Antibody-ALK5 inhibitor conjugates and their use

Abstract

To provide antibody-drug conjugates to target ALK5 inhibitors for cell types in which the inhibition of TGF-β signaling is therapeutically useful, while minimizing host tissue toxicity such as those observed in cardiac tissue and their uses.SOLUTION: The present invention provides an antibody-ALK5 inhibitor conjugate (ADC) comprising an ALK5 inhibitor operably linked to an antibody or an antigen binding fragment that binds to a T cell surface molecule. The present invention also provides a method of treating cancer, comprising administering to a subject in need thereof the ADC.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. Cross-reference to related applications This application is a PCT application filed on July 9, 2018, as published in U.S. Patent Application Publication No. 2018. Specification No. / 041291 and the US Patent Application for PCT Filing on June 19, 2019 Claiming the benefit of priority from the specification of the approved application publication No. 2019 / 037978, and the respective contents The entire text is incorporated herein by reference. [Background technology]

[0002] 2.Background Cytokine transforming growth factor-beta (TGF-β) family The cells undergo a wide variety of biological processes, both during normal tissue development and in disease states. It is a multifunctional protein that regulates inflammation and wounds. Members of the TGF-β family are involved in inflammation and wounds. Wound healing, accumulation of extracellular matrix, bone formation, tissue development, cell differentiation, cardiac valve remodeling It is involved in tissue fibrosis and tumor progression. (Barnard et al., 1990, Biochi 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 isotypes have been identified so far: TGF-β 1. TGF-β2 and TGF-β3 (Massague, 1990, Annu Rev Cell Biol 6:597-6) 41). As another member of the transforming growth factor superfamily, Acti Bin, inhibin, bone morphogenetic proteins, growth and differentiation factors, and Müller inhibitors A substance is one example.

[0003] TGF-βI is a receptor for two highly conserved single transmembrane serine / threonine kinases. The body converts signals via type I (ALK5) and type II TGF-β receptors. When ligand-inducible binding and oligomerization occur, the type II receptor becomes the GS of ALK5. In this region, serine / threonine residues are phosphorylated, thereby activating ALK5. This results in the generation of new SMAD introduction sites. SMADs are introduced from the extracellular environment into the cell nucleus. It is an intracellular protein that specializes in converting the signal of TGF-β. ALK5, with its C-terminal SSXS motif, phosphorylates Smad2 and Smad3. Therefore, this leads to their dissociation from the receptor and the formation of a complex with Smad4. Then, the Smad complex moves into the nucleus and assembles with cell-specific DNA-binding cofactors, and then... It modifies the expression of genes that regulate growth, differentiation, and development.

[0004] Activin converts signals in a similar way to TGF-β. Activin is used in celery ₂ / threonine kinase binds to activin type II receptor (ActRIIB) and activates it. The type II receptor, in the GS region of ALK4, exhibits hyperphosphatemia of serine / threonine residues. It transforms into hyperphosphorylate. Activated ALK4 then sequentially transforms into Smad2 and Sma d3 is phosphorylated. As a result, it forms a hetero-Smad complex with Smad4. This leads to the regulation of gene transcription induced by activin.

[0005] TGF-β signaling is involved in T lymphocytes and B lymphocytes, NK cells, and dendritic cells. By regulating both innate and adaptive immune cells, including antigen-presenting cells such as cysts, It is essential for maintaining immune homeostasis. TGF-β is generally used in the thymus. Immunosuppressive cells play an essential role in cell development and maintaining peripheral tolerance. It is considered to be in. TGF-β is CD4 + and CD8 + Proliferation of both T cells, site It inhibits the production of kine, cytotoxicity, and differentiation into T helper subsets (Li et al. ., 2008, Cell 134:392-404). TGF-β is also a natural regulatory T cell that arises in the thymus. The development of nTregs, as well as their peripheral development in response to various diseases such as inflammation and cancer. It plays an important role in the development of inducible Tregs (iTregs) (Tran et al., 2012, J (Mol Cell Bio 4:29-37, 2012). nTregs are typically CD25+ FoxP3+. Therefore, it actively suppresses the activation of T cells, which helps maintain peripheral T cell tolerance. It is a subset of CD4+ T cells that do not make up a certain proportion. TGF-β is used in peripheral nT reg of It is important for survival and expansion (Marie et al., 2005, J Exp Med 201:1061-67). Under inflammatory conditions, TGF-β affects naive CD4 + T cells, FoxP3 + iT reg It converts to suppress localized tissue-resident T cells. High levels of iT reg In many cases, It has been found that within the tumor itself, T cell-mediated tumor clearance is prevented (Whites ide, 2014, Expert Opin Biol Ther 14:1411-25).

