels of secreted IFN-γ following 36 h incubation of activated mouse CD3+ T cells in the presence of T3A #2-#5. [Figure 17] Quantitative T cell proliferation following 72 hour incubation of activated mouse CD3+ T cells in the presence of T3A #2-#5. [Figure 18] FIG. 1 shows internalization of CD7 into activated primary human CD3+ T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] 4. Detailed Description The present disclosure provides an antibody-drug conjugate (ADC) useful for treating cancer, comprising: An antibody comprising an antibody component covalently linked, either directly or through a linker, to an ALK5 inhibitor. A general description of the ADCs of the present disclosure is provided in Section 4.1. The antibody component of the ADC can be an intact antibody or a fragment thereof. Antibodies that can be used in the ADCs of the disclosure are described in detail in Section 4.2. ALK5 inhibitors capable of this are described in detail in Section 4.3. The ADCs of the present disclosure are typically The ADCs of the present disclosure contain a linker between the antibody and the ALK5 inhibitor. Exemplary linkers that can be used are detailed in Section 4.4. The ADCs of the disclosure may include Drug loading is described in detail in Section 4.5. The present disclosure further provides pharmaceutical formulations comprising the ADCs of the present disclosure. Pharmaceutical formulations containing the ADCs are described in Section 4.6. The present disclosure relates to a variety of therapeutic applications using the ADCs of the disclosure. Further provided are methods of treating various cancers, including as a monotherapy for the treatment of cancer, or Methods of using the ADCs of the disclosure as part of a combination therapy are described in Section 4.7.

[0017] 4.1. Antibody-drug conjugates The ADCs of the disclosure generally are typically substituted with aryl amines, such that the covalent bond does not interfere with binding of the antibody to the target. Typically, it consists of an ALK5 inhibitor covalently bonded to an antibody via a linker.

[0018] Techniques for conjugating drugs to antibodies are well known in the art (e.g., For example, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., at pp. 623-53 (Robinson et al., eds., 1987), Thorpe et al., 1982, Immunol. Rev. 62:119-58, Dubowchik e t al., 1999, Pharmacology and Therapeutics 83:67-123, and Zhou, 2017, Biomedic (See, e.g., J. Med. Ines 5(4):E64.) The ALK5 inhibitor is preferably a site-specific conjugate. The ALK5 inhibitor is linked to the antibody component in the ADC of the disclosure via a fusion protein. One or more naturally occurring or engineered cysteine, lysine, or glutamine residues group, one or more unnatural amino acids (e.g., p-acetylphenylalanine (pA cF), p-azidomethyl-L-phenylalanine (pAMF), or selenocysteine Sec), one or more glycans (e.g., fucose, 6-thiofucose , galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine amino acid (GlcNAc), or sialic acid (SA), or one of four to six amino acids The antibody component can be conjugated via one or more short peptide tags. See, e.g., Zhou, 2017, Biomedicines 5(4):E64, the contents of which are incorporated by reference. is incorporated herein in its entirety.

[0019] In one example, the antibody or fragment thereof is capable of binding to another protein (or a portion thereof, e.g., a TA at least 10, 20, or 50 amino acid portions of the protein In contrast, the antibody is linked to the N-terminus or C-terminus of the antibody via a covalent bond (e.g., a peptide bond). The antibody, or a fragment thereof, may be fused to another protein, either intracellularly or intracellularly. The constant domain can be linked at its N-terminus. Recombinant DNA procedures can be used to create such fusions. For example, the method described in WO 86 / 01533 can be used to prepare and in European Patent Application Publication No. 0 392 745. Thus, the effector molecule may increase the in vivo half-life and / or Alternatively, it may be possible to improve the delivery of antibodies to the immune system across the epithelial barrier. Examples of suitable effector molecules include polymers, albumin, and albumin-binding proteins. or albumin binding compounds, such as those described in PCT Publication No. WO 2005 / 023366, This is described in Brochure No. 117984.

[0020] A metabolic process or reaction is an enzymatic process, e.g., the catalysis of a peptide linker in an ADC. by enzyme-degrading cleavage or addition of functional groups, such as hydrazones, esters, or amides. Intracellular metabolism can include, but is not limited to, cell entry, diffusion, uptake, etc. These include antibodies and free drugs that are cleaved intracellularly or after transport.

[0021] The terms "cleaved intracellularly" and "intracellular cleavage" refer to the process by which the antibody-drug conjugate ( This refers to the metabolic process or reaction within a cell that results in the release of the drug moiety (D). The covalent bond between the antibody (Ab), i.e. the linker, is broken, resulting in the release of the antibody into the cell. The cleaved portion of the ADC is then released into the body as an intracellular metabolite, resulting in the release of the drug. be.

[0022] 4.2. Antibody components The present disclosure provides antibody drug conjugates in which an antibody component binds to a T cell surface molecule. Unless otherwise indicated, the term "antibody" (Ab) refers to an antibody that specifically binds to a particular antigen. The term refers to a group of antibodies that react immunologically with other molecules, such as polyclonal antibodies, monoclonal antibodies, and immunoglobulin molecules. The antibodies include, but are not limited to, monoclonal antibodies, genetically engineered antibodies, and other modified forms of antibodies. However, chimeric, humanized, and heteroconjugate antibodies (e.g., bispecific antibodies) may be used. Bispecific antibodies, diabodies, triabodies, and tetrabodies specific antibodies), as well as, for example, Fab', F(ab')2, Fab, Fv, rIgG, and antigen-binding fragments of antibodies, including scFv fragments. Insofar as the term "monoclonal antibody" (mAb) is used, it is understood that the term "monoclonal antibody" refers to an antibody that specifically binds to a protein. It is possible to produce complete molecules, as well as antibody fragments (e.g., Fab and F(ab')2 fragments). The term Fab and F(ab')2 fragments refer to the F(ab')2 fragments of an intact antibody. They lack the c fragment and are cleared more rapidly from the circulation of animals or plants than complete antibodies. may have less nonspecific tissue binding (Wahl et al., 1983, J. Nucl. Med. 24:316).

[0023] The term "scFv" refers to a combination of heavy and light chain variable domains derived from a conventional antibody. This refers to a single-chain Fv antibody, which consists of two chains.

[0024] References to "VH" refer to the immunoglobulin G (Ig) of an antibody, including the heavy chain of an Fv, scFv, or Fab. References to "VL" include Fv, scFv, dsFv, or Fab light chain. Immunoglobulins (Ig) are glycoproteins that have the same structural characteristics. Antibodies and target-specific ligands are the same. Natural antibodies and immunoglobulins include both antibodies that lack isomerism and other antibody-like molecules. , consisting of two identical light chains (L) and two identical heavy chains (H), usually about 150,0 It is a heterotetrameric glycoprotein of 10,000 daltons. Each heavy chain has a variable domain at the amino terminus. Each light chain has a VH domain followed by several constant domains. and a constant domain at the carboxy terminus.

[0025] For optimal delivery of the ALK5 inhibitor within the cell, the antibody is preferably internalized. The localized antibodies bind to their target molecules on the cell surface and are then absorbed by the cell as a result of the binding. The effect of this is that the ADC is taken up by the cell. Processes that allow for the determination of antibody internalization after antibody synthesis are known to those skilled in the art and are, for example, CT Publication WO 2007 / 070538 pamphlet, page 80, and the following Once internalized, e.g., as described in Section 4.4, When the ALK5 inhibitor is attached to the antibody using a cleavable linker, the ALK5 inhibitor can be Can be released from the antibody by lysosomal cleavage or by other cellular mechanisms .

[0026] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of fragments include Fab, Fab', F(ab')2, and Fv fragments. An "Fv" fragment is the minimum 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 in tight, non-covalent association. In this structure, the three CDRs of each variable domain interact with each other to form a VH-VL dimer. The VH-VL dimer defines an antigen-binding site on its surface. In most cases, the six CDRs are However, in some instances, a single variable domain may be used to confer target binding specificity to the host. The Fv fragment (or half of an Fv containing only the three target-specific CDRs) recognizes the target and "Single-chain Fv" or "scFv" antibody fragments have the ability to bind to a single In the polypeptide chain of the Fv polypeptide, the VH and VL domains of the antibody are included. The peptides are VH and VH peptides that enable the scFv to form the desired structure for target binding. A "single domain antibody" further comprises a polypeptide linker between the VL domain and the VL domain. It is composed of a single VH or VL domain that exhibits sufficient affinity for NF-α. In a specific embodiment, the single domain antibody is a camelid antibody (e.g., Riechman (see, e.g., J. Immunological Methods 231:25-38, 1999).

[0027] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments contain a heavy chain that contains one or more cysteines from the antibody hinge region. At the carboxyl terminus of the CH1 domain, it differs from the Fab fragment by the addition of several residues. F(ab') fragments are formed by cleavage of the hinge cysteines of the F(ab')2 pepsin digest. Further chemical coupling of antibody fragments is known to those skilled in the art. It is being done.

[0028] In certain embodiments, the antibodies of the present disclosure are monoclonal antibodies. The term "monoclonal antibody" as used herein refers to an antibody produced through hybridoma technology. The term "monoclonal antibody" refers to any eukaryotic, prokaryotic, or filamentous antibody. Refers to an antibody that is derived from a single clone, including a diclone, but does not include the method by which it is produced. Monoclonal antibodies useful in connection with the present disclosure include hybridoma, recombinant, and Any method known in the art, including the use of phage display technology, or a combination thereof. Antibodies of the present disclosure can be prepared using a wide variety of techniques. Antibodies include primatized, humanized, or human antibodies.

[0029] The antibody of the present disclosure may be a chimeric antibody. As used herein, the term "chimeric" antibody comprises variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and , a human immunoglobulin constant region typically selected from a human immunoglobulin template Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechni ques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715, 4,816,567, and 5,807,715, See US Pat. No. 4,816,397, the contents of which are incorporated herein by reference in their entirety. is incorporated into.

[0030] The antibodies of the present disclosure may be humanized. "Humanized" forms of non-human (e.g., murine) antibodies include: Chimeric immunoglobulins, immunoglobulins containing minimal sequences derived from non-human immunoglobulins A purine chain, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other target-binding subdomain). Generally, a humanized antibody will contain at least one Typically it will contain substantially all of the two variable domains, where all of the CDR regions, or Substantially all correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions All human immunoglobulin sequences are human. Humanized antibodies are also of immunoglobulin sequence. Contains at least a portion of the constant region (Fc), typically that of a human immunoglobulin consensus sequence Methods for humanizing antibodies are known in the art. See, for example, Riechman et al. N et al., 1988, Nature 332:323-7; U.S. Patent No. 5,530,100 to Queen et al. No. 1, U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761 Specification, U.S. Patent No. 5,693,762 Specification, U.S. Patent No. 6,180,370 Specification EP 239400, PCT Publication WO 91 / 0996 No. 7, U.S. Pat. No. 5,225,539, European Patent Application Publication No. 592 106, EP 519596, Padlan, 1991, Mol. Immun. ol., 28:489-498, Studnicka et al., 1994, Prot. Eng. 7:805-814, Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; U.S. Pat. No. 5,565,332. No. 6,399,433, all of which are incorporated herein by reference in their entireties.

[0031] The antibodies of the present disclosure can be human antibodies. Fully "human" antibodies are suitable for the therapeutic treatment of human patients. As used herein, a "human antibody" refers to an antibody that is produced by a human immunoglobulin G (HIG). a human immunoglobulin library, comprising an antibody having an amino acid sequence of an immunoglobulin; or Isolated from animals that are transgenic for one or more human immunoglobulins Human antibodies include antibodies that are produced by human immunoglobulin A and do not express endogenous immunoglobulins. The present invention relates to a method for the preparation of a nucleic acid sequence of a nucleic acid molecule, comprising the steps of: The compositions can be prepared by a variety of methods known in the art, for example, U.S. Pat. No. 7 and U.S. Pat. No. 4,716,111, as well as the International Publication of PCT Publications. International Publication No. 98 / 46645, International Publication No. 98 / 50433, International Publication No. 98 / 24893, International Publication No. 98 / 16654 , International Publication No. 96 / 34096 Brochure, International Publication No. 96 / 33735 Brochure See, for example, WO 91 / 10741, each of which is incorporated herein by reference. Human antibodies are also derived from cells expressing functional endogenous immunoglobulins. However, it is not possible to produce a transgenic mouse model of the human immunoglobulin gene expression. For example, the present invention can be produced using a transgenic mouse. Pamphlet No. 8 / 24893, Pamphlet No. 92 / 01047, Pamphlet No. No. 96 / 34096, International Publication No. 96 / 33735, U.S. Patent No. 5,413,923, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,569,825, U.S. Pat. ,661,016, U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,8 No. 14,318, U.S. Pat. No. 5,885,793, U.S. Pat. No. 5,916 No. 5,939,598, the contents of which are incorporated herein by reference. , which is incorporated herein by reference in its entirety. eton, NJ), Astellas Pharma (Deerfield, Ill. .), Amgen (Thousand Oaks, Calif.), and Regene ron (Tarrytown, NY) using similar techniques to those described above. It is possible to provide human antibodies directed against a selected antigen. Fully human antibodies that recognize the target gene were generated using a technique called "guided selection." In this approach, a non-human monoclonal antibody of choice can be used to generate A human antibody, such as a mouse antibody, is used to induce the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., 1988, Biotechnology 12:899-903).

[0032] The antibodies of the present disclosure may be primatized. The term "primatized antibody" refers to an antibody that is an antibody that contains monkey variable regions and It refers to an antibody that contains a human constant region. Methods for producing primatized antibodies are known in the art. For example, U.S. Pat. No. 5,658,570 and U.S. Pat. No. 1,722, and U.S. Pat. No. 5,693,780, the contents of which are incorporated herein by reference. is incorporated herein by reference in its entirety.

[0033] Antibodies of the present disclosure include derivatized antibodies. For example, but not limited to, derivatized antibodies are typically Typically, glycosylation, acetylation, pegylation, phosphorylation, amidation, known protecting groups / blocking groups, etc. Derivatization with locking groups, proteolytic cleavage, cellular ligands or other proteins (see Section 4.1 for a discussion of antibody conjugates) Any of a number of chemical modifications may be used, including but not limited to specific chemical cleavage, acetylation, etc. This can be accomplished by known techniques, including cleavage, formylation, metabolic synthesis of tunicamycin, etc. In addition, derivatives can be made by incorporating one or more non-natural amines, for example using ambrx technology. The amino acid may be a cyclic amino acid (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2). reference).

[0034] In yet another embodiment of the present disclosure, the antibody or fragment thereof is To modify the function of at least one constant region-mediated biological effector on The antibody may be an antibody or antibody fragment, the sequence of which has been modified. For example, in some embodiments, The antibodies of the disclosure are modified to have at least one constant region-mediated It can reduce the function of biological effectors, e.g., Fc receptors (FcγRs) or C1q binding. FcγR and C1q binding can be reduced by Specific regions required for cγR or C1q interaction, immunoglobulin constant region segments of antibodies This can be reduced by mutating the ment (e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491, Lund et al., 1991, J. Immunol. 147:2657-2662, Lo. et al See Wang et al., 2018, Protein Cell 9:63-73. ).

[0035] The reduction in FcγR binding ability of antibodies may also be due to the presence of other effectors that are dependent on FcγR interactions. - Functions such as opsonization, phagocytosis, and antibody-dependent cellular cytotoxicity ("ADCC") are also low. Although reduced C1q binding may reduce complement dependent cytotoxicity ("CDCC"). Thus, by reducing or eliminating effector function, targeting by the ADCs of the present disclosure may be possible. This can prevent the T cells that are activated from being destroyed via ADCC or CDC. In some embodiments, the effector functions of the antibody are mediated by selective activation of the Fc portion of the antibody. Mutationally modified, so that they maintain antigen specificity and internalization capacity, but are not ubiquitous in ADCs. C / CDC function is eliminated.

[0036] Numerous mutations that reduce FcγR and C1q binding have been described in the art. Such mutations may be included in the ADCs of the present disclosure. In the specification of 737,056, positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, Fc region at a single position at 376, 414, 416, 419, 435, 438, or 439 Amino acid modifications in the IL-1 domain result in reduced binding to FcγRII and FcγRII In U.S. Patent No. 9,790,268, it is disclosed that the amino acid at position 29 is an asparagine residue at position 8, and a serine or threonine residue at amino acid position 300 It has been disclosed that the antibody reduces FcγR binding. In the 190441 brochure, L234D / L235E:L234R / L235R / E233K, L234D / L235E / D265S:E233K / L234R / L235 R / D265S, L234D / L235E / E269K:E233K / L234R / L2 35R / E269K, L234D / L235E / K322A:E233K / L234R / L235R / K322A, L234D / L235E / P329W:E233K / L234 R / L235R / P329W, L234D / L235E / E269K / D265S / K3 22A:E233K / L234R / L235R / E269K / D265S / K322A, L234D / L235E / E269K / D265S / K322E / E333K:E233 K / L234R / L235R / E269K / D265S / K322E / E333KSudden change Modified Fc domains with reduced FcγR binding having a mutation have been described, wherein The set of mutations preceding the semicolon are in the first Fc polypeptide, The following mutations are in the second Fc polypeptide of the Fc dimer. FcγR receptor binding As well as mutations that can reduce C1q binding, N297A, N297Q, N297G, D265A / N297A, D265A / N297G, L235E, L234 A / L235A, and L234A / L235A / P329A (Lo. et al. , 2017, J Biol Chem 292: 3900-08, Wang et al., 2018, Protein Cell 9:63-73).

[0037] The constant region is mutated to reduce effector function, e.g., the Fc domain described above. Instead of mutating the antibody fragment, effector functions can be mutated by modifying the antibody fragment (e.g., Fab, Fab' , or F(ab')2 fragments).

[0038] In another embodiment of the present disclosure, the antibody or fragment thereof is modified to have a specific affinity to the unmodified antibody. Gaining or improving the function of at least one constant region-mediated biological effector. For example, FcγR interactions can be improved (see, e.g., U.S. Pat. No. 5,333,363). For example, the antibodies of the present disclosure may bind to FcγR IIA, FcγRIIB, and / or FcγRIIIA, respectively, with the corresponding wild-type constant regions It may have a constant region that binds with higher affinity.

[0039] Thus, the antibodies of the present disclosure have bioactivities that result in reduced opsonization, phagocytosis, or ADCC. Such alterations may have altered biological activity. Such alterations are known in the art. For example, Modifications in antibodies that reduce DCC activity are described in U.S. Pat. No. 5,834,597. It is listed.

[0040] In yet another embodiment, the antibody or fragment thereof is selected from the group consisting of antibodies, e.g., those involved in FcRn interactions. The immunoglobulin constant region segments are modified by mutating the immunoglobulin constant region segments in specific regions, Antibodies or antibodies that increase or decrease their binding affinity to the fetal Fc receptor, FcRn. or a fragment thereof (see, for example, WO 2005 / 123780). Such mutations can enhance the binding of antibodies to FcRn and block the binding of antibodies to FcRn. This protects it from solution and extends its half-life.

[0041] In yet other embodiments, the antibody is prepared as described, for example, in Jung and Pluckthun, 1997, Protein Engineering neering 10(9):959-966, Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9, and its ultra- It has one or more amino acids inserted into one or more of the variable regions.

[0042] The target of the antibody depends on the desired therapeutic use of the ADC. Typically, the target is an ALK5 inhibitor. A molecule present on the surface of a desired cell, such as a T cell, to which a cytotoxic agent is delivered. An antibody is Preferably, upon binding to the target, the antibody is internalized. Internalizing antibodies are described, for example, in Franke et al., 2 000, 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 It is described in.

[0043] Uses in which the ADC is intended to stimulate the immune system by reducing TGF-β activity For the purpose of this invention, it is desirable to generate antibodies that bind to T cell surface molecules. Without the need for ALK5 inhibitors, delivery of ALK5 inhibitors to T cells, particularly CD4 + and / or CDs 8 + It can activate T cell activity and inhibit regulatory T cell activity, both of which are important in the treatment of tumors. Thus, the T cell surface molecules in the ADCs of the present disclosure are believed to contribute to immune tolerance of tumors. The use of antibodies that bind to the IL-1 ... In various embodiments, the antibody is a CD4 + T cells, CD8 + T cells, T RE G In some embodiments, the antibody binds to a cell, Binds to pan-T cell surface molecules. Examples of T cell surface molecules suitable for targeting include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD2 5, CD28, CD70, CD71, CD103, CD184, Tim3, LAG3, C These include T-cell surface molecules, TLA4, and PD1. Examples of antibodies that have been used include OKT6 (anti-CD1, ATCC Accession No. CRL8020), OK T11 (anti-CD2, ATCC accession number CRL8027), OKT3 (anti-CD3, ATCC Accession number CRL8001), OKT4 (anti-CD4, ATCC accession number CRL8002), OKT8 (anti-CD8, ATCC accession number CRL8014), 7D4 (anti-CD25, ATC C accession number CRL1698), OKT9 (anti-CD71, ATCC accession number CRL8021 ), CD28.2 (anti-CD28, BD Biosciences Catalog No. 55662 0), UCHT1 (anti-CD3, BioXCell Catalog No. BE0231), M290 (anti-CD103, BioXCell catalog number BE0026), and FR70 (anti-C D70, BioXCell catalog number BE0022).

[0044] In some embodiments, the targeted T cell surface molecule is delivered via an endosome. It is a T cell surface molecule that can recycle to the cell surface after internalization (Goldenring, (See 2015 Curr. Opin. Cell Biol., 35:117-22.) Recycling via endosomes Exemplary T cell surface molecules believed to be capable of regulating T cell proliferation include CD5 and CD7. Without being bound by theory, they can be recycled through endosomes. Targeting T cell surface molecules also allows ALK5 to recycle through endosomes. This is thought to facilitate the delivery of ALK5 inhibitors to ALK5. Thus, targeting T cell surface molecules that can be recycled through endosomes is Doing so may help bring the ALK5 inhibitor into close proximity to ALK5.

[0045] ALK5 inhibitors The ALK5 inhibitors of the present disclosure preferably competitively and reversibly inhibit the cytoplasmic activity of the ALK5 receptor. A small molecule that binds to the ATP-binding site in the kinase domain and prevents downstream R-Smad phosphorylation. He is a child.

[0046] ALK5 inhibitors inhibit the binding of ALK5 to other TGF-β family receptors, such as ALK4 and and / or ALK7 and / or TGF-β receptor II specific or It may be, but need not be, selective. The inhibitors have activity against both ALK5 and TGF-β receptor II. While it is preferred that the inhibitor has limited inhibitory activity against the BMP II receptor, This means that the ADCs disclosed herein target T cells that have minimal or no BMP II activity. This is not necessary because

[0047] from at least 3 subjects, at least 5 subjects, or at least 10 subjects As measured in an in vitro cellular assay using T cells, the ALK5 inhibitors of the present disclosure IC of harmful agent 50 is preferably 100 nM or less, more preferably 50 nM or less, and most preferably or 20 nM or less. An exemplary cellular assay is described in Section 5.6 below. If DCs target human but not mouse T cell surface molecules, they will target human instead of mouse cells. Uses mouse cells and antibodies that recognize human instead of mouse CD28 and CD3 It is possible.

[0048] Specific examples of ALK5 inhibitors suitable for use in the antibody-drug conjugates of the present disclosure include: Imidazole-benzodioxole compounds, imidazole-quinoxaline compounds, pyrazolidine Examples of suitable aryl-pyrrolo compounds include aryl-pyrrolo compounds, and thiazole-based compounds.

[0049] According to one embodiment of the disclosure, the imidazole-benzodioxole ALK5 inhibitor is , which has the formula:

[0050] [ka]

[0051] In the formula, R 1 is hydrogen or lower alkyl having 1 to about 5 carbon atoms; R 2 is hydrogen or lower alkyl having 1 to about 5 carbon atoms; R 3 is an amide, nitrile, alkynyl having 1 to about 3 carbon atoms, carboxyl, or A is a direct bond or an alkanol having from 1 to about 5 carbon atoms. and B is a direct bond or an alkyl group having 1 to about 5 carbon atoms. In another preferred embodiment of the present disclosure, R 2 is hydrogen or methyl A has 1 carbon atom, B is a direct bond to a benzyl group, and R 3 is an amide In a combined preferred embodiment of the present disclosure, R 2 is hydrogen or methyl where A has 1 carbon atom and B is a direct bond to the benzyl group.

[0052] In accordance with another aspect of the present disclosure, the imidazole-quinoxaline ALK5 inhibitor is It has the following formula:

[0053] [ka]

[0054] In the formula, R 1 is hydrogen or lower alkyl having 1 to about 5 carbon atoms; R 2 is hydrogen, halogen, or lower alkyl having 1 to about 5 carbon atoms; R 3 teeth , amide, nitrile, alkynyl having 1 to about 3 carbon atoms, carboxyl, or A is an alkanol having 1 to about 5 carbon atoms, and A is a direct bond or and B is a direct bond or an alkyl group having 1 to about 5 carbon atoms. In another preferred embodiment of the present disclosure, R 2 is hydrogen or methyl halogen includes fluorine or chlorine; A has 1 carbon atom; B is , a direct bond to the benzyl group, and R 3 is an amide. In one embodiment, R 2 is hydrogen or methyl, A has 1 carbon atom, B is a direct bond to the benzyl group.

[0055] According to another embodiment of the disclosure, the pyrazole ALK5 inhibitor has the formula:

[0056] [ka]

[0057] In the formula, R 2 is hydrogen, halogen, or lower alkyl having 1 to about 5 carbon atoms. Yes, R 4is hydrogen, halogen, lower alkyl having 1 to about 5 carbon atoms, Alkoxy, haloalkyl, carboxyl, carboxyalkylene having 10 carbon atoms The alkyl group may be a ester, a nitrile, an alkylamine, or a group having the formula:

[0058] [ka]

[0059] In the formula, R 5 is a lower alkyl having 1 to about 5 carbon atoms, a halogen, or a morpho Reno, R 6 are pyrrole, cyclohexyl, morpholino, pyrazole, pyran, and isopropyl. midazole, oxane, pyrrolidinyl, or alkylamine; A is a direct bond; Or alkyl having 1 to about 5 carbon atoms.

[0060] In accordance with another embodiment of the present disclosure, the pyrazole-pyrrolo ALK5 inhibitor has the formula: Yes.

[0061] [ka]

[0062] In the formula, R 7 is hydrogen, halogen, lower alkyl having 1 to about 5 carbon atoms, alkoxy is an alkylamine, morpholino, or alkylamine; R 2 is hydrogen, halogen, or 1 is a lower alkyl having about 5 carbon atoms; R 8 is hydrogen, hydroxyl, amino , a halogen, or a group having the formula:

[0063] [ka]

[0064] In the formula, R 5 is piperazinyl, R 6 are morpholino, piperidinyl, and piperazinyl alkyl, alkoxy, hydroxyl, oxane, halogen, thioalkyl, or alkyla and A is a lower alkyl having 1 to about 5 carbon atoms.

[0065] According to another embodiment of the disclosure, the thiazole ALK5 inhibitor has the formula:

[0066] [ka]

[0067] In the formula, R 9 is hydrogen, halogen, or lower alkyl having 1 to about 5 carbon atoms. Yes, R 10 is hydrogen or lower alkyl having 1 to about 5 carbon atoms.

[0068] In certain embodiments, the ALK5 inhibitor is a compound designated A-N in Table 1 below. The compound is selected from any one of the following:

[0069] [Table 1-1]

[0070] [Table 1-2]

[0071] [Table 1-3]

[0072] In a more specific embodiment, the ALK5 inhibitor is one of the compounds designated 1-283 in Table 2 below. The compound is selected from any of the compounds listed above.

[0073] [Table 2-1]

[0074] [Table 2-2]

[0075] [Table 2-3]

[0076] [Table 2-4]

[0077] [Table 2-5]

[0078] [Table 2-6]

[0079] [Table 2-7]

[0080] [Table 2-8]

[0081] [Table 2-9]

[0082] [Table 2-10]

[0083] [Table 2-11]

[0084] [Table 2-12]

[0085] [Table 2-13]

[0086] [Table 2-14]

[0087] [Table 2-15]

[0088] [Table 2-16]

[0089] [Table 2-17]

[0090] [Table 2-18]

[0091] The preparation and use of ALK5 inhibitors is well known in the scientific and patent literature, This is well documented. PCT Publication WO 2000 / 61576, and U.S. Patent Application Publication No. 2003 / 0149277, which discloses triaryl imidazolidins. The present application discloses azole derivatives and their use as ALK5 inhibitors. In WO 2001 / 62756, pyridinyl imidazole derivatives and and its use as an ALK5 inhibitor. 2 / 055077 brochure, Imidazolyl cyclic acetamides as ALK5 inhibitors The use of phenyl derivatives is disclosed in PCT Publication No. WO 2003 / 087304. In FRET, tri-substituted heteroaryls and ALK5 and / or ALK4 inhibition Its use as an anti-inflammatory agent is disclosed in WO 2005 / 103028. No. 2008 / 0319012, and U.S. Pat. No. 7,958 discloses 2-pyridyl ALK5 and / or ALK4 inhibitors. Substituted imidazoles are disclosed. One representative compound, IN-1130, has several It exhibits ALK5 and / or ALK4 inhibitor activity in several animal models. The patent and publications provide additional examples of ALK5 inhibitors, including exemplary synthesis schemes, and Methods of using ALK5 inhibitors are provided: U.S. Pat. No. 6,465,493; US Patent No. 6,906,089, US Patent No. 7,365,066, US Patent No. No. 7,087,626, U.S. Pat. No. 7,368,445, U.S. Pat. No. 7,265,225, U.S. Pat. No. 7,405,299, U.S. Pat. 407,958, U.S. Pat. No. 7,511,056, U.S. Pat. No. 7,61 No. 2,094, U.S. Pat. No. 7,691,865, U.S. Pat. No. 7,863, 288, U.S. Pat. No. 8,410,146, U.S. Pat. No. 8,410,14 No. 6, U.S. Patent No. 8,420,685, U.S. Patent No. 8,513,222 Specification, U.S. Patent No. 8,614,226 Specification, U.S. Patent No. 8,791,113 Specification No. 8,815,893; No. 8,846,931; U.S. Patent No. 8,912,216, U.S. Patent No. 8,987,301, U.S. Patent No. 9,051,307, U.S. Pat. No. 9,051,318, U.S. Pat. No. 9,073,918 and PCT Publication No. WO 2004 / 06539 2, International Publication No. 2009 / 050183, International Publication No. Pamphlet No. 09 / 133070, Pamphlet No. WO 2011 / 146287 and WO 2013 / 009140. The aforementioned patents and patent publications is incorporated by reference in its entirety.

[0092] Several ALK5 inhibitors are commercially available, including SB-525334 (CAS 356 559-20-1), SB-505124(CAS 694433-59-5), SB- 431542 (CAS 301836-41-9), SB-202474 (EMD4 B iosciences Merck KGaA, Darmstadt, Germany) , LY-364947(CAS 396129-53-6), IN-1130, GW-7 88388, and D4476 (EMD4 Biosciences Merck KG aA, Darmstadt, Germany).

[0093] The structures and names of the ALK5 inhibitors described herein may be used in combination with antibodies and / or linkers. This refers to a molecule before it is bound to a ligand.

[0094] Preferred ALK5 inhibitors are those that are substituted with a free NH or NH2 group, preferably an alkyl, heteroaryl, NH or NH2 groups bonded to alkyl, heteroaryl, or aryl groups Attached to the linker via the aryl or NH or NH2 moiety of the aryl group (For example, compounds 1 to 23, 26 to 29, and 31 shown in Table 2) , 35, 37, 39, 40, 42, 43, 45-48, 50-85, 87-90, 93, 96, 98~104, 106, 108, 109, 111, 112, 114, 116~12 0, 132, 146, 149, 156, 184, 187, 193, 218, 260-27 7, 282, and 283). ALK5 inhibitors can be derivatized to free NH or NH2 groups. The design of the derivatized ALK5 inhibitors preferably involves the addition of a transactivation inhibitor. Although it can be determined empirically, adding NH or NH2 groups abolishes inhibitor activity. To reduce the possibility of Exemplary derivatized counterparts of some of the compounds shown in Table 1 are shown in Table 3 below. will be done.

[0095] [Table 3]

[0096] Linker Typically, the ADC includes a linker between the ALK5 inhibitor and the antibody. A moiety that contains a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. In the form, the linker may be an alkyldiyl, an aryldiyl, a heteroaryldiyl, Divalent radicals such as -(CR2) n O(CR2) n -, alkyloxy (e.g. polyethylene ethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethylene Amino, Jeffamine™ repeat units, and succinate moieties. , succinamide, diglycolate, malonate, and caproamide Examples include esters and amides.

[0097] The linker may be one or more linker moieties, such as a stretcher and a spacer moiety. For example, a peptidyl linker can include two or more amino acids, as well as, optionally, and containing one or more stretcher and / or spacer peptidyl moieties. A variety of linker moieties are known in the art, some of which include , as described below.

[0098] The linker may be a "cleavable linker" that facilitates release of the drug within the cell. For example, , acid labile linkers (e.g. hydrazones), protease sensitive linkers (e.g. peptidyltransferases), linker, photolabile linker, dimethyl linker, or disulfide-containing phosphorus linker. Chari et al., 1992, Cancer Research 52:127-131; U.S. Pat. No. 5,208,022 0) can be used.

[0099] Examples of linkers and linker moieties known in the art include areimidocaproyl (mc); Maleimidocaproyl-p-aminobenzylcarbamate; Maleimidocaproyl yl-peptide-aminobenzylcarbamate linkers, e.g., maleimidocaproyl -L-phenylalanine-L-lysine-p-aminobenzylcarbamate, and maleate Midcaproyl-L-valine-L-citrulline-p-aminobenzylcarbamate (vc );N-Succinimidyl 3-(2-pyridyldithio)proprionate (N-Succinimidyl also known as imidyl 4-(2-pyridyldithio)pentanoate or SPP); 4-Succinimidyl-oxycarbonyl-2-methyl-2-(2-pyridyldithio)- Toluene (SMPT); N-Succinimidyl 3-(2-pyridyldithio)propionate N-Succinimidyl 4-(2-pyridyldithio)butyrate (SPDP); B); 2-Iminothiolane; S-Acetyl succinic anhydride; Disulfide benzyl carbame ester;carbonate;hydrazone linker;N-(α-maleimidoacetoxy)succinate Imidoester;N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pi Lysyldithio)propionamide (AMAS); N[β-maleimidopropyloxy]s Succinimide ester (BMPS); [N-ε-Maleimidocaproyloxy]succinimide Imido ester (EMCS); N-[γ-maleimidobutyryloxy]succinimide ester Succinimidyl-4-[N-maleimidomethyl]cyclohexane (GMBS); -1-Carboxy-[6-amidocaproate] (LC-SMCC); Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate (LC-SPD P); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-Succinimidyl [4-iodoacetyl]aminobenzoate (SIAB); Maleimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SM CC); N-Succinimidyl 3-[2-pyridyldithio]-propionamide (SPD P); [N-ε-maleimidocaproyloxy]sulfosuccinimide ester (sulfo -EMCS); N-[γ-maleimidobutyryloxy]sulfosuccinimide ester ( Sulfo-GMBS; 4-Sulfosuccinimidyl-6-methyl-α-(2-pyridyldi Thio)toluamide]hexanoate-)(sulfo-LC-SMPT); Sulfosuccini 6-(3'-[2-pyridyldithio]-propionamido)hexanoate(sulfonyl) e-LC-SPDP); m-Maleimidobenzoyl-N-hydroxysulfosuccinimide N-Sulfosuccinimidyl [4-iodoacetyl] ester (sulfo-MBS); Sulfosuccinimidyl 4-[N-maleimide Methyl]cyclohexane-1-carboxylate (Sulfo-SMCC); Sulfosuccinic acid ethylene glycol 4-[p-maleimidophenyl]butyrate (sulfo-SMPB); Licor-bis(succinic acid N-hydroxysuccinimide ester) (EGS); DST (Dysprosyltransferase); 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid (DOTA);Diethylenetriamine-pentaacetic acid (DTPA); thiourea linkers; and oxime-containing linkers.