[0006] Generally, high levels of TGF-β expression are associated with poor clinical prognosis. Many cases, tumors incorporate and utilize the TGF-β pathway to avoid T cell-mediated tumor clearance (Yang et al., Trends Immunol 31:220-7, 2010, Tu et al., Cytokine Growth Factor Rev 25:423-35, 2014). This occurs in two ways. The first is that TGF-β directly inhibits the expansion of CD4+ and CD8+ T cells, cytokine production, and tumor cell death. The second is that TGF-β is important for the survival and / or conversion of nT and iT respectively, which also suppresses immune-mediated tumor clearance. In a number of preclinical mouse models, it has been demonstrated that neutralization of TGF-β results in reduced tumor burden due to increased T cell-mediated tumor clearance. Importantly, inhibition of TGF-β signaling in T cells via expression of a dominant negative TGF-βRII or using a soluble TGF-β receptor is sufficient to restore effective immune-mediated tumor clearance in vivo. Gorelik et al., 2001, Nat Med 7:1118-22, Thomas et al., 2005, Cancer Cell 8:369-80. reg and iT reg respectively, which also suppresses immune-mediated tumor clearance. In a number of preclinical mouse models, it has been demonstrated that neutralization of TGF-β results in reduced tumor burden due to increased T cell-mediated tumor clearance. Importantly, inhibition of TGF-β signaling in T cells via expression of a dominant negative TGF-βRII or using a soluble TGF-β receptor is sufficient to restore effective immune-mediated tumor clearance in vivo. Gorelik et al., 2001, Nat Med 7:1118-22, Thomas et al., 2005, Cancer Cell 8:369-80. In addition to its effects on the immune system, TGF-β signaling plays an important but complex role in tumorigenesis. In preclinical studies, TGF-β has a paradoxical effect on the tumor itself

[0007] In addition to its effects on the immune system, TGF-β signaling plays an important but complex role in tumorigenesis. In preclinical studies, TGF-β has a paradoxical effect on the tumor itself​​​​​ It has been shown to have effects and confounding effects on surrounding stromal cells. In the early stages, TGF-β regulates tumor growth and It inhibits swelling. However, in the later stages, TGF-β loses its growth-inhibiting properties. Through the induction of epithelial-mesenchymal transition (EMT), as well as stromal fibroblasts, angiogenesis, It promotes tumor metastasis through its effects on the extracellular matrix (ECM) (Connolly e (t al., 2012, Int J Bio 8:964-78). If delivered at the wrong stage, TGF-β signaling Broad-spectrum inhibition of signal transduction carries the risk of promoting tumor metastasis and / or non-tumor metastasis. There is a risk of inhibiting the cerebral stromal cell population and indirectly worsening 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 inhibition effect, the inhibitors cause the tumor to become more invasive and metastatic. It is possible.