[0100] In some embodiments, the linker is cleavable under intracellular or extracellular conditions. Thus, cleavage of the linker releases the ALK5 inhibitor from the antibody in the appropriate environment. In yet other embodiments, the linker is not cleavable and the drug is, for example, It is released by degradation of the antibody in the endothelium (U.S. Patent Application Publication No. 2005 / 023864 No. 9, which is incorporated herein by reference in its entirety for all purposes. ).

[0101] Examples of non-cleavable linkers that can be used in the ADCs of the disclosure include N-maleimines. cyclohexane 1-carboxylate, maleimidocaproyl, or mercapto and acetamidocaproyl linkers.

[0102] In some embodiments, the linker is a polypeptide that is expressed in an intracellular environment (e.g., a lysosome or The linker can be cleaved by a cleavage agent present in the endosome or caveolae. Intracellular, including, but not limited to, lysosomal or endosomal proteases It may be a peptidyl linker that is cleaved by a peptidase or protease enzyme. In some embodiments, the peptidyl linker is at least 2 amino acids in length, or It contains a peptidyl moiety that is at least three amino acids in length, or longer.

[0103] Cleavage agents may include, but are not limited to, cathepsins B and D, and plasmin. All of these hydrolyze dipeptide drug derivatives that release the active drug within the target cell. It is known that For example, peptidyl linkers can be used to bind thiol-dependent proteases such as cathepsin. Linker B (e.g., Phe-Leu, or Gly-Phe-Leu-Gly linker) Other examples of such linkers are described, for example, in U.S. Pat. No. 45, the entirety of which is incorporated herein by reference for all purposes. Be absorbed.

[0104] In some embodiments, the peptidyl lysate is cleavable by an intracellular protease. The linker is a Val-Cit linker or a Phe-Lys linker (e.g., U.S. Patent No. 6,214,344 describes the synthesis of doxorubicin with an l-cit linker. (See specification no. 5).

[0105] In other embodiments, the cleavable linker is pH sensitive, i.e., at a certain pH Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. Degradable. For example, an acid-labile linker ( For example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amines, orthoesters, acetals, ketals, etc.) can be used (e.g., U.S. Patent No. 5,122,368, U.S. Patent No. 5,824,805, U.S. Patent No. 5,622,929, Dubowchik and Walker, 1999, Pharm. Therapeutic (See, e.g., J. Med. Soc. 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions such as those in blood, but are stable under the pH of lysosomes. In certain embodiments, the hydrolyzed Degradable linkers include thioether linkers (e.g., those that can be degraded via an acylhydrazone bond). thioethers that bind to the agent (see, e.g., U.S. Pat. No. 5,622,929). reference).

[0106] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., A variety of disulfide linkers are known in the art, For example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP ( N-Succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N- Succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N- Succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl- dithio)-toluene)-, SPDB, and SMPT can be used to form (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 (C.W. Vogel ed., Oxford U. Press, 1987; see also U.S. Pat. No. 4,880 (See also specification 935)

[0107] In other embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anti Cancer Res. 15:1387-93), maleimidobenzoyl linker (Lau et al., 1995, Bioorg -Med-Chem. 3(10):1299-1304), or the 3'-N-amide analogue (Lau et al., 1995, Bibliography). oorg-Med-Chem. 3(10):1305-12).

[0108] In many cases, the linker is not substantially sensitive to the extracellular environment. In this case, "substantially insensitive to the extracellular environment" in the context of a linker means that the ADC Approximately 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, or Approximately 1% or less of the linkers are cleaved when the ADC is present in an extracellular environment (e.g., plasma). This means that

[0109] Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by assaying the ADC in plasma. Incubate for a given period of time (e.g., 2, 4, 8, 16, or 24 hours) and then The amount of free drug present in the solution can be determined by quantifying the amount of free drug present in the solution.

[0110] In other, non-mutually exclusive, embodiments, the linker is capable of promoting cellular internalization. In certain embodiments, the linker can be (i.e., in the context of the linker-therapeutic agent portion of the ADCs described herein), In yet another embodiment, the linker is a linker that is capable of binding to the ALK5 inhibitor and It promotes cellular internalization when conjugated to both a ribozyme and an antibody.

[0111] In many embodiments, the linker is self-immolative. The term "self-immolative" refers to a compound that is covalently linked to two spaced apart chemical moieties to form a stable triad. It refers to a bifunctional chemical moiety that can be broken down into two groups when its bond to the first moiety is cleaved. In some cases, the compound spontaneously separates from the second chemical moiety. No. 7 / 059404, International Publication No. 2006 / 110476, International Publication No. 2005 / 112919, International Publication No. 2010 / 062171 No. 2009 / 017394, International Publication No. 200 No. 7 / 089149, International Publication No. 2007 / 018431, International Publication No. 2004 / 043493 and International Publication No. 2002 / 083 See Brochure No. 180, which describes how drugs and cleavable substances are voluntarily self-immolating agents. The present study covers drug-cleavable substance conjugates linked through an anchor, all of which are The present invention is incorporated by reference in its entirety herein. Examples of self-immolative spacer units are set forth in Formula I below.

[0112] Various exemplary linkers that can be used in the present compositions and methods are described in PCT Publication No. International Publication No. 2004 / 010957, U.S. Patent Application Publication No. 2006 / 00 No. 74008, U.S. Patent Application Publication No. 2005 / 0238649, and U.S. No. 2006 / 0024317 (each of which is incorporated herein by reference in its entirety). (which is incorporated herein by reference in its entirety for this purpose).

[0113] The ADCs of the disclosure may be of Formula I below, where the antibody (Ab) is linked to an optional linker (L ) is conjugated to one or more ALK5 inhibitor drug moieties (D). Ab-(LD) p I

[0114] Thus, antibodies can be conjugated with drugs, either directly or through a linker. In Formula I, p represents the number of drugs per antibody (i.e., ALK5 Inhibitor) moieties, which may 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. Possibly. Further details of drug loading are provided in Section 4.5 below.

[0115] In some embodiments, the linker moiety is linked to another A linker component or "stretcher" may be included that links the antibody to the drug moiety. A suitable Stretcher is shown below, where the wavy line on the left indicates the site of covalent attachment to the antibody. and the wavy line on the right indicates the site of covalent attachment to another linker component or drug moiety.

[0116] [ka] U.S. Pat. No. 9,109,035; Ducry et al., 2010, Bioconjugate Chem. 21: See 5-13.

[0117] In some embodiments, the linker component may include an amino acid unit. In one embodiment, the amino acid unit allows for cleavage of the linker by a protease, This allows the release of drugs from the ADC upon exposure to intracellular proteases, such as lysosomal enzymes. The release of substances is promoted. 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 the like. Exemplary dipeptides include valine, tripeptides, and pentapeptides. - Citrulline (VC or val-cit), Alanine-Phenylalanine (AF or phenylalanine-lysine (FK or phe-lys), Examples include N-methyl-valine-citrulline (Me-val-cit). Examples of amino acids include glycine-valine-citrulline (gly-val-cit) and The amino acid units include: gly-gly-gly. Contains naturally occurring amino acid residues, as well as a few amino acids and non-natural amino acid analogs. For example, the citrulline amino acid unit acts as a stimulator of certain enzymes, such as cathepsins B, C, and Designed and optimized for selection against enzymatic cleavage by proteases D and D, or plasmin It can be made into

[0118] In some embodiments, the linker moiety is either directly or in combination with a stretcher and "Spacer" units that link the antibody to the drug moiety, either by the " units. Spacer units can be "self-immolative" or "non-self-immolative". A "non-self-immolative" spacer unit is one in which some or all of the spacer units are attached to an enzyme (e.g., A spacer that remains attached to the drug moiety upon cleavage by a protease, for example. Exemplary units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. "Self-immolative" spacer units are spacer units that do not require a separate hydrolysis step. In certain embodiments, the linker's spectrometer can be used to selectively release the drug moiety without the need for a cyclodextrin. In one such embodiment, the catalyst unit comprises a p-aminobenzyl unit. Aminobenzyl alcohol is linked to the amino acid unit via an amide bond and carbamate , methyl carbamate, or carbonate are formed between benzyl alcohol and cytotoxic agents. See, for example, Hamann et al., 2005, Expert Opin. Ther. Patents 15:1087-1103. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl ( In certain embodiments, the phenylene moiety of the p-aminobenzyl unit is Minutes are Q m where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl -aryl), -halogen, -nitro, or -cyano, and m is an integer ranging from 0 to 4. Examples of self-immolative spacer units include, but are not limited to, p-aminobenzyl alcohol. and aromatic compounds that are electronically similar to the aromatic compounds described in U.S. Patent Application Publication No. 2005 / 025603. No. 0), e.g. 2-aminoimidazole-5-methanol derivatives (Hay et al. l., 1999, Bioorg. Med. Chem. Lett. 9:2237), and ortho- or para-aminobenzamide. Further examples include substituted and unsubstituted 4-aminobutyric acid amides (Rodrig et al. ues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1 ] and the bicyclo[2.2.2] ring system (Storm et al., 1972, Amer. Chem. Soc. 94:5815 ), and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Chem. 55:5867) is used, which undergoes ring formation upon hydrolysis of the amide bond. The exclusion of amine-containing drugs that are substituted at the a-position of glycine (Kingsbury et al. l., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer useful in ADCs. be.

[0119] In one embodiment, the spacer unit is a branched bis(hydroxymethyl) (BHMS) units, which are used to incorporate and release multiple drugs. It is possible.

[0120] [ka] where Ab and D are defined above for Formula I, and A is a stretcher where a is an integer ranging from 0 to 1, W is an amino acid unit, and w is an integer ranging from 0 to 1. 2, and Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; and n is , 0 or 1, and p ranges from 1 to about 20.

[0121] The linker may include any one or more of the linker components described above. In the form, the linker is shown in brackets in the formula of the ADC below: Ab-(-[Aa-Ww-Yy]-D) p II where Ab, A, a, W, w, D, and p are defined in the previous paragraph, and Y is (wherein y is a pacer unit and y is 0, 1, or 2). Exemplary of such linkers are Embodiments are described in U.S. Patent Application Publication No. 2005 / 0238649, which , which is incorporated herein by reference.

[0122] Exemplary linker moieties and combinations thereof are shown below in the context of an ADC of Formula II:

[0123] [ka]

[0124] Linker components, including stretcher, spacer, and amino acid units, can be prepared using methods known in the art. Methods known in the art, such as those described in U.S. Patent Application Publication No. 2005 / 0238649. It can be synthesized by

[0125] 4.5. Drug loading Drug loading is represented by p, the average number of ALK5 inhibitor moieties per antibody in the molecule. The drug loading ("p") is approximately 1 per antibody, although the average number is often a fraction or decimal. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20 or more moieties (D). On average, there are 2 to 8 drug moieties per antibody, more preferably 2 to 4 drug moieties per antibody. This results in approximately 5 to 7 drug moieties per antibody.

[0126] As will be appreciated by those of skill in the art, in many instances reference to ADCs (sometimes referred to as pharmaceutical compositions) is intended to ADC is shorthand for a collection or population of ADC molecules, each of which may contain one or more or multiple ALK5 inhibitor moieties are covalently linked to an antibody, with the ratio of distinct molecular bases being Although drug loading ratios can vary from ADC molecule to ADC molecule in a population, the drug loading ratios are based on the average drug loading ratio of a population or population. In some embodiments, the population or assemblage represents a loading of 1-30 drug moieties. In some embodiments, 1 to 20, 1 to 15, 2 to 12, or an ADC molecule comprising an antibody covalently attached to any of 2-8 drug moieties. Preferably, the population average is as described in the previous paragraph, e.g., for antibodies. More preferably, there are 2 to 8 drug moieties per antibody, or 4 to 8 drug moieties per antibody. Each has 5 to 7 drug moieties.

[0127] Several ADC populations include the ADCs described herein and antibody fragments lacking a drug moiety. The antibody may be in the form of a composition comprising a somatic molecule, for example an antibody that fails to bind to an ALK5 antibody.

[0128] In preparing an ADC from a conjugation reaction, the ratio of ALK5 inhibitor moieties per antibody The average number is characterized by conventional means such as mass spectroscopy and ELISA assays. do.

[0129] The quantitative distribution of the ADC with respect to p can also be determined. Isolation, purification, and characterization of a single ADC with other ALK5 inhibitors A certain value from 0.01 to 0.1 can be achieved by means such as electrophoresis.

[0130] For some antibody-drug conjugates, p is limited by the number of binding sites on the antibody. For example, when the bond is a cysteine ​​thiol, the above exemplary embodiment can be wherein the antibody may have only one or more cysteine ​​thiol groups, or , one or more sufficiently reactive thiol groups to which a linker can be attached. In certain embodiments, at higher drug loadings, e.g., p>5, Certain antibody-drug conjugates may become aggregated, insoluble, toxic, or have reduced cell permeability. In certain embodiments, drug loading on the ADCs of the disclosure can range from 1 to about 8 , about 2 to about 6, about 3 to about 5, about 3 to about 4, about 3.1 to about 3.9, about 3.2 to about 3.8, about 3.2 to about 3.7, about 3.2 to about 3.6, about 3.3 to about 3.8, or about 3.3 to about 3. In fact, for a given ADC, the optimal ratio of drug moieties per antibody is It has been shown that the molecular weight can be less than 8 and can be from about 2 to about 5. See US Pat. No. 5,399,649, which is incorporated herein by reference in its entirety. .

[0131] In certain embodiments, less than the theoretical maximum drug moiety is present in the conjugation reaction. The antibody is conjugated to the antibody during the - may contain lysine residues that do not react with the intermediate or linker reagent. does not contain many free reactive cysteine ​​thiol groups that can be linked to a drug moiety; Indeed, many cysteine ​​thiol residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody is reacted with dithiothreitol under partial or total reducing conditions. A reducing agent such as dichloromethane (DTT) or tricarbonylethylphosphine (TCEP) to generate a reactive cysteine ​​thiol group. In the method, the antibody is subjected to denaturing conditions to remove reactive nucleophilic groups such as lysine or cysteine. Represents.

[0132] The loading of ADCs (drug / antibody ratio) can be achieved by various techniques, e.g. (i) drug-linked antibody (ii) limiting the molar excess of the linker intermediate or linker reagent; (iii) limiting the reaction time or temperature; and (iv) limiting the number and location of cysteine ​​residues; Linker-drug bonds (see, e.g., PCT Publication WO 2006 / 034488) ThioMab as disclosed in Lett., which is incorporated herein by reference in its entirety. or ThioFab) to control the number and / or location of the This can be controlled by engineering the amino acid sequence of the antibody.

[0133] Two or more nucleophilic groups react with a drug-linker intermediate or linker reagent, followed by When reacted with a biological moiety reagent, the resulting product then comprises one or more It should be understood that the ADC compound is a mixture of ADC compounds having a distribution of drug moieties. The average number of drugs per mixture can be calculated by dual ELISA antibody assay. This is antibody-specific and drug-specific. Individual ADC molecules are They can be identified in a mixture by spectroscopy and HPLC, e.g., hydrophobic interaction chromatography. can be separated by

[0134] In some embodiments, homogeneous ADCs with a single loading value can be obtained by electrophoresis or It can be isolated from a complex mixture by chromatography.

[0135] 4.6. Formulation and Administration Suitable routes of administration of the ADC include, but are not limited to, oral, parenteral, rectal, transmucosal, intestinal Intrathecal, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intracavity, intraperitoneal, or intratumoral injection The preferred route of administration is parenteral, more preferably intravenous. Alternatively, the compound may be administered in a localized manner rather than systemically, for example, to a solid or blood tumor. The compounds can be administered via direct injection into the subject.

[0136] The immunoconjugates can be formulated according to known methods for preparing pharma- ceutically useful compositions. The ADC can be prepared by combining the mixture with a pharma- ceutical useful excipient. Sterile phosphate buffered saline is one example of a pharma- ceutical useful excipient. Additives are well known to those skilled in the art. See, for example, Ansel et al., Pharmaceutical Dosage Form rms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), and Gennaro ( ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990) and its revised edition.

[0137] In a preferred embodiment, the ADC is N-(2-acetamido)-2-aminoethane Sulfonic acid (ACES); N-(2-acetamido)iminodiacetic acid (ADA); N,N- Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES); 2-(N-morpho 3-(N-morpholino)ethanesulfonic acid (MES); 3-(N-morpholino)propanesulfonic acid (MO PS; 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO) and piperazine-N,N'-bis(2-ethanesulfonic acid) [Pipes] Formulated in Good's biological buffer (pH 6-7) using a buffer selected from the group More preferred buffer solutions are those with a concentration in the range of 20 to 100 mM, more preferably about MES or MOPS at a concentration of 25 mM. Most preferred is 2 mM MES at pH 6.5. The formulation contained 25 mM trehalose as an additive, and 0.01 % v / v polysorbate 80, and as a result of the added additives, the final buffer The concentration is adjusted to 22.25 mM. The preferred method of storage is lyophilization of the conjugate. As a formulation, it is stored at a temperature ranging from -20°C to 2°C, most preferably at 2°C to 8°C. It is stored in.

[0138] The ADC may be administered intravenously, for example, via bolus injection, slow infusion, or continuous infusion. Preferably, the ADC can be formulated for administration for less than about 4 hours, more preferably for administration for less than about Infused over less than 3 hours. For example, the first 25-50 mg should be infused within 30 minutes, preferably Inject 15 minutes at a time and inject the remainder over the next 2 to 3 hours. The formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may be in any form, such as suspensions, solutions, or emulsions in oily or aqueous vehicles. The pharmaceutical composition may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be dissolved in a suitable vehicle, such as a sterile pipette, prior to use. It may be in powder form for constitution with halogen-free water.