[0008] Despite the paradoxical effects on the tumor itself and the widespread expression of TGF-β receptors Inhibition of the TGF-β pathway as a cancer treatment has long been a subject of interest. Inhibitors are TGF -β-neutralizing antibody, TGF-β2 antisense RNA, and small molecule ATP-competitive ALK5 quinol This includes enzyme inhibitors. Some of the classic ALK5 inhibitors being developed are pyrazole-based. These are imidazole-based and triazole-based drugs (Bonafoux et al., 2009, Expert Opin). Ther Patents 19:1759-69, Ling et al., 2011, Current Pharma Biotech 12:2190-2202 Many ALK5 inhibitors are used in in vitro cell-based assays, as well as in in vitro cell-based assays. It has been tested in both xenograft and syngeneic tumor models in ivo mice, demonstrating significant efficacy. (Neuzillet et al., 2015, Pharm & Therapeutics 147:22-31). However, T Because GF-β receptors are expressed in an eccentric manner, there are issues of host toxicity and the intention to promote tumor growth. Due to the risk of promoting the disease, many TGF-β inhibitors, especially ALK5 inhibitors, have been shown to be preclinically effective. It remains in the preliminary stage. For example, in preclinical toxicological studies in rats, two different The ALK5 inhibitor series is characterized by bleeding, inflammation, degeneration, and proliferation of valve interstitial cells. A lesion of the heart valve was found (Anderton et al., 2011, Tox Path 39:916-24). [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Therefore, while minimizing host tissue toxicity, such as that observed in cardiac tissue, ALK5 inhibitors are targeted at cell types where inhibiting TGF-β signaling is therapeutically beneficial. It is necessary to target it. [Means for solving the problem]

[0010] 3. Overview To avoid targeted host toxicity and to prevent unintended tumor progression with ALK5 inhibitor therapy. To prevent deterioration, the inventor directs the compound only to these cells that will yield therapeutic benefits. We developed a novel approach for this purpose.

[0011] For cancer treatment, this approach involves guiding ALK5 inhibitors to the T cell compartment via antibodies. This promotes T-cell-mediated tumor clearance and long-term effects without causing systemic toxicity. It includes ensuring complete remission. Although not bound by theory, TG in T cells Inhibition of F-β signaling not only directly improves T cell-mediated clearance, T inducible T cells reg It inhibits the conversion of natural T in tumors. reg Lower the survival rate Therefore, inhibition of TGF-β signaling within T cells is thought to affect CD4 + Oh CD8 + In addition to restoring T cell activity, T cells reg Remove "brake" This effectively reconnects the immune system. More importantly, TGF- Inhibition of β-signaling has broad-spectrum implications, both from a tumor perspective and in terms of host tissue toxicity. It is safer than TGF-β inhibitors.

[0012] Therefore, this disclosure relates to an antibody-drug conjugate (A) in which the drug is an ALK5 inhibitor. The ADC provides T cell surface molecules (e.g., human T cell surface molecules). ) may be an antibody or antigen-binding fragment that binds to ). Section 5.2 describes the use of the ADCs of this disclosure Examples of antibody components that can be used are described. In some embodiments, AL K5 inhibitors include imidazole-benzodioxole compounds and imidazole-quinoxaline. These are compounds, pyrazole-pyrrolo compounds, or thiazole compounds. An example is ALK. The 5 inhibitors are listed in Section 5.3 and Tables 1-3.

[0013] ALK5 inhibitors can be directly conjugated with antibody components, or The linker can be linked to the antibody component. The linker is a non-cleavable linker. Alternatively, it may be a severable linker. An example of a non-severable linker is shown below. The cleavable linker is described in Section 5.4. For each antibody or antigen-binding fragment The average number of bound ALK5 inhibitor molecules can vary, and generally, antibody or antigen binding is involved. Each fragment contains between 2 and 8 ALK5 inhibitor molecules. Drug loading is detailed in Section 5.5. It is stated.

[0014] This disclosure further provides pharmaceutical compositions including the ADC of this disclosure. Examples of pharmaceutical additives that can be used to formulate pharmaceutical compositions are shown in section 5. It is described in section 6.