[0139] Additional pharmaceutical methods can be utilized to control the duration of action of the ADC. Release preparations can be prepared through the use of polymers to complex or adsorb the ADC. For example, poly(ethylene-co-vinyl acetate) can be used as a biocompatible polymer. A matrix of stearic acid dimer and a polyanhydride copolymer of sebacic acid. Sherwood et al., 1992, Bio / Technology 10:1446. The release rate of an ADC from a matrix such as It depends on the amount of DC and the size of the dispersed particles. Saltzman et al., 1989, Biophys. J. 5 5:163, Sherwood et al., supra. Other solid dosage forms are described in Ansel et al., Pharmaceutical osage forms and drug delivery systems, 5th edition (Lea & Febiger 1990), and G ennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company 1990) and its revised editions.

[0140] In general, the dose of an ADC administered to a human depends on the patient's age, weight, height, sex, and total body mass. Approximately 0.3mg / kg to 5mg, depending on factors such as health status and past medical history. Doses of the ADC in the range of 1 / kg can be provided to recipients as a single intravenous infusion. Lower or higher doses may be administered depending on the circumstances, although this may be desirable. For example, a dose of 0.3 to 5 mg / kg for a 70 kg patient is 21 ~350 mg, which is 12–20 mg for a 1.7 m patient. 6 mg / m 2 At a dosage of Dosage may be adjusted as needed, e.g., once weekly for 2-10 weeks, once weekly for 8 weeks, or once weekly for 10 weeks. This can be repeated once a week for 12 weeks or once a week for 4 weeks. Depending on the situation, less frequently, for example, every other week for several months or It may be given monthly or quarterly for up to 12 weeks. Not specified, but 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. A more preferred dosage is 0.6 mg / kg per week. / kg and 1.2 mg / kg for less frequent administration. Any amount ranging from 1 g / kg may be used. Dosage is preferably 1 g / kg per week. The drug is administered once or multiple times for 4 weeks, more preferably for 8 weeks, more preferably for 16 weeks, or can use a longer minimum dose schedule, the frequency of administration being The schedule of administration depends on adverse side effects and recovery from them, most commonly associated with hematologic toxicity. The courses were: (i) once a week; (ii) every other week; (iii) one week of therapy followed by two weeks (iv) 2 weeks of therapy followed by 1 week, 2 weeks, 3 weeks, or 4 weeks of rest; (v) 3 weeks of therapy followed by 1 week, 2 weeks, 3 weeks, or 4 weeks of rest; or a 5-week break; (vi) 4 weeks of therapy followed by 1, 2, 3, or 4 weeks or a 5-week break; (vii) 5 weeks of therapy followed by 1, 2, 3, or 4 weeks (viii) once a month, or with a 5-week break; The cycle may include administration once or twice weekly at 100 mg / kg / day. This can be repeated 10, 12 or more times.

[0141] Alternatively, the ADC should be administered in one dose every two or three weeks for a total of at least three doses. It can be administered repeatedly or twice weekly for 4 to 6 weeks. Doses can be administered once every other week, or even less frequently, allowing patients may reverse any drug-related toxicity. can be shortened, i.e., every 2 or 3 weeks for 2 to 3 months. The dosing schedule can optionally be repeated at other intervals, and the dosages can be adjusted according to the dose and Can be administered via a variety of parenteral routes with appropriate schedule adjustments .

[0142] 4.7. Treatment Method The ADCs of the disclosure can be used to treat a variety of cancers. Associated with care agents or regimens, either as monotherapy or as part of a combination therapy regimen Suitable antibodies for inclusion in ADCs for the treatment of cancer include those that inhibit T cell Exemplary antibodies that target surface antigens are described in Section 4.2.

[0143] Examples of cancers that can be treated using the ADCs of the disclosure include, but are not limited to, pancreatic cancer. Cancer, glioblastoma, myelodysplastic syndrome, prostate cancer, liver cancer (e.g. hepatocellular carcinoma), Cancers of the bladder, urethra, and ureter are some of the most common cancers. Can be obtained.

[0144] For the treatment of melanoma carrying a BRAF mutation, the ADCs of the disclosure are BRAF mutation-specific anti-cancer drugs such as venurafenib, dabrafenib, and trametinib It can be used in combination with specifically targeting drugs.

[0145] For the treatment of malignant melanoma, the ADCs of the disclosure may be administered in combination with ipilimumab or nivolumab or Can be used in combination with checkpoint inhibitors such as pembrolizumab .

[0146] For the treatment of non-small cell lung cancer (NSCLC), the ADCs of the disclosure may be administered in combination with cisplatin, carboplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, Used in combination with standard-of-care chemotherapy treatments such as toposide or vinblastine In addition, the ADC can be used to treat targeted It can be used in combination with therapy.

[0147] For the treatment of bladder cancer, the ADCs of the disclosure may be used in combination with cisplatin, mitomycin C, calcitonin, Boplatin, docetaxel, paclitaxel, doxorubicin, 5-FU, methotrexate including, but not limited to, vinblastine, ifosfamide, and pemetrexed It can be used in combination with standard of care treatments.

[0148] For the treatment of renal cancer, the ADCs of the disclosure may be used in combination with standard of care treatments, such as angiogenesis and and / or drugs that block specific tyrosine kinases, e.g., sorafenib, sunitinib , in combination with temsirolimus, everolimus, pazopanib, and axitinib It is possible.

[0149] For the treatment of breast cancer, the ADCs of the disclosure may be used in combination with anthracyclines (doxorubicin or epirubicin), and taxanes (paclitaxel or docetaxel), as well as Standard-of-care chemotherapies such as fluorouracil, cyclophosphamide, and carboplatin In addition, the ADCs of the disclosure can be used in combination with targeted therapies. It can be used in combination with other drugs as a targeted therapy for HER2 / neu positive tumors. These include trastuzumab and pertuzumab, which are used in estrogen receptor (ER)-positive tumors. Targeted therapies for this include tamoxifen, toremifene, and fulvestrant. can be done.

[0150] For pancreatic cancer, the disclosed ADCs are cisplatin, oxaliplatin, paclitaxel, capecitabine, cisplatin, or docetaxel It can be used in combination with standard of care chemotherapy agents such as cisplatin. ADCs are used in combination with targeted therapies such as erlotinib, which inhibits EGFR. It is possible.

[0151] For glioblastoma, the ADCs of the disclosure are Standard of care, such as etoposide, lomustine, methotrexate, or procarbazine The present invention can be used in combination with other chemotherapeutic agents.

[0152] For prostate cancer, the ADCs of the disclosure comprise docetaxel, optionally with a steroid. In combination with standard-of-care chemotherapy agents, including prednisone or cabazitaxel It can be used. EXAMPLES

[0153] The following abbreviations are found throughout the examples. Boc - tert-butyloxycarbonyl DCM - dichloromethane DMA - Dimethylamine DMF - Dimethylformamide DIPEA - N,N-Diisopropylethylamine EtOAc - Ethyl acetate EtOH - Ethanol Fmoc - Fluorenylmethyloxycarbonyl HOBt - Hydroxybenzotriazole MeOH - Methanol NaHMDS - Sodium hexamethyldisilazide RT - Room temperature, approximately 21°C TBTU - O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl Thiuronium tetrafluoroborate TEA - Triethylamine THF - Tetrahydrofuran TFA - Trifluoroacetic acid TMS-Imidazole - 1-(Trimethylsilyl)imidazole

[0154] 5.1. [Example 1] 4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridin-2-yl)-1 Synthesis of ,3-thiazol-2-amine (compound A) Compound A was prepared according to the general method in Scheme 1 below.

[0155] [ka]

[0156] 5.1.1. 2-Methyl-1,5-naphthyridine (A1) Concentrated sulfuric acid (2.5 ml), sodium m-nitrobenzenesulfonate (2.08 g, 9. 24 mmol), boronic acid (445 mg, 7.21 mmol), and ferrous sulfate heptahydrate. A mixture of glycerol (1. 5 ml) followed by 5-amino-2-methylpyridine (A-SM) (500 mg, 4.6 2 mmol) and water (2.5 ml) were added to the reaction mixture and heated at 135° C. for 18 hours. After the reaction was complete as determined by TLC, the reaction mixture was cooled to approximately 21° C. and diluted with 4N N The mixture was basified using aOH and extracted with EtOAc (2 x 100 ml). The organic extracts were combined. The combined product was washed with water (200 ml), dried over Na2SO4, and evaporated under reduced pressure to give crude Compound A1 was obtained. The crude product was purified by silica gel column chromatography using (2% MeOH / CH2Cl2). Compound A1 (200 ml) was purified by column chromatography to give a light brown crystalline solid. g, 30%).

[0157] 1 H NMR (500 MHz, CDCl3): δ 8.92 (d, J = 3.0 Hz, 1H), 8.35 (d, J = 9.0 Hz, 1H) , 8.31 (d, J = 5.9 Hz, 1H), 7.62 (dd, J = 8.5, 4.5 Hz, 1H), 7.54 (d, J = 5.9 Hz, 1H), 2.8 (s, 3H)

[0158] LC-MS(ESI):m / z 145[M+H] +

[0159] 5.1.2. 1-(6-methylpyridin-2-yl)-2-(1,5-naphthyridine- 2-yl)ethan-1-one (A2) A1 (200 mg, 1.38 mmol) and methyl 6-methylpicolinate (209 A solution of 1.38 mg (1.38 mmol) in anhydrous THF (10 ml) was placed under a N2 atmosphere. Cool to -78°C. Potassium bis(trimethylsilyl)amide (0.5M in toluene) , 6.9 ml, 3.47 mmol) was added dropwise over a period of 5 minutes. The reaction mixture was The mixture was stirred at -78°C for 1 hour, then warmed to approximately 21°C and maintained for 20 hours. (determined by TLC), the reaction mixture was quenched with saturated ammonium chloride solution (20 ml). The aqueous layer was extracted with EtOAc (2×20 ml). The combined organic extracts were washed with water ( 100 ml), dried over Na2SO4 and evaporated to give crude compound A2. The material was purified by column chromatography (1% MeOH / CH2Cl2) to give Compound A2 (110 mg, 30.5%) was obtained as an orange-yellow solid.

[0160] 1 H NMR (400 MHz, CDCl3: enol form): δ 15.74 (brs,-OH), 8.69 (t, J = 3.6, 1H), 8.12 (d, J = 9.2 Hz, 1H), 8.06 (dd, J = 8.4, 4.4 Hz, 2H), 7.82 (t, J = 7.6 Hz, 1H), 7.55 (dd, J = 8.4, 4.8 Hz, 1H) 7.45 (d, J= 9.6 Hz,1H), 7.3 (dd, J = 7.6, 4.0 Hz, 1H), 7.16 (s, 1H), 2.75(s, 3H)

[0161] LC-MS(ESI):m / z 264[M+H] +

[0162] 5.1.3. 4-(6-methylpyridin-2-yl)-5-(1,5-naphthyridine- 2-yl)-1,3-thiazol-2-amine (Compound A) 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). Stir at approximately 21° C. for 1 h and then concentrate under reduced pressure to give crude A3 (120 mg). This was used in the next step without further purification. Crude A3 (120 mg) was The mixture was dissolved in 15 ml of thiourea (3.5 mg, 0.046 mmol). was added and the reaction mixture was stirred for 78 h (until complete consumption of starting material was observed by TLC). The reaction mixture was cooled to approximately 21° C. and ammonia solution (25%, 1.5 ml) was added with gentle stirring. The solvent was evaporated and the residue was dissolved in CH2 It was dissolved in Cl2 (2 x 20 ml) and washed with water (50.0 ml). The organic layer was washed with 1N HCl (30 ml x 2). The combined aqueous layers were washed with 35% hydroxide It was basified with aqueous sodium (20 ml) and extracted with CH2Cl2 (2 x 20 ml). The organic layer was dried over sodium sulfate and evaporated to give crude compound A. Compound A (35%) was purified by recrystallization from triethylamine (2 ml) as a yellow crystalline solid. mg, 49% yield over two steps).

[0163] 1 H NMR (400 MHz, CDCl3): δ 8.86 (dd, J = 4.4, 1.6 Hz, 1H), 8.29 (t, J = 8.4 H z, 1H), 8.06 (d, J = 9.2 Hz,1H), 7.64 (t, J = 7.6 Hz, 1H), 7.60-7.55 (m, 2H), 7 .46 (d, J = 8 Hz, 1H), 7.20 (d, J = 7.6, 1H), 5.32 (brs, 2H), 2.57 (s, 3H)

[0164] LC-MS(ESI):m / z 320[M+H] +

[0165] UPLC purity: 97.6%

[0166] 5.2. [Example 2] N-Methyl-2-(4-{4-[3-(pyridin-2-yl)-1H-pyrazole-4- Synthesis of (phenyl)pyridin-2-yl}phenoxy)ethan-1-amine (compound B) Compound B was prepared according to the general method in Scheme 2 below.

[0167] [ka]

[0168] 5.2.1. tert-Butyl (2-chloroethyl) (methyl)carbamate (B7) To a stirred solution of Boc-anhydride (1.7 ml, 7.30 mmol) in THF (4 ml) At the same time, a solution of B6 (1 g, 7.69 mmol) in water (4 ml) and TEA (1 A solution of 1 ml of 1,2-dichloromethane (7.69 mmol) in THF (4 ml) was added over 1 h. The resulting mixture was stirred for 16 hours at approximately 21° C. The reaction mixture was diluted with saturated NaCl solution (20 ml) and extracted with DCM (3×50 ml). The combined organic extracts were diluted with Na Drying over 2SO4 and concentration in vacuo gave the crude compound which was dissolved in 10% EtOAc / Hexane. The product was purified by silica gel column chromatography using hexane to give a pale yellow liquid. Compound B7 (1 g, 5.18 mmol, 71%) was obtained.

[0169] 1 H NMR (400 MHz, CDCl3): δ 3.58-3.52 (m, 4H), 2.93 (s, 3H), 1.46 (s, 9H)

[0170] 5.2.2. tert-Butyl methyl (2-(4-(4,4,5,5-tetramethyl- 1,3,2-Dioxaborolan-2-yl)phenoxy)ethyl)carbamate (Int -B) 4-Hydroxyphenylboronic acid pinacol ester (789 mg, 3.58 mmol) To a stirred solution of B7 (900 mg, 4.66 mmol), KI (18 mg, 0.10 mmol), and Cs2CO3 (2.57 g, 7.88 mmol ) was added under an argon atmosphere. 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 give the crude material, which was then extracted with 7% EtOAc / hexanes. The resulting mixture was purified by column chromatography using 100 mL of 1000 sachets to give Int-B (58 0 mg, 1.53 mmol, 43%) was obtained.

[0171] 1 H NMR (400 MHz, CDCl3):δ 7.74 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2 H) , 4.16-4.06 (m, 2H), 3.65-3.59 (m, 2H), 2.97 (s, 3H), 1.45 (s, 9H), 1.33 (s, 12H) )

[0172] 5.2.3. 2-(2-bromopyridin-4-yl)-1-(pyridin-2-yl) ester Tan-1-one (B2) 2-Bromo-4-methylpyridine (B1) (2 g, 11.62 mmol) in THF (3 To a stirred solution of 100 ml of NaHMDS (2M in THF, 1 A solution of 2.7 ml of 25.58 mmol) was added dropwise. The yellow solution was incubated at -78°C for 30 The mixture was stirred for 1 min. Then, ethyl picolinate (1.72 ml, 12.79 mmol) was added to TH A solution of F (10 ml) was added and the reaction mixture was warmed to approximately 21° C. and stirred for 16 h. The solvent was evaporated under reduced pressure and the solid residue was triturated with diethyl ether, filtered and The solid was then diluted with saturated NH4Cl solution (30 ml) and The organic phase was extracted with EtOAc (2 x 200 ml). The organic layer was dried over Na2SO4 and concentrated. The crude product was purified by silica gel column chromatography using 10% EtOAc / hexane. Compound B2 (2.06 g, 7.46 mmol) was purified by HPLC as a yellow solid. l, 64.3%).

[0173] 1 H NMR (400 MHz, CDCl3):δ 8.75 (d, J = 5.2 Hz, 1H), 8.32 (d, J= 5.2 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.89 (t, J =7.6 Hz 1H), 7.56-7.51(m, 2H), 7.28-7.25 (m , 1H), 4.55 (s, 2H)

[0174] LC-MS(ESI):m / z 277[M] +

[0175] 5.2.4. 2-Bromo-4-[3-(pyridin-2-yl)-1H-pyrazole-4 -yl]pyridine (B3) A solution of B2 (850 mg, 3.07 mmol) in dry DMF (3.4 ml) was added to Al The mixture was treated with glacial acetic acid (0.45 ml, 7.39 mmol) in DMF under atmospheric pressure. 0.6 ml, 4.61 mmol) was added dropwise and the mixture was incubated at approximately 21° C. for 2 hours in argon. The mixture was stirred under nitrogen atmosphere. Hydrazine monohydrate (1.15 ml, 23.09 mmol) was added dropwise. The resulting mixture was heated at 50° C. for 3 hours and at approximately 21° C. for 16 hours. The reaction mixture was poured into water (30 ml) and extracted with CH2Cl2 (3 x 30 ml). The organic layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure to give the crude compound. The crude product was purified by silica gel column chromatography using 30% EtOAc / hexane. Compound B3 (560 mg, 1.86 mmol) was purified by chromatography as a yellow solid. , 60.6%).

[0176] 1 H NMR (500 MHz, CDCl3):δ 8.74 (brs, 1H), 8.34 (d, J = 5.0 Hz, 1H), 7.83 (brs , 1H), 7.81 (t, J = 6.0 Hz, 1H), 7.56 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39-7. 84 (m, 1H), 7.31-7.26 (m, 1H)

[0177] LC-MS(ESI):m / z 301[M] +

[0178] 5.2.5. 2-Bromo-4-(3-(pyridin-2-yl)-1-trityl-1H- Pyrazol-4-yl)pyridine (B4) To a stirred solution of B3 (500 mg, 1.66 mmol) in acetone (10 ml), CO3 (1.37 g, 9.99 mmol) and trityl chloride (464 mg, 2.4 9 mmol) was added. The reaction mixture was then heated to reflux and stirred for 24 hours. The mixture was filtered, and the filtrate was concentrated and then washed with CH2Cl2 (20 mL) and water (10 mL). The organic phase was dried over Na2SO4 and concentrated. The crude solid was partitioned between 2% MeO Purification by silica gel column chromatography using 1H / CH2Cl2 gave a pale yellow Compound B4 (402 mg, 0.74 mmol, 44%) was obtained as a yellow solid.