[0015] This disclosure provides for administering the ADCs or pharmaceutical compositions of this disclosure to subjects who require them. This further provides methods for treating cancer. ADCs and pharmaceutical compositions of this disclosure These substances can be used as monotherapy or as part of combination therapy, for example, immune checkpoint inhibitors. It can be administered in combination with the agent. Treatment with the ADC and pharmaceutical composition of this disclosure Exemplary cancers for which this treatment is possible, as well as exemplary combination therapies, are described in Section 5.7. [Brief explanation of the drawing]

[0016] 4. Brief explanation of the drawing [Figure 1]This study demonstrates the effects of TGF-β on CD4+ and CD8+ T cells. During tumor progression, TGF-β, which can originate from both the tumor and the T cells themselves, inhibits CD4+ T cell functions such as cytokine production, proliferation, and Th differentiation. Simultaneously, TGF-β also inhibits tumor death by inhibiting the expression of granzymes and perforins in cytotoxic CD8+ T cells. By inhibiting both CD4+ and CD8+ T cell populations, it completely suppresses T cell-mediated tumor clearance. [Figure 2] This study demonstrates the effects of TGF-β on Treg cells during tumor progression. During tumor progression, nTreg and iTreg cells are typically found within the tumor and regulate in situ T cell-mediated function. TGF-β suppresses T cell-mediated tumor clearance by promoting nTreg cell viability and iTreg cell conversion. The increase in Treg cells at the tumor site ensures that T cells infiltrating the tumor also prevent the tumor from being cleared. [Figure 3] This disclosure demonstrates the mechanism of action of the ADC in CD4+ and CD8+ T cells. Inhibition of T cell targeting of TGF-β signaling restores CD4+ T cell activity and CD8+ T cell-mediated tumor death. [Figure 4] This disclosure demonstrates the mechanism of action of ADCs in Treg cells. Inhibition of T cell targeting of TGF-β signaling also blocks Treg-mediated suppression of immune-mediated tumor clearance in situ. [Figure 5A-B] The inhibition of TGF-β-induced luciferase activity in HEK293T cells by compounds A and B is shown. Figure 5A: Compound A, Figure 5B: Compound B. [Figure 5C-D] The inhibition of TGF-β-induced luciferase activity in HEK293T cells by compounds C to D is shown. 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 using the 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] Figure 7A shows LC-MS data for an exemplary ADC (ADC2) of this disclosure. Figure 7B shows LC-MS data for the ADC heavy chain and for the ADC light chain. [Figure 8] This is a chromatogram of ADC2 purified by SEC and prepared with an S-4FB / Ab ratio of 6. SEC analysis of the purified ADC2 shows that aggregation is less than 5%. [Figure 9A-B] This disclosure demonstrates that an exemplary antibody (anti-transferrin receptor antibody R17217) induces the internalization of the transferrin receptor (TfR), the antibody's target, 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] This disclosure demonstrates that an exemplary antibody (anti-transferrin receptor antibody R17217) induces the internalization of the transferrin receptor (TfR), the antibody's target, 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]This disclosure demonstrates that an exemplary antibody (anti-transferrin receptor antibody R17217) induces the internalization of the transferrin receptor (TfR), the antibody's target, in primary mouse CD4+ T cells. Figure 9E: 180-minute incubation with anti-transferrin receptor antibody; Figure 9F: Mean fluorescence intensity (MFI) over a 3-hour time course. [Figure 10] This disclosure demonstrates the reversal of TGF-β-mediated inhibition of proliferation in mouse CTLL2 cells by an exemplary ADC (ADC1). [Figure 11] This disclosure demonstrates the desuppression of granzyme B expression in TGF-β-activated primary CD8+ T cells by an exemplary ADC (ADC1). ADC1 partially restores granzyme B expression to a degree comparable to that of a free ALK5 inhibitor. [Figure 12] This disclosure demonstrates that an exemplary ADC (ADC1) reduces iTreg production, similar to a 100 mM free ALK5 inhibitor. [Figure 13A-B] This shows the internalization of CD5 (Figure 13A) and CD2 (Figure 13B) into activated primary mouse CD3+ T cells. [Figure 13C-D] This shows the internalization of CD5 (Figure 13C) and CD2 (Figure 13D) into activated primary mouse CD3+ T cells. [Figure 14] The levels of CD8+ T cells expressing granzyme (GzmB) are shown following 36-hour incubation of activated mouse CD3+ T cells in the presence of T3A #2~#5. [Figure 15] This shows the levels of secreted IL-2 following 36 hours of incubation of activated mouse CD3+ T cells in the presence of T3A #2~#5. [Figure 16] This shows the levels of secreted IFN-γ following 36 hours of incubation of activated mouse CD3+ T cells in the presence of T3A #2~#5. [Figure 17] This shows the amount of T cell proliferation following 72 hours of incubation of activated mouse CD3+ T cells in the presence of T3A #2~#5. [Figure 18]This demonstrates the internalization of CD7 into activated human primary CD3+ T cells. [Figure 19] The levels of IFN-γ secreted from CMV-responsive PBMCs cultured in the presence of CMV antigen, as well as (i) 1 nM TGF-β ("+"), (ii) 1 nM TGF-β and T3A #5 at 1 ng / ml ("T3A"), (iii) 1 nM TGF-β and pembrolizumab at 1 ng / ml ("ICI"), (iv) 1 nM TGF-β, T3A #5, and pembrolizumab at 1 ng / ml ("T3A ICI"), or (v) 1 nM TGF-β and isotype control antibody ("-control"). [Modes for carrying out the invention]