[0179] 1 H NMR (500 MHz, CDCl3): δ 8.53 (d, J = 4.5 Hz, 1H), 8.20 (d, J = 5.5 Hz, 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)

[0180] 5.2.6. tert-Butylmethyl(2-(4-(4-(3-(pyridin-2-yl) )-1-Trityl-1H-pyrazol-4-yl)pyridin-2-yl)phenoxy)e Chill) Carbamate (B5) To a stirred solution of B4 (100 mg, 0.18 mmol) in toluene (2 ml), EtO Int-B (185 mg, 0.49 mmol) in H (0.75 ml) was then added to 2 M A solution of Na2CO3 (0.45 ml) was added under an argon atmosphere. The mixture was degassed with argon for 20 min, and then Pd(PPh3)4 (16 mg, 0.01 mmol) was added. After complete consumption of the starting material (monitored by TLC), the reaction The mixture was poured into water and extracted with toluene (3×15 ml). The organic layer was washed with Na2SO4 and concentrated under reduced pressure to give the crude product which was extracted with 30% EtOAc / Hexanes. The compound was purified by silica gel column chromatography using B5 (70 mg, 0.09 mmol, 53%) was obtained.

[0181] 1 H NMR (400 MHz, CDCl3): δ 8.53 (s, 1H), 8.49 (d, J= 4.8 Hz, 1H),7.82 (d, J = 8.8 Hz, 2H) 7.74-7.76 (m, 3H), 7.60 (s, 1H), 7.40-7.34 (s, 8H), 7.31-7.30 (m, 2 H), 7.24-7.19 (m, 4H), 7.12- 7.10 (m, 1H), 6.93(d, J = 8.8 Hz, 2H), 4.19-4.12 (m , 2H), 3.66-3.58 (m, 2H), 2.98 (s, 3H), 1.46 (s, 9H)

[0182] 5.2.7. N-methyl-2-(4-(4-(3-(pyridin-2-yl)-1H-pyridinyl) (4-pyridinyl)pyridin-2-yl)phenoxy)ethan-1-amine hydrochloride (chemical Compound B) To a stirred solution of B5 (70 mg, 0.09 mmol) in CH2Cl2 (6 ml), 1, 4N HCl in 4-dioxane (0.5 ml) was added at 0° C. The reaction mixture was cooled to room temperature and cooled to room temperature. The mixture was stirred under argon for 1 h. After complete consumption of the starting material (monitored by TLC), The solvent was evaporated under reduced pressure to give the crude compound, which was triturated with n-pentane (2 x 1 ml). Crushed and dried to give the HCl salt of compound B as a colorless solid (25 mg, 0.06 mmHg). ol, 69%).

[0183] 1 H NMR (400 MHz, DMSO-d6):δ 8.94 (brs, 2H), 8.62-8.56 (m, 3H), 8.30 (brs, 1H) , 8.03-7.96 (m, 3H), 7.86 (d, J = 7.6 Hz, 1H),7.69 (brs, 1H), 7.49 (dd, J =7.2, 5.6 Hz, 1H), 7.29 (d, J=7.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 4.36 (t, J = 4.8 Hz, 2H), 3.39-3.35 (m, 2H), 2.67-2.63 (m, 3H)

[0184] LC-MS(ESI):m / z 372[M+H] +

[0185] 5.3. [Example 3] N-Methyl-2-(4-{4-[3-(6-methylpyridin-2-yl)-1H-pyrazo (4-yl)pyridin-2-yl}phenoxy)ethan-1-amine (Compound C) synthesis Compound C was prepared according to the general method in Scheme 3 below.

[0186] [ka]

[0187] 5.3.1. 2-(2-bromopyridin-4-yl)-1-(6-methylpyridine-2 -yl)ethan-1-one (C2) 2-Bromo-4-methylpyridine (B1) (1 g, 5.81 mmol) in THF (15 To a stirred solution of 1000 mL of NaHMDS (2M in THF, 6. A solution of 100 ml (39 mL, 12.8 mmol) was added dropwise. The yellow solution was incubated at -78°C for 30 min. Then, 6-methylpicolinic acid methyl ester (1.19 ml, 8.72 mm A solution of 100 ml of 1 ml of ethyl acetate in THF (7 ml) was added and the reaction mixture was warmed up to approximately 21° C. The mixture was stirred for 16 h. The solvent was evaporated under reduced pressure and the solid residue was triturated with diethyl ether. The solid was then dissolved in saturated NH4Cl solution (20 ml) and washed with diethyl ether. The organic layer was diluted with EtOAc (2×150 ml) and the aqueous phase was extracted with NaSO The crude product was purified by column chromatography on silica gel using 10% EtOAc / Hexanes. Purification by gel column chromatography gave compound C2 (1.1 g, The yield was 3.79 mmol, 65.4%).

[0188] 1 H NMR (500 MHz, CDCl3): δ 8.30 (d, J = 5.0 Hz, 1H), 7.86 (d, J = 8 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.51 (s, 1H), 7.36 (d, J = 8 Hz, 1H), 7.24 (d, J = 5 Hz, 1H), 4.52 (s, 2H), 2.64 (s, 3H)

[0189] LC-MS(ESI):m / z 291[M] +

[0190] 5.3.2. 2-Bromo-4-[3-(6-methylpyridin-2-yl)-1H-pyrazol-2-yl] 4-[4-yl]pyridine (C3) A solution of C2 (300 mg, 1.03 mmol) in dry DMF (1 ml) was pressurized under argon The mixture was treated with glacial acetic acid (0.14 ml, 2.48 mmol) in DMF under reduced pressure. 2 ml, 1.55 mmol) was added dropwise and the mixture was incubated at approximately 21° C. for 1 h under argon. The mixture was stirred under atmospheric pressure. Hydrazine monohydrate (0.37 ml, 7.75 mmol) was added dropwise. The resulting mixture was heated at 50° C. for 3 hours and at approximately 21° C. for 16 hours. The mixture was poured into water (20 ml) and extracted with CH2Cl2 (3 x 20 ml). The layer was dried over Na2SO4 and filtered. The solvent was evaporated under reduced pressure to give crude C3. C3 was purified by silica gel column chromatography using 2% MeOH / DCM. Purification afforded purified C3 (172 mg, 0.54 mmol, 53%) as a yellow solid. Ta.

[0191] 1 H NMR (500 MHz, CDCl3):δ 11.40 (brs, 1H), 8.37 (d, J = 5.0 Hz, 1H), 7.74 (s, 1H), 7.64 (s, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.34 (d, J= 6.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 2.60 (s, 3H)

[0192] LC-MS(ESI):m / z 315[M+H] +

[0193] 5.3.3. 2-Bromo-4-(3-(6-methylpyridin-2-yl)-1-trimethylpyridine (1H-pyrazol-4-yl)pyridine (C4) To a stirred solution of C3 (40 mg, 0.12 mmol) in acetone (2 ml), 3 (53 mg, 0.38 mmol) and trityl chloride (53 mg, 0.19 mmol) l) was added. The reaction mixture was then heated to reflux and stirred for 24 hours. Filter, concentrate the filtrate, then partition between CH2Cl2 (5 mL) and water (5 mL). The organic phase was dried over Na2SO4 and concentrated. The crude solid was purified by 2% MeOH / CH2Cl Purification by silica gel column chromatography using l2 gave a pale yellow solid. Compound C4 (30 mg, 0.05 mmol, 41%) was obtained.

[0194] 1 H NMR (400 MHz, CDCl3):δ 8.22 (d, J = 4.8 Hz, 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 = 12 Hz, 1H), 2.53 (s, 3H)

[0195] LC-MS(ESI):m / z 558[M+H] +

[0196] 5.3.4. tert-Butyl methyl (2-(4-(4-(3-(6-methylpyridine -2-yl)-1-trityl-1H-pyrazol-4-yl)pyridin-2-yl)phenyl (N-oxy)ethyl)carbamate (C5) To a stirred solution of C4 (150 mg, 0.26 mmol) in toluene (5 ml), EtO Int-B (152 mg, 0.40 mmol) in 1 ml H, followed by 2 M Na A solution of CO3 (0.7 ml) was added under an argon atmosphere. The reaction mixture was then purged with argon. Degas for 20 min, then add Pd(PPh3)4 (25 mg, 0.02 mmol) Reflux for 6 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was The mixture was poured into water and extracted with toluene (3 x 10 ml). The organic layer was dried over Na2SO4. The mixture was then concentrated under reduced pressure to give crude C5, which was then purified by elution with 30% EtOAc / hexanes. Purification by silica gel column chromatography gave purified C5 (5 Yield: 1 mg, 0.07 mmol, 26%)

[0197] 1 H NMR (400 MHz, CDCl3): δ 8.48 (d, J = 5.2 Hz, 1H), 7.82 (d, J = 8.8 Hz, 3H) , 7.74 (s, 1H), 7.60 (s, 1H), 7.56 (d, J = 15.2Hz, J = 7.6Hz, 2H), 7.35-7.33 (m , 8H), 7.28-7.27 (m, 6H), 7.08 (d, J = 6.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4. 16-4.08 (m, 2H), 3.63-3.58 (m, 2H), 2.98 (s, 3H), 2.41 (s, 3H), 1.46 (s, 9H)

[0198] 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) To a stirred solution of C5 (51 mg, 0.07 mmol) in CH2Cl2 (5 ml), 1, 4N HCl in 4-dioxane (0.3 ml) was added at 0° C. Then the reaction mixture The mixture was 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 give crude compound C. The crude compound C was then Triturate with ethanol (2 × 1 ml) and dry to give compound C as the HCl salt as a brown solid. Obtained (20mg, 0.05mmol, 74%)

[0199] 1 H NMR (400 MHz, DMSO-d6):δ 8.93 (brs, 2H), 8.61 (d, J = 5.6 Hz, 1H),8.56 (br s, 1H), 8.33 (brs, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.88 (t, J = 7.6 Hz, 1H), 7.78 -7.74 (m,1H), 7.65 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 7.20 (d, J = 8 .4 Hz, 2H), 4.36 (t, J = 5.2 Hz, 2H), 3.36 (t, J = 5.2 Hz, 2H), 2.66-2.63 (m, 3H ), 2.50-2.46 (m, 3H)

[0200] LC-MS(ESI):m / z 386[M+H] +

[0201] 5.4. [Example 4] (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonyl) (phenyl)amino)(phenyl)methylene)-2-oxoindoline-6- Synthesis of carboxamide (compound D) Compound D was prepared according to the general method in Scheme 4 below.

[0202] [ka]

[0203] 5.4.1. Methyl 1-acetyl-2-oxoindoline-6-carboxylate (D 2) Methyl 2-oxoindoline-6-carboxylate (D1) (2.0 g, 10.47 A stirred solution of 10 mmol of acetic acid in acetic anhydride (16 ml) was heated to 130 °C under an inert atmosphere for 6 h. After complete consumption of the starting material (monitored by TLC), the reaction mixture was cooled to 10° C. The precipitate was filtered, washed with n-hexane (2 x 50 ml) and concentrated under vacuum. The mixture was dried at 40° C. for 1 hour to give compound D2 (1.5 g, 61.5%) as a yellow solid.

[0204] 1 H NMR (400 MHz, DMSO-d6):δ 8.66 (s, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 3.91 (s, 2H), 3.87 (s, 3H), 2.57 (s, 3H)

[0205] 5.4.2. Methyl (Z)-1-acetyl-3-(hydroxy(phenyl)methylene) -2-Oxoindoline-6-carboxylate (D3) To a stirred solution of compound D2 (1.5 g, 6.43 mmol) in DMF (10 ml), T BTU (2.69g, 8.36mmol), benzoic acid (903mg, 7.40mmol) , and triethylamine (2.2 ml) were added at 0° C. under an inert atmosphere. The mixture was warmed to approximately 21° C. and stirred for 16 h. After complete consumption of the starting material (TLC The reaction mixture was quenched with ice-cold water (30 ml) and × 40 ml). The combined organic extracts were dried over Na2SO4, filtered and vacuum Concentration at 4° C. gave crude product D3, which was purified by silica gel column chromatography using 80% EtOAc / hexanes. Compound D3 (900 ml) was purified by gel column chromatography to give a yellow solid. g, 42%).

[0206] 1 H NMR (400 MHz, CDCl3): δ 14.01 (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 = 8.0 Hz, 1H), 3.90 (s, 3H), 2. 83 (s, 3H)

[0207] LC-MS(ESI):m / z 338.3[M+H] +

[0208] 5.4.3. (Z)-3-(Hydroxy(phenyl)methylene)-2-oxoindole 6-Carboxylic Acid (D4) To a stirred solution of compound D3 (900 mg, 2.67 mmol) in MeOH (15 ml) Then, 1N aqueous NaOH solution (15 ml) was added at approximately 21° C. The reaction mixture was diluted with 100 C. and stirred for 6 h. After complete consumption of the starting material (monitored by TLC), The reaction mixture was cooled to approximately 21° C. and quenched with 1N aqueous HCl (13 ml); Stirred for 30 min. The precipitated solid was filtered and washed with 20% EtOAc / Hexanes to give Compound D4 (580 mg, 77%) was obtained as an off-white solid, which was further purified. This was used in the next step without dilution.

[0209] 1 H NMR (400 MHz, DMSO-d6):δ 12.76 (brs, 1H), 11.61 (brs, 1H), 7.77-7.50 (m, 8 H), 7.13 (brs, 1H)

[0210] 5.4.4. (Z)-N-ethyl-3-(hydroxy(phenyl)methylene)-2-methyl- Xoindoline-6-carboxamidelate (Fragment A) To a stirred solution of compound D4 (580 mg, 2.06 mmol) in DMF (10 ml), TBTU (729mg, 2.27mmol), HOBt (306mg, 2.27mmol) ), and N,N-diisopropylethylamine (1.9 ml, 10.32 mmol) After 30 min, THF (2.1 ml, 4.12 mL) was added at approximately 21° C. under an inert atmosphere. 2N ethylamine in 100 mmol) was added at 0° C. and stirred for 1 h. Then, the reaction mixture was The mixture was warmed to approximately 21° C. and stirred for an additional 16 h. After complete consumption of the starting material (T The volatiles were removed in vacuo. The residue was diluted with water (15 ml). It was filtered and washed with 20% EtOAc / Hexane (2×10 ml) to give the crude product. This was purified by silica gel column chromatography using 10% MeOH / CH2Cl2. Purification by HPLC gave Fragment A (410 mg, 64.5%) as an off-white solid. .

[0211] 1H NMR (400 MHz, DMSO-d6):δ 13.62 (brs, 1H), 11.39 (brs, 1H), 8.35-8.33 (m, 1 H), 7.76-7.52 (m, 5H), 7.44-7.36 (m, 3H), 3.29-3.22 (m, 2H), 1.10 (t, J = 7.2 Hz , 3H)

[0212] LC-MS(ESI): m / z 307.1(MH + )

[0213] 5.4.5. N-(2-(dimethylamino)ethyl)-N-(4-nitrophenyl)methyl Tansulfonamide (D8) To a stirred solution of compound D7 (800 mg, 3.70 mmol) in acetone (15 ml) , potassium carbonate (1.32 g, 9.62 mmol), sodium iodide (110 mg, 0 Compound B6 (799 mg, 5.55 mmol) was added to an inert atmosphere. The mixture was added at 0° C. under atmospheric pressure. The reaction mixture was heated to 50° C. and stirred for 20 hours. After complete consumption (monitored by TLC), the volatiles were removed in vacuo. The residue was washed with water. (20 ml) and extracted with EtOAc (2×40 ml). The combined organic extracts were Drying over Na2SO4, filtering and concentrating in vacuo gave the crude product which was dissolved in 5% Me The crude product was purified by silica gel column chromatography using OH / CH2Cl2 to give a pale yellow color. Compound D8 (460 mg, 43%) was obtained as a yellow solid.

[0214] 1 H NMR (500 MHz, DMSO-d6): δ 8.27 (d, J = 9.5 Hz, 2H), 7.68 (d, J = 9.5 Hz, 2H ), 3.85 (t, J = 6.5 Hz, 2H), 3.13 (s, 3H), 2.31 (t, J = 6.5 Hz, 2H), 2.12 (s, 6H) )

[0215] LC-MS(ESI):m / z 288.3[M+H] +

[0216] 5.4.6. N-(4-aminophenyl)-N-(2-(dimethylamino)ethyl)methyl Tansulfonamide (fragment B) To a stirred solution of compound D8 (460 mg, 1.60 mmol) in MeOH (10 ml) 10% Pd / C (40 mg) was added and the mixture was heated at approximately 21° C. under a hydrogen atmosphere (balloon pressure). After complete consumption of the starting material (monitored by TLC), the reaction mixture was stirred at rt for 3 h. Filter through a pad of Celite® and wash with MeOH (10 ml). The filtrate was concentrated in vacuo to give the crude product, which was extracted with 10% MeOH / CH2Cl2. Fragment B was purified by silica gel column chromatography as a pale yellow solid. (300 mg, 73%)

[0217] 1 H NMR (400 MHz, DMSO-d6): δ 6.99 (d, J = 8.8 Hz, 2H), 6.54 (d, J = 8.8 Hz, 2H ), 5.25 (s, 2H), 3.55 (t, J = 7.2 Hz, 2H), 2.91 (s, 3H), 2.24 (t, J = 7.2 Hz, 2H ), 2.12 (s, 6H)

[0218] LC-MS(ESI):m / z 258.2[M+H] +

[0219] 5.4.7. (Z)-3-(((4-(N-(2-(dimethylamino)ethyl)methyl Sulfonamido)phenyl)amino)(phenyl)methylene)-N-ethyl-2-oxo Indoline-6-carboxamide (D5) Fragment A (200mg, 0.64mmol), Fragment B (500mg, 1.94mmol) and TMS-imidazole (455 mg, 3.24 mmol) in THF (5 ml) The solution was heated to 170° C. in a microwave for 1 h. Consumption of starting material (TLC and L After purification (monitored by C-MS), the volatiles were removed in vacuo. The residue was diluted with water (10 ml). and extracted with EtOAc (3×25 ml) to give the crude product, which was purified by preparative HPLC. C to give compound D5 (150 mg, 42%) as a pale yellow solid.