[0017] 5. Detailed explanation This disclosure relates to antibody-drug conjugates (ADCs) useful for treating cancer, Antibodies containing antibody components that are covalently bound to ALK5 inhibitors, either directly or through a linker. - Provides drug conjugates (ADCs). An overview of the ADCs of this disclosure is provided in Section 5.1. The antibody component of the ADC may be a complete antibody or a fragment thereof. The antibodies that can be used are detailed in Section 5.2. ALK5 inhibitors capable of this are detailed in Section 5.3. The ADCs of this disclosure are typically It contains a linker between the antibody and the ALK5 inhibitor. Exemplary linkers that can be used are detailed in Section 5.4. The ADCs of this disclosure per antibody It can contain various numbers of ALK5 inhibitor moieties. Drug loading is detailed in Section 5.5. This disclosure further provides pharmaceutical formulations, including the ADC of this disclosure. The pharmaceutical formulations included are described in Section 5.6. This disclosure describes how to use the ADCs of this disclosure. Further methods to treat various cancers are provided. As monotherapy for the treatment of cancer, or The use of the ADCs of this disclosure as part of a combination therapy is described in Section 5.7.

[0018] 5.1. Antibody-drug conjugates The ADCs of this disclosure are generally designed so that the covalent bond does not interfere with the binding of the antibody to the target. It is primarily composed of an ALK5 inhibitor covalently bound to an antibody via a linker.

[0019] Techniques for conjugating drugs into antibodies are well known in the relevant field (e.g.) For example, Hellstrom et al., Controlled Drug Delivery, 2nd Ed., at pp. 623-53 (Robins on et al., eds., 1987), Thorpe et al., 1982, Immunol. Rev. 62:119-58, Dubowchik et al., 1999, Pharmacology and Therapeutics 83:67-123, and Zhou, 2017, Biomed See icines 5(4):E64). ALK5 inhibitors are preferably site-specific conjugates. It binds to the antibody component in the ADC of this disclosure via fermentation. For example, an ALK5 inhibitor One or more natural or designed cysteine, lysine, or glutamine residues A group, one or more unnatural amino acids (e.g., p-acetylphenylalanine (pA) cF), p-azidomethyl-L-phenylalanine (pAMF), or selenoside (Sec), one or more glycans (e.g., fucose, 6-thiofucose) Galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine 1 of 4-6 amino acids (GlcNAc, or sialic acid (SA)), or 1 of 4-6 amino acids It can be conjugated with antibody components via one or more short peptide tags. For example, see Zhou, 2017, Biomedicines 5(4):E64, and its contents are as follows: The entirety of this is incorporated herein.

[0020] In one example, an antibody or a fragment thereof may be another protein (or a portion thereof, for example, a The amino acid sequence of at least 10, 20, or 50 amino acids in the protein In contrast, via covalent bonds (e.g., peptide bonds), the N-terminus or C-terminus of the antibody can be transmitted. To fuse, either internally or externally. The antibody, or a fragment thereof, with other proteins, and the antibody The constant domain can be ligated at the N-terminus. Recombinant DNA procedures involve such fusions. It can be used to create, for example, the pamphlet for International Publication No. 86 / 01533. In another example, Therefore, effector molecules can increase the in vivo half-life, and / or Alternatively, it can improve the delivery of antibodies to the immune system after crossing the epithelial barrier. Suitable effector molecules include polymers, albumin, and albumin-binding proteins. Examples include albumin-binding compounds, for example, PCT Publication No. 2005 / This information is included in pamphlet number 117984.

[0021] A metabolic process or metabolic reaction is an enzymatic process, for example, the peptide linker of ADC. Protein-degradable cleavage, or addition of functional groups, such as hydrazones, esters, or amides. It may be involved in water splitting. As an intracellular metabolite, it is not limited to, but is involved in cell entry, diffusion, and uptake. Examples include antibodies and free drugs that are cleaved intracellularly after transport or transport.

[0022] The terms "cleaved within the cell" and "intracellular cleavage" refer to antibody-drug conjugates. This refers to the intracellular metabolic process or reaction of ADCs, thereby affecting the drug portion (D). The covalent bond between the antibody (Ab) and the cell, i.e., the linker, is destroyed, and as a result, the antibody enters the cell. It releases free drugs that have been dissociated from the body. Therefore, the cleaved portion of ADC is an intracellular metabolite. be.

[0023] 5.2. Antibody components This disclosure provides an antibody-drug conjugate in which an antibody component binds to a T cell surface molecule. Unless otherwise specified, the term “antibody” (Ab) means an antibody that specifically binds to a particular antigen. This refers to immunoglobulin molecules that react immunologically, such as polyclonal antibodies or monoclonal antibodies. Examples include ronal antibodies, genetically modified antibodies, and other modified forms of antibodies, and are limited to... However, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (for example, bispecific antibodies) Bispecific antibody, diabody antibody, triplicate antibody, and quadruple antibody Specific antibodies), as well as, for example, Fab', F(ab')2, Fab, Fv, rIgG, Examples include antigen-binding fragments of antibodies containing scFv fragments. Furthermore, unless otherwise indicated, To the extent of the term, "monoclonal antibody" (mAb) refers to an antibody that specifically binds to a protein. This is possible for complete molecules, as well as antibody fragments (e.g., Fab and F(ab')2 fragments). This means that it contains both fragments. The Fab and F(ab')2 fragments are the F of the complete antibody. The c fragment is missing, and it is removed more rapidly from the circulation of animals or plants, and the complete antibody... It may have less nonspecific tissue binding (Wahl et al., 1983, J. Nucl. Med. 24:316).

[0024] The term "scFv" refers to a compound formed by combining the variable domains of the heavy and light chains derived from conventional antibodies. This refers to single-stranded Fv antibodies that form a chain.

[0025] References to "VH" refer to antibody immunoglobulins containing the heavy chains of Fv, scFv, or Fab. This refers to the variable region of the robulin heavy chain. References to "VL" include Fv, scFv, dsFv, Alternatively, it refers to the variable region of the immunoglobulin light chain, including the Fab light chain. Antibodies (Ab) and Immunoglobulins (Ig) are glycoproteins that share the same structural characteristics. Antibodies are specific. While antibodies exhibit binding specificity to specific targets, immunoglobulins exhibit binding specificity to antibodies and target specificity. It includes both other antibody-like molecules that lack the opposite sex. Natural antibodies and immunoglobulins are It consists of two identical light chains (L) and two identical heavy chains (H), typically about 150,0 It is a 00 Dalton heterotetrameric glycoprotein. Each heavy chain has a variable dormancy at the amino terminus. It has a (VH) group followed by several constant domains. Each light chain has an amino terminus. It has a variable domain (VL) and a constant domain at the carboxyl terminus.