[0220] 1 H NMR (400 MHz, DMSO-d6):δ 12.14 (s, 1H), 10.91 (s, 1H), 8.17 (t, J = 5.6 Hz , 1H), 7.64-7.57 (m, 3H), 7.53-7.51 (m, 2H), 7.34 (s, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.8 Hz, 2H), 5.73 (d, J = 8.4 Hz, 1H ), 3.58 (t, J = 6.8 Hz, 2H), 3.23-3.20 (m, 2H), 2.93 (s, 3H), 2.13 (t, J = 6.8 H z, 2H), 1.90 (s, 6H), 1.06 (t, J = 7.2 Hz, 3H)

[0221] LC-MS(ESI):m / z 548.6[M+H] +

[0222] 5.4.8. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl) (phenyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxo Endrine-6-carboxamide (Compound D) To a stirred solution of compound D5 (70 mg, 0.12 mmol) in dry toluene (3 ml), , 2,2,2-trichloroethoxycarbonyl chloride (0.04 ml, 0.19 mmol) 1) was added at approximately 21° C. under an inert atmosphere. The reaction mixture was heated to reflux (120° C.). ) and maintained for 16 hours. After complete consumption of the starting material (monitored by TLC), The reaction mixture was cooled to approximately 21° C., diluted with EtOAc (30 mL) and diluted with 1N HC1 The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. Condensation gave the monodemethylated, di-troc protected compound (40 mg).

[0223] The crude product from the above reaction was dissolved in acetic acid (3 ml) and zinc powder (9 mg, 0.1 3 mmol) was added at approximately 21° C. under an inert atmosphere. The reaction mixture was heated to 50° C. The reaction mixture was heated and stirred for 8 h. After complete consumption of the starting material (monitored by TLC), The mixture was cooled to approximately 21° C. and the volatiles were removed in vacuo. The residue was diluted with water (20 ml). The mixture was diluted with EtOAc (2×25 ml) and extracted with EtOAc (2×25 ml). The combined organic extracts were washed with saturated NaHC Wash with O3 solution (20 ml), dry over Na2SO4, filter and concentrate under reduced pressure to give Crude compound D was obtained, which was then eluted on a silica gel column using 5-6% MeOH / CH2Cl2. The mixture was purified by column chromatography to give 12 mg of compound D with an HPLC purity of 83%. Ta.

[0224] The reaction was repeated on a 60 mg scale and the resulting crude product was combined with the previous batch. and purified by preparative HPLC to give compound D (8.0 mg, 6.3%) as a pale yellow solid. Got it.

[0225] 1 H NMR (400 MHz, CD3OD):δ 7.65-7.59 (m, 3H), 7.52.7.50 (m, 2H), 7.40 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5 .95 (d, J = 8.4 Hz, 1H), 3.95 (t, J = 5.6 Hz, 2H), 3.39-3.32 (m, 2H), 3.05 (t, J = 5.6 Hz, 2H), 2.93 (s, 3H), 2.71 (s, 3H), 1.19 (t, J = 7.2 Hz, 3H)

[0226] LC-MS(ESI):m / z 534.6[M+H] +

[0227] UPLC purity: 99.18%

[0228] 5.5. [Example 5] (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfonyl) (phenyl)amino)(phenyl)methylene)-2-oxoindoline-6- Alternative synthesis of carboxamide (compound D) Compound D was also prepared according to the general method in Scheme 5 below.

[0229] [ka]

[0230] 5.5.1. N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfone Amide (D9) To a stirred solution of compound D7 (1.0 g, 4.65 mmol) in DMF (10 ml), water Sodium hydroxide (60% in mineral oil, 320 mg, 7.99 mmol) was added under an inert atmosphere. The mixture was added at 0° C. and stirred for 30 minutes at approximately 21° C. To this mixture, 1,2-dibromoethylene At approximately 21° C., ethanol (2.18 g, 11.60 mmol) was added. The mixture was stirred for 90 The reaction mixture was heated to ° C. and stirred for 24 hours. The reaction was monitored by TLC. Cool to approximately 21° C., quench with ice-cold water (30 ml), and add EtOAc (2×40 ml). The combined organic extracts were dried over Na2SO4, filtered and concentrated in vacuo to give The crude product was purified by silica gel column chromatography using 5% MeOH / CH2Cl2. Chromatographic purification afforded 1.2 as a mixture containing 40% unreacted starting material. g of D9 was obtained. The resulting mixture was used directly in the next reaction without further purification.

[0231] 1 H NMR (500 MHz, CDCl3):δ 8.29 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 4.12 (t, J = 7.0 Hz, 2H), 3.44 (t, J = 7.0 Hz, 2H), 3.01 (s, 3H)

[0232] 5.5.2. N-(2-(methylamino)ethyl)-N-(4-nitrophenyl)meth Sulfonamide (D10) To a stirred solution of compound D9 (1.2 g, impure) in THF (10 ml) was added triethylamine. (1.6 ml) and methylamine (2 M in THF, 9.3 ml, 18.63 mmol). l) was added in a sealed tube under an inert atmosphere at approximately 21° C. The reaction mixture was heated to 80° C. The reaction was heated and maintained for 16 hours. After complete consumption of the starting material (monitored by TLC), the reaction The reaction mixture was cooled to approximately 21° C. and concentrated under reduced pressure to give crude D10. By silica gel column chromatography using 15% MeOH / CH2Cl2 Purification gave compound D10 (500 mg, 39% overall over two steps) as a yellow solid. Yield) was obtained.

[0233] 1 H NMR (500 MHz, DMSO-d6): δ 8.94 (brs, 1H), 8.31 (d, J = 9.0 Hz, 2H), 7.80 (d , J = 8.5 Hz, 2H), 4.06 (t, J = 6.0 Hz, 2H), 3.15 (s, 3H), 3.00 (t, J = 6.0 Hz, 2H), 2.55 (s, 3H)

[0234] 5.5.3. tert-Butylmethyl (2-(N-(4-nitrophenyl)methylsulfonyl) (Homo)ethyl)carbamate (D11) To a stirred solution of D10 (500 mg, 1.83 mmol) in CH2Cl2 (10 ml) , triethylamine (0.4 ml, 2.61 mmol) and Boc anhydride (659 mg, 3.02 mmol) was added at approximately 21° C. under an inert atmosphere and maintained for 5 hours. After complete consumption of the material (monitored by TLC), the volatiles were removed in vacuo to give the crude product. The product was purified by silica gel column chromatography using 5% MeOH / CH2Cl2. D11 (320 mg, 47%) was obtained as a colorless thick syrup by raffinization. Got it.

[0235] 1 H NMR (400 MHz, DMSO-d6): δ 8.27 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H ), 3.91 (t, J = 6.4 Hz, 2H), 3.28-3.25 (m, 2H), 3.07 (s, 3H), 2.72-2.70 (m, 3H), 1.33-1.27 (m, 9H)

[0236] LC-MS(ESI): m / z 274.2(M + -B℃)

[0237] 5.5.4. tert-Butyl (2-(N-(4-aminophenyl)methylsulfonyl) (mido)ethyl)(methyl)carbamate (Boc variant of fragment B) To a solution of compound D11 (250 mg, 0.67 mmol) in EtOH (10 ml), Raney-Ni (40 mg) was added and incubated at approximately 21° C. for 1 h under a hydrogen atmosphere (balloon pressure). After complete consumption of the starting material (monitored by TLC), the reaction mixture was diluted with C The mixture was filtered through a pad of elite® and washed with EtOH (10 ml). The combined filtrate was concentrated in vacuo to give the crude product, which was then diluted with 10% MeOH / CH2Cl2 The fraction was purified by silica gel column chromatography using The Boc variant of B (180 mg, 77%) was obtained.

[0238] H NMR (400 MHz, DMSO-d6):δ 7.01 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 8.4 HZ, 2H) , 5.24 (s, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.18 (t, J = 6.4 HZ, 2H), 2.88 (s, 3H) , 2.75-2.71 (m, 3H), 1.36-1.33 (m, 9H)

[0239] LC-MS(ESI): m / z 244.2(M + -B℃)

[0240] 5.5.5. tert-Butyl (Z)-(2-(N-(4-(((6-(ethylcarbamoyl) (phenyl)methyl)amino)phenyl (methylsulfonamido)ethyl)(methyl)carbamate (D10) Fragment A (70 mg, 0.22 mmol), Boc variant of fragment B (155 mg, 0.4 5 mmol) and TMS-imidazole (159 mg, 1.13 mmol) in THF( The solution in 3 ml) was heated in a microwave at 170° C. for 160 min. Consumption of starting material. (monitored by TLC and LC-MS), the volatiles were removed in vacuo to give a residue This was purified by preparative HPLC to give compound D10 (50 mg) as a pale yellow solid. , 36%).

[0241] 1 H NMR (400 MHz, CDCl3):δ 12.13 (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 = 8.4 HZ, 1H), 6.75 (d, J = 8. 4 HZ, 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 = 7.2 HZ, 3H)

[0242] LC-MS(ESI): m / z 634.6[M+H] +

[0243] 5.5.6. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl) (phenyl)methylsulfonamido)phenyl)amino)(phenyl)methylene)-2-oxo Endrine-6-carboxamide hydrochloride (compound D as the HCl salt) Compound D10 (20 mg, 0.03 mmol) stirred in diethyl ether (3 ml) The solution was added with 4N HCl in 1,4-dioxane (0.3 ml) at 0° C. under an inert atmosphere. The reaction mixture was stirred at approximately 21° C. for 1 h. Complete consumption of the starting material (T After HPLC monitoring, the volatiles were removed in vacuo to give the crude product, which was Trituration with pentane (2 × 4 ml) afforded compound D (1H HCl salt) as a pale yellow solid. 2 mg, 71%) was obtained.

[0244] 1 H NMR (400 MHz, CD3OD):δ 7.65-7.59 (m, 3H), 7.52.7.50 (m, 2H), 7.40 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5 .95 (d, J = 8.4 Hz, 1H), 3.95 (t, J = 5.6 Hz, 2H), 3.39-3.32 (m, 2H), 3.05 (t, J = 5.6 Hz, 2H), 2.93 (s, 3H), 2.71 (s, 3H), 1.19 (t, J = 7.2 Hz, 3H).

[0245] LC-MS(ESI):m / z 534.7[M+H] +

[0246] UPLC purity: 96.26%

[0247] 5.6. [Example 6] In vitro assays for testing the activity of compounds A-D 5.6.1. N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfone Amide (2) Compounds A to D were tested to determine whether they inhibited TGF-α in HEK293T cells in vitro. We determined whether β-induced luciferase activity could be inhibited by β-lactamase.

[0248] 30,000 HEK293T cells were seeded overnight in a 96-well white flat-bottom plate. The next day, 100 ng of SMAD luciferase reporter plasmid was added per well. The following day, the cells were transfected with Lipofectamine for 24 hours. The cells were treated with compounds A-D and 100 pM TGFβ for 24 hours. The enzyme activity was measured using the Dual-Glo® Luciferase Assay Kit (Promega). a) was used. The assay was performed in duplicate for compounds A, B, and D. Three replicates were performed for compound C. The results are shown in Table 4.

[0249] [Table 4]

[0250] Activity data from experiment 1 is shown in FIG.

[0251] Compounds A to C showed the greatest inhibitory activity.

[0252] 5.6.2. MTS proliferation assay Compounds A to D were tested and found to be effective in inhibiting primary mouse CD4 + TGF-β signaling in T cells We determined whether transmission could be inhibited.

[0253] Primary mouse CD4 + T cells were separated using the RoboSep™ cell separation system (Stemcell The cells were isolated from the spleens of C57 / B6 mice using a cellular technology 0.5 μg / ml of hamster anti-mouse CD3e antibody (145-2C11, eBio Science) was coated overnight on a 96-well flat-bottom plate. 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 After 72 hours, cell proliferation was monitored using the manufacturer's Measurements were performed using the MTS assay (Promega) according to the manufacturer's instructions. Shown in 5.

[0254] [Table 5]

[0255] Data from experiment 1 are shown in FIG.

[0256] In two separate experiments, IC 50 No value was obtained for compound D. Compound A was also In addition, mouse CD4 + However, compounds B and C showed no consistent effect on T cells. and C reversed TGF-β-mediated inhibition of T cell proliferation.

[0257] Based on the two assays, compound C was selected for conjugation to the ADC. .

[0258] 5.7. [Example 7] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2H-pyrrole-1(5 H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido benzylmethyl(2-(4-(4-(3-(6-methylpyridin-2-yl)-1H -Pyrazol-4-yl)pyridin-2-yl)phenoxy)ethyl)carbamate Growth Compound C can be prepared by the synthesis of valine-citrulline according to the general method in Scheme 6 below. Linked to a linker.

[0259] [ka]

[0260] L1 (122 mg, 0.165 mmol, 1.1 equiv.) and TEA (52 μl, 0. 375 mmol, 2.5 equiv.) to compound C (58 mg, 0.150 mmol, 1.0 equiv.). A solution of 100 ml of 1,000 sachets of 100% ethanol in DMF (2 ml) was added at 0° C., and the reaction mixture was incubated at approximately 21° C. for 2 h. Stirring gave crude ADC-1. The crude ADC-1 was purified by preparative HPLC to give a white solid. The purified ADC-1 (34 mg, 24% yield) was obtained as a crude product.

[0261] 5.8. [Example 8] Generation of antibody drug conjugate 1 (ADC1) Anti-mouse transferrin receptor antibody R17217, and rat anti-mouse IgG2A Isotype control antibodies (BioXCell) were incubated in complex buffer (25 mM borate in 25 mM NaCl, 0.3 mM EDTA, final pH 7.4) The antibody was dialyzed overnight using tris(2-carboxyethyl)phosphine (TCEP). The mixture was then reduced for 2 h at a reduction ratio of 10–30. ADC-1 was added to a final concentration of 10 mM in DMSO. The antibody was dissolved in DMSO and then conjugated to the antibody at a conjugation ratio of 5–30 in the presence of 15% DMSO. All reactions were performed at approximately 21°C. In contrast, 50% propylene glycol was used as the organic solvent during the conjugation step. The final ADC was dialyzed overnight in PBS and filtered through a 0.22 μm filter. The mixture was filtered and analyzed via HPLC-HIC to determine the DAR and via HPLC-SEC to determine the DAR. For HPLC-HIC, samples were run at a flow rate of 0 Phase A was run on a TSKgel® Butyl-NPR column at 0.5 ml / min. , 25 mM sodium phosphate, and 1.5 M ammonium sulfate at pH 6.95. Phase B was 75% 25 mM sodium phosphate at pH 6.95, and 2 5% isopropyl alcohol. For HPLC-SEC analysis, TSKgel (registered trademark) G3000SW column (Tosoh Bioscience) was loaded at a flow rate of 0. Used 25 ml / min, 280 nM for 25 min.

[0262] 5.9. [Example 9] Synthesis of compound C (ADC-2) linked to a disulfide linker Compound C can be converted to a disulfide according to the general method in Schemes 7A-B below. Linked to a linker.

[0263] [ka]

[0264] [ka]

[0265] 5.9.1. Synthesis of intermediate A 2-Chlorotrityl chloride resin (L2) (4 g, 4 mmol) was dissolved in DCM (2 × 40 1 ml), swell in 50 ml DCM for 10 min, then drain. -Cys(Trt)-OH(L3) (7.03 g, 12 mmol) in 40 mL of DCM and add to the vessel containing 2-chlorotrityl chloride resin. IPEA (6.8 ml, 40 mmol) was added to the vessel and the mixture was incubated at approximately 21° C. for 2 hours. Stir. Then add 10 ml of methanol to the mixture and stir for 30 minutes. The resin (L4) obtained is then drained and washed five times with DMF. To protect, approximately 40 ml of 20% piperidine in DMF is added to resin L4, and the mixture is The resin is shaken and then drained to produce resin L5. Another 40 ml of 20% piperidine is added to the resin and shaken for 15 minutes. The resin L5 is drained and washed with DMF (6×40 ml).

[0266] The solution of Fmoc-amino acids was prepared by the procedure of Fmoc-Asp(OtBu)-OH (4.93 g, 1 2mmol), Fmoc-Asp(OtBu)-OH (4.93g, 12mmol), F moc-Arg(Pbf)-OH(7.79g, 12mmol), Fmoc-Asp(O tBu)-OH (4.93 g, 12 mmol), and Fmoc-Glu-OtBu (5 .1g, 12mmol) to HBTU / HOBT (4.55g, 12mmol / 1.62 g, 12 mmol) and DIPEA (2 ml, 12 mmol) were separately prepared. To manufacture.

[0267] The Fmoc-Asp(OtBu)-OH solution was added to resin L5 and shaken for 60 minutes. This produces resin L6. Resin L6 is washed with DMF (6×40 ml) and then purified as described above. , and deprotected with 20% piperidine in DMF. Resins L7, L8, L9, and L10 was prepared by performing successive couplings using Fmoc-amino acid solutions. The same procedure is used to prepare resin L6 from resin L5.

[0268] In an exemplary synthesis, dry resin L10 (8 g) was added to a flask and 80 ml of cleavage solution was added. The solution was added (TFA:TES:EDT:HO=90:5:3:2, v / v / v / v The reaction was allowed to proceed for 1.5 hours. The resin was then removed from the reaction mixture by filtration under pressure. The resin was then washed twice with TFA. The filtrates were combined and diluted with 10x the volume of cold M TBE was added dropwise. The precipitated peptide (Intermediate A) was then centrifuged and diluted with cold MTBF. Intermediate A was then dried under reduced pressure and purified by preparative HPLC to give Obtained 1.1 g of intermediate A (37% yield) as a white solid. LC-MS (ESI)m / z:752[M+H]+.