[0026] For optimal delivery of ALK5 inhibitors within cells, antibodies are preferably internalized. Resident antibodies, after binding to their target molecules on the cell surface, then, as a result of this binding, are transmitted by the cell It is internalized. This effect is that ADCs are taken up by cells. It binds to the antigen. A process that enables the determination of antibody internalization is known to those skilled in the art, for example, P Page 80 of the International Publication No. 2007 / 070538 of the CT Publication, and the following: It is described in section 6.11. When internalized, for example, as described in section 5.4, When an ALK5 inhibitor binds to an antibody using a cleavable linker, the ALK5 inhibitor will It can be released from the antibody by cleavage in lysosomes or by other cellular mechanisms. .

[0027] The term "antibody fragment" refers to a portion of a full-length antibody, generally known as the target-binding or variable region. Examples of fragments include Fab, Fab', F(ab')2, and Fv. The "Fv" fragment is a minimal antibody fragment containing complete target recognition and binding sites. The region consists of a dimer of a variable domain consisting of one heavy chain and one light chain that are tightly non-covalently bonded. (VH-VL dimer). In this structure, the three CDRs of each variable domain interact, The surface of the VH-VL dimer defines antigen-binding sites. In many cases, six CDRs are anti It gives target binding specificity to the body. However, in some cases, a single variable domain The (or half of the Fv containing only the three target-specific CDRs) recognizes the target and connects It can have the ability to combine. A single-stranded Fv or scFv antibody fragment is one The polypeptide chain contains the VH and VL domains of the antibody. Generally, Fv polypept Buttido allows scFv to form the desired structure for target binding, VH It further includes a polypeptide linker between the VL domains. A "single-domain antibody" is a T It consists of a single VH or VL domain that exhibits sufficient affinity for NF-α. In a specific embodiment, the single-domain antibody is a camel antibody (e.g., Richman See n, 1999, Journal of Immunological Methods 231:25-38.

[0028] The Fab fragment contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment contains one or more cysteine ​​heavy chains from the antibody hinge region. The addition of several residues at the carboxyl terminus of the CH1 domain differentiates it from the Fab fragment. The F(ab') fragment is a hinge cysteine ​​of the F(ab')2 pepsin digestion product. It is produced by the cleavage of sulfide bonds. Further chemical bonding of antibody fragments is known to those skilled in the art. It is being done.

[0029] In certain embodiments, the antibody of this disclosure is a monoclonal antibody. The term "monoclonal antibody" used here refers to an antibody produced through hybridoma technology. It is not limited to the body. The term "monoclonal antibody" refers to an antibody derived from a single clone. This refers to eukaryotic, prokaryotic, or phage clones, but does not include methods for producing them. Monoclonal antibodies useful in connection with this disclosure include hybridoma, recombinant, and f Many known in the art, including the use of rage display technology or a combination thereof. They can be prepared using a variety of techniques. The antibodies disclosed herein include chimeric and primate-like antibodies. Examples include (primatized), humanized, or human antibodies.

[0030] The antibodies disclosed herein may be chimeric antibodies. The term “chimeric” as used herein refers to an antibody and This includes variable sequences derived from non-human immunoglobulins such as rat or mouse antibodies, and Typically selected from human immunoglobulin templates, the constant region of human immunoglobulins This refers to antibodies that have a specific region. Methods for producing chimeric antibodies are known in the art. 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. Patent U.S. Patent No. 5,807,715, U.S. Patent No. 4,816,567, and U.S. Patent See Patent No. 4,816,397, the contents of which are hereby referred to in their entirety. It will be incorporated into it.