[0269] 5.9.2. 2-(Pyridin-2-yldisulfanyl)ethylmethyl (2-(4-( 4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridine -2-yl)phenoxy)ethyl)carbamate (L12) Compound C (40 mg, 0.1038 mmol) and 4-nitrophenyl 2-(pyridinyl) (2-yldisulfanyl)ethyl carbamate (L11) (80 mg, 0.2272 A solution of 1 mmol) in DMF (5 ml) was added with DIPEA (0.5 ml) and HOBt ( The mixture was incubated at approximately 21 °C under N2 for 16 h. The crude L12 was purified by preparative HPLC to give L12 as a white solid. This gave 35 mg of purified L12 (56% yield).

[0270] 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-(4-(6-methylpyridin-2-yl)-1H-pyra) (3-yl)pyridin-2-yl)phenoxy)ethyl)carbamoyloxy) (ethyl)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6, 9,12,15-Pentaazaicosane-1,20-dioic acid (L13) Dissolve L12 (35 mg, 0.058 mmol) in THF / H2O (5 ml / 5 ml). To the solution was added intermediate A (80 mg, 0.106 mmol) under N2. The mixture was stirred at 21° C. for 16 hours to produce L13. The crude L13 was purified by preparative HPLC. This afforded 23 mg of purified L13 (31% yield) as a white solid.

[0271] 5.9.4. (2R,5S,8S,11S,14S,19S)-19-(2-(ter t-Butoxycarbonylaminooxy)acetamido)-5,8,14-tris(carbo 2-(2-(methyl)-11-(3-guanidinopropyl)-2-((2-(methyl)-2-(4 -(4-(4-(6-methylpyridin-2-yl)-1H-pyrazol-3-yl)pyridine phenoxy)ethyl)carbamoyloxy)ethyl)disulfanyl) Methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-penta Azaicosane-1,20-dioic acid (L15) A solution of L13 (32 mg, 0.025 mmol) in DMF (3 ml) was added to 2,5-dichlorophenyl Oxopyrrolidin-1-yl 2-(tert-butoxycarbonylaminooxy)acetate Add 100 mL of L14 (28 mg, 0.097 mmol) followed by TEA (0.5 ml). The reaction mixture was stirred at approximately 21 °C under N2 atmosphere for 16 h to produce L15. The crude L15 was purified by preparative HPLC to give 12 mg of purified L15 as a white solid. 15 (33% yield) was produced.

[0272] 5.9.5. (2R,5S,8S,11S,14S,19S)-19-(2-(amino Oxy)acetamido)-5,8,14-tris(carboxymethyl)-11-(3-glutamyl anidinopropyl)-2-(((2-(methyl(2-(4-(4(4-(6-methylpyridine 1H-pyrazol-3-yl)pyridin-2-yl)phenoxy)e ethyl)carbamoyloxy)ethyl)disulfanyl)methyl)-4,7,10,13, 16-Pentaoxo-3,6,9,12,15-pentaazaicosane-1,20-dioic acid ( ADC-2) To a mixture of L15 (12 mg, 0.0085 mmol) in DCM (5 ml), TFA (1 ml) was added. The mixture was stirred at approximately 21° C. for 30 minutes to produce ADC-2. The crude ADC-2 was concentrated and purified by preparative HPLC to give 3.5 mg of a white solid. of purified ADC-2 (31% yield).

[0273] 5.10. [Example 10] Generation of antibody drug conjugate 2 (ADC2) ADC-2 was prepared by converting antibody lysine residues to phenylalanine according to the general method in Scheme 8 below. The anti-TfR antibody bound via the IL-1 domain.

[0274] [ka]

[0275] The heterobifunctional polymeric linker S-4FB was purchased from Solulink. Anti-mouse IgG2a and anti-mouse transferrin receptor antibody R17217 were incubated at pH S-4FB was dialyzed in PBS at pH 7.4 at different molar ratios. The antibody was added and incubated at approximately 21° C. for 3 hours. S-4FB modified antibody solution , in a 2-hydrazinopyridine solution (0.5 mM in 100 mM MES buffer, pH 5. 0) at various conjugation ratios ranging from 5 to 50 and incubated at 37°C for 30 min. The S4FB / Ab molar substitution ratio was determined by UV-Vis at A354. The solution was desalted using a Zeba™ spin desalting column, 50 mM phosphate buffer (pH 6.5, 15 The resulting mixture was purified using buffer exchange to 0 mM NaCl (0.1 mM NaCl) and then the linker-SS-drug The compound ADC-2 (10 mM in DMSO) was mixed with the compound ADC-2 (10 mM in DMSO) at different molar ratios for 24 h at 37°C. The next day, the ADC2 sample was dialyzed overnight against PBS. The samples were filtered and then analyzed via HPLC-SEC, SDS-PAGE, and LC-MS. ADC2 was prepared at an S-4FB / Ab ratio of 6 and an ADC-2 / Ab ratio of 20. Exemplary LC-MS data for the ADC2 samples tested are shown in FIG. The average DAR was 4.99, with heavy chain DAR of 1.97 and light chain DAR of 0.53. This indicates that.

[0276] If ADC2 aggregation at 5% is detected by HPLC-SEC, the aggregated components The SEC column (GE Healthcare Life Sciences, Sup AKTA with erdex 200 increase 10 / 300GL) The mixture was purified by SEC to remove aggregates and analyzed again by HPLC-SEC. The chromatogram of ADC2 is shown in FIG.

[0277] 5.11. [Example 11] Antibody-induced receptor internalization assay 96-well flat-bottom plates were coated with anti-mouse CD3e antibody overnight at 4°C. CD4+ T cells were isolated using the RoboSep™ cell separation system (Stemcell™ The cells were isolated from mouse spleens using the 30-kDa antibody (30-kDa chromosome-specific antibody). Approximately 2 × 10 5 pcs Cells were plated per well with soluble anti-CD28 antibody for 24-48 h at 37 °C. Once activated, CD4 + T cells were harvested, washed and incubated at the indicated time points at 37 °C. Then, the cells were replated with 5 μg / ml of primary (anti-transferrin receptor) antibody to detect the intracellular The reaction was stopped with ice-cold staining buffer and kept on ice to stop internalization. At the end of the assay, cells were washed twice with ice-cold staining buffer to remove unbound antibody. The cells were pelleted and then stained with PE and conjugated with a goat anti-rat secondary antibody. The cells were then washed with staining buffer and then FACS As shown in Figure 9, TfR expression increased within 1 h on primary CD4 + T TfR began to be internalized by cells, and within 3 hours, more than 70% of TfR was converted to anti-transferrin receptor antibodies. It is internalized by the body and R17217.

[0278] 5.12. [Example 12] In vitro assays Proliferation Assays Murine CTLL2 cells were cultured at 1 × 10 cells in 0.2 ng / ml IL2. 5 Pieces / well Each well was cultured with 1 nM TGF-β, 1 μg / ml ADC, and / or Or 100 nM of the ALK5 inhibitor Compound C was added to the wells for 24 hours. Quantification was performed by adding BrdU reagent (Abcam) to the wells for an additional 12 h. The results were analyzed by ELISA.

[0279] As shown in FIG. 10, treatment of CTLL2 cells with TGF-β increased proliferation by approximately 60%. However, the addition of ADC1 (DAR2-4, 4-6, or 6-8) inhibited Thus, TGF-β inhibition was almost completely reversed, as was treatment of cells with an ALK5 inhibitor alone. Rat anti-mouse IgG2A isotype control Cells treated with the ALK5 ADC did not restore CTLL2 proliferation. In cells treated with ADC1 without TFR or with naked Tfr antibody alone However, no inhibition of proliferation occurred, indicating that ADC1 does not affect proliferation unless TGF-β is present. (data not shown).

[0280] 5.12.2. Granzyme B Expression Assay Mouse CD3 + T cells were isolated using the EasySep™ Mouse T Cell Isolation Kit (negative The mice were isolated from the spleens of mice using the Stemcell Technology (STMT) It was purified from CD3 + T cells were immunolabeled using plate-bound anti-CD3e and soluble anti-CD28. The T cells were activated for 48 hours before use. The T cells were washed and resuspended in 5% serum and 1 nM TGF-β. -β- / +ADC-containing medium.

[0281] Golgi stop reagent was added for the final 4 hours and then cells were stained with surface CD8 (BD) and Immunostaining for intracellular GzmB (eBioscience) was performed using flow cytometry. Granzyme B (GzmB) is a marker for CD8 + T cells induce tumor cell proliferation GzmB is a serine protease that is released to kill the vesicle. The increase in CD8 + Demonstrates cytotoxic T cell activity.

[0282] As shown in Figure 11, TGF-β inhibited the proliferation of primary CD8 + Suppressing GzmB expression in T cells However, treatment with ADC1 for all three DARs, 2-4, 4-6, and 6-8, also The rat anti-mouse The IgG2A isotype control ALK5 ADC did not restore GzmB expression. I did.

[0283] 5.12.3. iTreg conversion assay Naïve CD4 T cells were isolated from mouse spleen cells using a negative selection kit. The cell density was 0.4 × 10 cells. 6 Adjust to 10 ng / ml. Mouse IL-2, 20ng / ml TGF-β, and 1μg / ml soluble anti-CD28 was added to the cell suspension.

[0284] Anti-mouse CD3 antibody at 10 μg / ml was coated onto a 24-well plate, Incubated overnight at 4° C. The antibody was then aspirated from the plate. 1 ml of cell suspension was added. The suspension was added to each well of a 24-well plate. ADC1 (DAR4-6), anti-transferrin receptor antibody, rat anti-mouse IgG2 A isotype control ALK5 ADC and A at 100 nM and 1 μM The LK5 inhibitor Compound C was added to separate wells of a 24-well plate. The cells were then The cells were cultured for 72 hours. TfR expression was examined at 48 hours (data not shown). The cells were stained with FoxP3 (eBioscience FoxP3 staining buffer) and incubated for 72 h. The cells were sorted by FACS at time points.

[0285] As shown in Figure 12, ADC1 (+CD71-ALK5 ADC) at 5 μg / ml The biomarkers were similar to those of 100 nM of free ALK5 inhibitor alone (+ALK5 inh 100 nM). In contrast, control ALK5 ADCs (+Iso-ALK5 ADC) and naked anti-TfR antibody (+anti-CD71) were g There was no effect on FoxP3 expression.

[0286] 5.13. [Example 13] Synthesis and characterization of compound N Compound N was synthesized according to the general method in Scheme 9 below.

[0287] [ka]

[0288] Compound N was compared to Compound C in several in vitro assays. Its IC50 activity and its K i The values ​​are summarized in Table 6. Table 6 also shows the effect of Compound C on TGF-β signaling in human HEK cells and mouse T cells. Compound C also exhibits inhibitory activity against null transmission, 10-fold more potent than compound N in recombinant assays. It turned out to be powerful.

[0289] [Table 6]

[0290] 5.14. [Example 14] Internalization of CD2 and CD5 into T cells Two different internalization studies were performed, one with anti-CD2 and one with anti-CD5 antibodies, respectively. Following T cell incubation, internalization of CD2 and CD5 was measured.

[0291] 5.14.1. Study 1: No antibody washout Murine CD3+ T cells were incubated with plate-bound anti-CD3 antibody (1 μg / ml) and soluble anti-CD3 antibody (1 μg / ml). The cells were activated with CD28 antibody (2 μg / ml) for 36 hours. The cells were washed and incubated with 1 μg / ml of La Mouse anti-mouse CD2 antibody (clone 12-15, Southern Biotech, Catalog no. 1525), rat anti-mouse CD5 antibody (clone 53-7.3, South ern Biotech, Cat. No. 1547) or rat isotype control roll antibody at the indicated time points (0, 15 min, or 0.5, 1, 3, or 6 h); The cells were incubated at 37° C. At each time point, the assay was performed by placing the cells on ice. Expression of CD2 and CD5 was monitored using fluorescently conjugated secondary antibodies. And detected it.

[0292] At 6 hours, more than 60% of CD5 and more than 50% of CD2 were mouse CD3+ T cells. The IgG4-dependent cytotoxicity was then internalized within the cytoplasm (FIGS. 13A and 13B, respectively).

[0293] 5.14.2. Study 2: Antibody washout Study 1 was repeated except that free antibody was incubated with the cells for 30 minutes at 4°C. All cell surface receptors were saturated. Any antibody remaining in the supernatant was washed away before the time course began. did.

[0294] At 6 hours, nearly 90% of CD5 and more than 50% of CD2 were mouse CD3+ T cells. and was internalized within the cells (FIGS. 13C and 13D, respectively).

[0295] 5.14.3. Discussion In study 1, new and recycled receptors, if present, were distributed to the cell surface over the time course. In Study 2, the time course was started by This allows the internalization of only the receptor present at the time of binding to be monitored. The results of Study 1 and Study 2 for CD2 were similar. These results suggest that CD2 does not reverse rapidly. In the Schout study (Study 2), internalization increased by approximately 20%, which is due to the presence of a novel receptor, 6 Recycle or increase via de novo synthesis over the time course? This indicates that a large amount of de novo synthesis occurred over a 6-hour time course. Since this is not expected to occur for a long time, recycling is considered a likely option. The results of Study 1 and Study 2 suggest that CD5 may recirculate to the cell surface to a greater extent than CD2. This suggests that there is a possibility.

[0296] 5.15. [Example 15] Generation and characterization of ADC targeting CD2 and CD5 5.15.1. Example 15: Generation of ADCs In this example, we have four A's, which we call T cell-targeted TGF-β antagonists (T3A). LK5-ADC was incubated with a rat anti-mouse CD2 antibody (clone 12-15, Southern Biotech, Cat. No. 1525), and rat anti-mouse CD5 antibody (Clontech, Cat. No. 1525). (SEQ ID NO: 53-7.3, Southern Biotech, Cat. No. 1547) Two linker-ALK5 inhibitor payloads were used to generate T3A. However, one of them is a cleavable Val-Cit (VC) linked to ALK5-compound C. linker, and the other is a non-cleavable maleimidocaproyl (M C) includes a linker.

[0297] The four T3A antibody, linker, and ALK5 payload combinations are shown in Table 7.

[0298] [Table 7]

[0299] T3A #2 to #5 were purified by size exclusion chromatography (SEC) and The antibody ratio was calculated by hydrophobic interaction chromatography (HIC). T3A #2 The % aggregation, % unbound antibody, and DAR values ​​for each of #1 through #5 are shown in Table 8.

[0300] [Table 8]

[0301] ADC Characterization To determine the efficacy of T3A #2-5 in reversing TGF-β-mediated immunosuppression Mouse CD3+ T cells were purified from the spleen and treated with 1 nM TGF-β and the small molecule ALK. 5. Inhibitor Compound C (positive control), T3A #2-5, or isotype control T Activated with anti-CD3 and anti-CD28 antibodies for 36–72 h in the presence of 3A (negative control). Thirty-six hours later, the levels of granzyme (GzmB)-expressing CD8+ T cells were assessed using cytotoxic As a marker of sexual function (Figure 14), levels of the secreted cytokine IL2 (Figure 15) and IFN-γ levels (Figure 16) were measured by ELISA. After a period of time, the amount of T cell proliferation was measured by Cell Titer Glo (Promega). All of these assays were associated with tumor clearance in vivo. There is a series.

[0302] The amount of function observed associated with activated T cells (set to 100%) is shown in Figures 14 to 17. T3A #5 restored GzmB expression and T cell proliferation, but not IFN The study only partially restored IL-γ expression. No effect on IL2 expression was observed.

[0303] 5.15.3. Discussion The data from the Examples above demonstrate that the level of target expression on T cells determines efficacy in primary T cell assays. Both CD2 and CD5 are expressed in 20–50% of activated T cells. Unlike CD71, which is only highly expressed, CD2 and CD5 are highly expressed in more than 85% of T cells. However, CD5-targeted ADCs were observed to have higher efficacy than CD2-targeted ADCs. Based on the pattern of receptor internalization observed for CD2 and CD5 in Example 14, Based on this, approximately 85% of CD5 was internalized by primary mouse T cells at 6 hours, whereas C Only 53% of CD2 was internalized. In addition, CD5 began internalization earlier than CD2. The data indicate that the amount internalized also influences efficacy.

[0304] The data also show that the linker attaching the ALK5 inhibitor to the antibody, and the release mechanism, both We also show that both are important for efficacy. The SynB cleavable VC linker was the most effective T3A. However, anti-CD5 antibody (T3 A #4) A non-cleavable MC in combination with a linker also binds to an anti-CD5 antibody. In this case, it had some activity.

[0305] Based on studies in primary mouse T cells, T3A can be ranked in potency as follows: Can be used with: 1) T3A #5, 2) T3A #4, 3) T3A #3, and 4) T3 A#2.

[0306] Without being bound by theory, it is believed that for high ADC activity, ADCs inhibit naive T cells. It is broadly expressed across cytoplasmic and activated T cells (e.g., expressed in >70% of cells) and rapidly Rapid internalization and established intracellular release mechanisms (e.g., proteolytic processing) , it is believed that T cell targets should be targeted.

[0307] 5.16. [Example 16] Internalization of CD7 into T cells An internalization study was performed following incubation of T cells with two different anti-CD7 antibodies. Subsequent internalization of CD7 was measured.

[0308] Human CD3+ T cells were incubated with plate-bound anti-CD3 antibody (1 μg / ml) and soluble anti-C The cells were activated with D28 antibody (2 μg / ml) for 40 hours. The cells were washed and incubated with 1 μg / ml of anti-Human CD7 antibodies (clones 124-D1 and 4H9, Caprico Biotech) , or rat isotype control antibody for 30 min at 4°C. The antibody remaining in the supernatant was washed away, and the cells were then incubated at 37°C for 1 h. The incubation was continued for 0 to 6 hours. At each time point (5, 15, 30, 60, 180, and 360 At 1 min (min), the assay was stopped by placing the cells on ice. Detection was performed using photoconjugated secondary antibodies.

[0309] At 6 hours, approximately 70-80% of CD7 cells were internalized (Figure 18). The suitability of CD7 as an ADC target, comparable to CD2 and CD5, was demonstrated.

[0310] 6. Specific embodiments The present disclosure is illustrated by the following specific embodiments. 1. ALK operably linked to an antibody or antigen-binding fragment that binds to a T cell surface molecule. 5 inhibitors, antibody-ALK5 inhibitor conjugates (ADCs). 2. IC of ALK5 inhibitors 50 is at least 20 nM. C. 3. The ALK5 inhibitor is an imidazole, pyrazole, or thiazole. The ADC of embodiment 1 or embodiment 2, which is a compound based on 4. The ADC of embodiment 3, wherein the ALK5 inhibitor is an imidazole-based compound. 5. The ADC of embodiment 3, wherein the ALK5 inhibitor is a pyrazole compound. 6. The ADC of embodiment 3, wherein the ALK5 inhibitor is a thiazole compound. 7. The ALK5 inhibitor is an imidazole-benzodioxole compound or an imidazole - the ADC of embodiment 3, which is an imidazole-based compound that is a quinoxaline compound . 8. The method of embodiment 7, wherein the ALK5 inhibitor is an imidazole-benzodioxole compound. The ADCs described herein. 9. The method of embodiment 7, wherein the ALK5 inhibitor is an imidazole-quinoxaline compound. ADC. 10. The ALK5 inhibitor is a pyrazole-pyrrolo compound, a pyrazole compound. , the ADC of embodiment 3. 11. ALK5 inhibitors are imidazole-benzodioxole compounds, imidazole-ki a pyrazole-pyrrolo compound, a thiazole-based compound, The ADC of Form 3. 12. The ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a linker. 12. The ADC of any one of embodiments 1 to 11. 13. The ADC of embodiment 12, wherein the linker is a non-cleavable linker. 14. The non-cleavable linker is N-maleimidomethylcyclohexane-1-carboxyle carboxylate, maleimidocaproyl, or mercaptoacetamidocaproyl linker. , The ADC described in embodiment 13. 15. The non-cleavable linker is N-maleimidomethylcyclohexane 1-carboxylate. The ADC of embodiment 14, which is a trincor. 16. The method according to embodiment 14, wherein the non-cleavable linker is a maleimidocaproyl linker. On-board ADC. 17. The non-cleavable linker is a mercaptoacetamidocaproyl linker, 15. The ADC of form 14. 18. The ADC of embodiment 12, wherein the linker is a cleavable linker. 19. The cleavable linker is a dipeptide linker, a disulfide linker, or a hydra The ADC of embodiment 18, wherein the ADC is a zone linker. 20. The ADC of embodiment 19, wherein the cleavable linker is a dipeptide linker. 21. The ADC of embodiment 19, wherein the cleavable linker is a disulfide linker. . 22. The ADC of embodiment 19, wherein the cleavable linker is a hydrazone linker. 23. The linker is a protease-sensitive valine-citrulline dipeptide linker. The ADC of embodiment 19. 24. The method according to embodiment 19, wherein the linker is a glutathione-sensitive disulfide linker. On-board ADC. 25. The ADC of embodiment 19, wherein the linker is an acid-sensitive disulfide linker. . 26. ALK5 inhibitors bind to antigens or antigen-binding sites via site-specific conjugation. 26. The ADC of any one of embodiments 1 to 25, wherein the ADC is conjugated to a functional fragment. 27. The ALK5 inhibitor binds to one or more cysteines on the antibody or antigen-binding fragment, A according to embodiment 26, which is conjugated via a lysine or glutamine residue. DC. 28. The ALK5 inhibitor binds to one or more cysteine ​​residues on the antibody or antigen-binding fragment. The ADC of embodiment 27, which is conjugated via a group. 29. The ALK5 inhibitor binds to one or more lysine residues on the antibody or antigen-binding fragment. 28. The ADC of embodiment 27, which is conjugated via 30. The ALK5 inhibitor binds to one or more glutamine residues on the antibody or antigen-binding fragment. The ADC of embodiment 27, which is conjugated via a group. 31. The ALK5 inhibitor comprises one or more non-natural amino acids on the antibody or antigen-binding fragment. The ADC of embodiment 26, which is conjugated via an acid residue. 32. One or more unnatural amino acid residues are p-acetylphenylalanine (pAc The ADC of embodiment 31, comprising: 33. One or more non-natural amino acid residues are p-azidomethyl-L-phenylalanine 32. The ADC of embodiment 31, comprising pAMF. 34. A method for the preparation of a peptide comprising the steps of: The ADC of embodiment 31. 35. ALK5 inhibitors bind to the antibody or antigen-binding fragment via one or more glycans 27. The ADC of embodiment 26, wherein the ADC is conjugated to 36. The ADC of embodiment 35, wherein one or more glycans comprises fucose. 37. The method of claim 35, wherein one or more glycans contain 6-thiofucose. DC. 38. The ADC of embodiment 35, wherein one or more glycans comprises galactose. . 39. One or more glycans contain N-acetylgalactosamine (GalNAc). 36. The ADC of embodiment 35. 40. One or more glycans contain N-acetylglucosamine (GlcNAc) , The ADC described in embodiment 35. 41. The method of claim 35, wherein one or more glycans comprises sialic acid (SA). DC. 42. The compound according to embodiment 26 to 4, in which the ALK5 inhibitor is conjugated via a linker. 2. The ADC according to any one of claims 1 to 11. 43. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 2 to 8. 43. The ADC of any one of embodiments 1 to 42, wherein 44. The method of any one of the preceding claims, wherein the antibody is a monoclonal antibody. C. 45. The ADC of embodiment 44, wherein the antibody is human or humanized. 46. ​​The ADC of embodiment 45, wherein the antibody is human. 47. The ADC of embodiment 45, wherein the antibody is humanized. 48. The antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. An ADC according to any one of embodiments 1 to 47. 49. The ADC of embodiment 48, wherein the antigen-binding fragment is a Fab. 50. The ADC of embodiment 48, wherein the antigen-binding fragment is a Fab'. 51. The ADC of embodiment 48, wherein the antigen-binding fragment is F(ab')2. 52. The ADC of embodiment 48, wherein the antigen-binding fragment is an Fv fragment. 53. Embodiment 48, in which the antigen-binding fragment is an antigen-binding fragment of a human or humanized antibody. 53. The ADC of any one of claims 1 to 52. 54. The method of claim 53, wherein the antigen-binding fragment is an antigen-binding fragment of a human antibody. C. 55. The antigen-binding fragment of embodiment 53, wherein the antigen-binding fragment is an antigen-binding fragment of a humanized antibody. DC. 56. The ADC of any of embodiments 1 to 47, comprising an antibody. 57. The ADC of any of embodiments 1 to 55, comprising an antigen-binding fragment. 58.T cell surface molecules include CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD70, CD71, CD103, CD184, Tim3 , LAG3, CTLA4, or PD1, ADC. 59. The ADC of embodiment 58, wherein the T cell surface molecule is CD1. 60. The ADC of embodiment 58, wherein the T cell surface molecule is CD2. 61. The ADC of embodiment 58, wherein the T cell surface molecule is CD3. 62. The ADC of embodiment 58, wherein the T cell surface molecule is CD4. 63. The ADC of embodiment 58, wherein the T cell surface molecule is CD5. 64. The ADC of embodiment 58, wherein the T cell surface molecule is CD6. 65. The ADC of embodiment 58, wherein the T cell surface molecule is CD7. 66. The ADC of embodiment 58, wherein the T cell surface molecule is CD8. 67. The ADC of embodiment 58, wherein the T cell surface molecule is CD25. 68. The ADC of embodiment 58, wherein the T cell surface molecule is CD28. 69. The ADC of embodiment 58, wherein the T cell surface molecule is CD70. 70. The ADC of embodiment 58, wherein the T cell surface molecule is CD71. 71. The ADC of embodiment 58, wherein the T cell surface molecule is CD103. 72. The ADC of embodiment 58, wherein the T cell surface molecule is CD184. 73. The ADC of embodiment 58, wherein the T cell surface molecule is Tim3. 74. The ADC of embodiment 58, wherein the T cell surface molecule is LAG3. 75. The ADC of embodiment 58, wherein the T cell surface molecule is CTLA4. 76. The ADC of embodiment 58, wherein the T cell surface molecule is PD1. 77. T cell surface molecules that can be recycled through endosomes 58. The ADC of any of embodiments 1 to 57, wherein 78. The ADC of embodiment 77, wherein the T cell surface molecule is CD5 or CD7. 79. The ADC of embodiment 78, wherein the T cell surface molecule is CD5. 80. The ADC of embodiment 78, wherein the T cell surface molecule is CD7. 81. Fc domain with one or more amino acid substitutions that reduce effector function 81. The ADC of any one of embodiments 1 to 80, comprising an 82. One or more substitutions are N297A, N297Q, N297G, D265A / N 297A, D265A / N297G, L235E, L234A / L235A, L234A / L235A / P329A, L234D / L235E:L234R / L235R / E23 3K, L234D / L235E / D265S:E233K / L234R / L235R / D 265S, L234D / L235E / E269K:E233K / L234R / L235R / E269K, L234D / L235E / K322A:E233K / L234R / L23 5R / K322A, L234D / L235E / P329W:E233K / L234R / L 235R / P329W, L234D / L235E / E269K / D265S / K322A :E233K / L234R / L235R / E269K / D265S / K322A, or L234D / L235E / E269K / D265S / K322E / E333K:E233 Including K / L234R / L235R / E269K / D265S / K322E / E333K , The ADC described in embodiment 81. 83. The ADC of embodiment 82, wherein the one or more substitutions comprises N297A. 84. The ADC of embodiment 82, wherein the one or more substitutions comprises N297Q. 85. The ADC of embodiment 82, wherein the one or more substitutions comprises N297G. 86. The method according to embodiment 82, wherein the one or more substitutions include D265A / N297A. ADC. 87. The method according to embodiment 82, wherein the one or more substitutions include D265A / N297G. ADC. 88. The ADC of embodiment 82, wherein the one or more substitutions comprises L235E. 89. The amino acid sequence according to embodiment 82, wherein the one or more substitutions include L234A / L235A. ADC. 90. The embodiment in which the one or more substitutions include L234A / L235A / P329A. 82. The ADC according to claim 82. 91. One or more substitutions are: L234D / L235E:L234R / L235R / E The ADC of embodiment 82, comprising 233K. 92. One or more substitutions are: L234D / L235E / D265S:E233K / L The ADC of embodiment 82, comprising 234R / L235R / D265S. 93. One or more substitutions are: L234D / L235E / E269K:E233K / L The ADC of embodiment 82, comprising 234R / L235R / E269K. 94. One or more substitutions are L234D / L235E / K322A:E233K / L The ADC of embodiment 82, comprising 234R / L235R / K322A. 95. One or more substitutions are: L234D / L235E / P329W:E233K / L The ADC of embodiment 82, comprising 234R / L235R / P329W. 96. One or more substitutions are: L234D / L235E / E269K / D265S / K 322A:E233K / L234R / L235R / E269K / D265S / K322A 83. The ADC of embodiment 82, comprising: 97. One or more substitutions are: L234D / L235E / E269K / D265S / K 322E / E333K:E233K / L234R / L235R / E269K / D265S The ADC of embodiment 82, comprising: / K322E / E333K. 98. An ADC according to any one of embodiments 1 to 97, and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising: 99. A method for treating cancer, comprising administering to a subject in need thereof any one of embodiments 1 to 97. 98. A method for treating a patient with a rheumatoid arthritis comprising administering to said patient an ADC ... a pharmaceutical composition comprising said ADC or a pharmaceutical composition comprising said pharmaceutical composition. method. 100. The method of embodiment 99, wherein the cancer is an immunogenic cancer. 101. The method of embodiment 100, wherein the cancer is a solid tumor that expresses a tumor antigen. 102. The method of embodiment 1, in which the tumor antigen is gp100, MelanA, or MAGE A1. The method described in 01. 103. The method of embodiment 102, wherein the tumor antigen is gp100. 104. The method of embodiment 102, wherein the tumor antigen is MelanA. 105. The method of embodiment 102, wherein the tumor antigen is MAGE A1. 106. The method of embodiment 99, wherein the cancer is a solid tumor containing immune infiltration. 107. Any of embodiments 99 to 106, wherein the cancer is treatable by immunotherapy. The method described. 108. Immunotherapy includes cytokine therapy, adoptive T cell therapy, and chimeric antigen receptor (CAR) 108. The method of embodiment 107, wherein the therapy is a T-cell checkpoint inhibitor therapy. 109. The method of embodiment 108, wherein the immunotherapy is a cytokine therapy. 110. The method of embodiment 108, wherein the immunotherapy is adoptive T cell therapy. 111. The method of embodiment 108, wherein the immunotherapy is chimeric antigen receptor (CAR) therapy. method. 112. As described in embodiment 108, the immunotherapy is a T-cell checkpoint inhibitor therapy. How to. 113. T cell checkpoint inhibitors inhibit PD1, PDL1, or CTLA4 The method of embodiment 108 or embodiment 112, wherein the 114. The method according to embodiment 113, wherein the T cell checkpoint inhibitor is an inhibitor of PD1. Method of posting. 115. The method according to embodiment 113, wherein the T cell checkpoint inhibitor is an inhibitor of PDL1. The method described. 116. The T cell checkpoint inhibitor is an inhibitor of CTLA4, embodiment 113. The method described above. 117. Cancer: Non-small cell lung cancer (NSCLC), liver cancer, urothelial cancer, renal cancer, breast 117. The method of any of embodiments 99-116, wherein the cancer is cancer, cancer, or melanoma. 118. The method of embodiment 117, wherein the cancer is NSCLC. 119. The method of embodiment 117, wherein the cancer is liver cancer. 120. The method of embodiment 120, wherein the liver cancer is hepatocellular carcinoma. 121. The method of embodiment 117, wherein the cancer is urothelial carcinoma. 122. The method of embodiment 121, wherein the cancer is bladder cancer. 123. The method of embodiment 117, wherein the cancer is renal cancer. 124. The method of embodiment 117, wherein the cancer is breast cancer. 125. The method of embodiment 117, wherein the cancer is melanoma. 126. Any of embodiments 99 to 125, wherein the cancer is treatable with an ALK5 inhibitor. The method according to any one of the above. 127. The ADC or pharmaceutical composition of embodiment 99 to 12, wherein the ADC or pharmaceutical composition is administered as monotherapy. 7. A method according to any one of claims 6 to 6. 128. An embodiment in which the ADC or pharmaceutical composition is administered as part of a combination therapy regimen 99. The method of claim 99, wherein the first and second substrates are fused to the first substrate. 129. The ADC or pharmaceutical composition is administered in combination with a standard of care therapy or treatment regimen. 129. The method of embodiment 128, wherein the

[0311] Various specific embodiments are illustrated and described, but they are not intended to depart from the spirit and scope of the disclosure. It will be understood that various changes may be made without departing from the spirit and scope of the invention.

[0312] 7. Citation of References All publications, patents, patent applications, and other references cited in this application are hereby expressly incorporated by reference in their entirety. Each patent, patent application, or other document is individually incorporated by reference for all purposes. and to the same extent as if each of the preceding claims were expressly granted in its entirety and for all purposes. Among the teachings of this specification and one or more of the references incorporated into this disclosure In the event of any conflict, the teachings of the present specification are intended.

Claims

1. ALK5 OPERATORY LINKED TO ANTIBODY OR ANTIGEN-BINDING FRAGMENT THAT BINDS TO A T-CELL SURFACE MOLECULE An antibody-ALK5 inhibitor conjugate (ADC) comprising the inhibitor.

2. IC of the ALK5 inhibitor ５０ is at least 20 nM. C.

3. The ALK5 inhibitor is an imidazole compound, a pyrazole compound, or a thiazolide compound.

2. The ADC of claim 1, which is a benzodiazepine-based compound.

4. The ALK5 inhibitor is an imidazole-benzodioxole compound, an imidazole- a pyrazole-pyrrolo compound, or a thiazole-based compound, Item 3. The ADC according to item 3.

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

6. N-Maleimidomethylcyclohexane-1-carboxylate, Maleimidocaproyl or via a non-cleavable linker which is a mercaptoacetamidocaproyl linker 6. The method of claim 5, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment. The ADC described herein.

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

8. The linker may be a protease-sensitive valine-citrulline dipeptide linker, a glutamate linker, The linker is a thione-sensitive disulfide linker or an acid-sensitive disulfide linker. Item 8. The ADC according to item 7.

9. The ALK5 inhibitor comprises one or more sites on the antibody or antigen-binding fragment. a tein residue, or one or more lysine residues on the antibody or antigen-binding fragment. and optionally said ALK5 inhibitor is conjugated via a linker.

2. The ADC of claim 1 .

10. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is in the range of 2 to 8.

2. The ADC of claim 1 ,

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 antigen-binding fragment is Fab, Fab', F(ab') ２ or an Fv fragment, The ADC of claim 1.

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

15. 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 T cell surface molecule is capable of recycling through endosomes.

2. The ADC of claim 1 ,

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

18. A pharmaceutical composition comprising the ADC of claim 1 and a pharma- ceutically acceptable carrier.

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

20. The cancer is (a) Is the cancer immunogenic? (b) Is it a solid tumor that contains an immune infiltrate? (c) is a solid tumor that is treatable by immunotherapy; or 20. The method of claim 19, wherein (d) the patient is treatable with an ALK5 inhibitor.

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

22. The cancer is treatable by immunotherapy, the immunotherapy being selected from the group consisting of cytokine therapy, trophoblast therapy, and immunotherapy. T cell therapy, chimeric antigen receptor (CAR) therapy, or T cell checkpoint inhibitors The method of claim 20 which is a therapy.

23. The ADC is administered as a monotherapy or as part of a combination therapy regimen.

20. The method according to claim 19.

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