[0031] The antibodies described herein may be humanized. The “humanization” of non-human (e.g., mouse) antibodies is: Chimeric immunoglobulins containing minimal sequences derived from non-human immunoglobulins, immunoglobulins Bulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 of an antibody) or other target-binding subdomains). Generally, humanized antibodies have at least one, Typically, it includes virtually all of the two variable domains, where all of the CDR area, or In effect, all of them correspond to non-human immunoglobulins, and all of the FR region, or substantially All of these are human immunoglobulin sequences. Humanized antibodies also determine immunoglobulins. It includes at least a portion of the normal region (Fc), typically a common sequence of human immunoglobulins. This is possible. Methods for humanizing antibodies are known in the art. For example, Richman n et al., 1988, Nature 332:323-7, U.S. Patent No. 5,530,10 U.S. Patent No. 1, U.S. Patent No. 5,585,089, U.S. Patent No. 5,693,761 Specification, U.S. Patent No. 5,693,762, U.S. Patent No. 6,180,370 The document, European Patent Application Publication No. 239400, International Publication No. 91 / 0996 of the PCT. Brochure No. 7, U.S. Patent No. 5,225,539, European Patent Application Publication No. 592 Specification No. 106, European Patent Application Publication No. 519596, Padlan, 1991, Mol. Immun ol., 28:489-498, Studnicka et al., 1994, Prot. Eng. 7:805-814, Roguska et al., 1 See 994, Proc. Natl. Acad. Sci. 91:969-973, U.S. Patent No. 5,565,332. All of these are incorporated herein by reference in their entirety.

[0032] The antibodies disclosed herein may be human antibodies. Fully "human" antibodies are used in the therapeutic treatment of human patients. It may be desirable for the placement. As used herein, "human antibody" means human immunoglobulin The antibody contains the amino acid sequence of robulin, and is part of a human immunoglobulin library, or Isolation from animals that are transgenic with respect to one or more human immunoglobulins. It includes antibodies that do not express endogenous immunoglobulins. Human antibodies are human immunoglobulins The technical field includes a phage display method using a sequence-derived antibody library. It can be manufactured by various methods known to the public. For example, U.S. Patent No. 4,444,888 U.S. Patent No. 7 and U.S. Patent No. 4,716,111, as well as international publications of the PCT. Pamphlet No. 98 / 46645, International Publication No. 98 / 50433, Country International Publication No. 98 / 24893, International Publication No. 98 / 16654 International Publication No. 96 / 34096, International Publication No. 96 / 33735 See International Publication No. 91 / 10741, each of which is by reference to the The entirety is incorporated herein. Human antibodies also express functional endogenous immunoglobulins. It is impossible to do so, but it is possible to express human immunoglobulin genes, trans It can be produced using genetically modified mice. For example, in the International Publication No. 9 of the PCT Publication. Pamphlet No. 8 / 24893, International Publication No. 92 / 01047, International Publication Pamphlet No. 96 / 34096, International Publication Pamphlet No. 96 / 33735, United States Japanese Patent No. 5,413,923, U.S. Patent No. 5,625,126, U.S. Patent U.S. Patent No. 5,633,425, U.S. Patent No. 5,569,825, U.S. Patent No. 5 U.S. Patent No. 661,016, U.S. Patent No. 5,545,806, U.S. Patent No. 5,8 U.S. Patent No. 14,318, U.S. Patent No. 5,885,793, U.S. Patent No. 5,916 See U.S. Patent No. 771 and U.S. Patent No. 5,939,598, which contain the following information. The entirety of that is incorporated herein by reference. In addition, Medarex (Princ eton, NJ), Astellas Pharma (Deerfield, Ill. .), Amgen (Thousand Oaks, Calif.), and Regene Using the same technology as described above, companies like ron (Tarrytown, NY) It is possible to provide human antibodies targeted to the selected antigen. Fully human antibodies that recognize the virus use a technique called "guided selection." This can be generated. In this approach, selected non-human monoclonal antibodies Using a human body, such as a mouse antibody, we induce the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., 1988, Biotechnology 12:899-903).

[0033] The antibodies disclosed herein can be primate-modified. The term "primate-modified antibody" refers to the monkey variable region and This refers to antibodies that include a human constant region. Methods for producing primate-like antibodies are described in the relevant technical field. It is known to the United States Patent No. 5,658,570, and U.S. Patent No. 5,68 Refer to U.S. Patent No. 1,722 and U.S. Patent No. 5,693,780, and their contents. The entire text is incorporated herein by reference.

[0034] The antibodies described herein include derivatized antibodies. For example, but not limited to, derivatized antibodies are... In terms of type, glycosylation, acetylation, pegylation, phosphorylation, amidation, known protecting groups / br Derivatization by locking groups, proteolytic cleavage, cell ligands or other proteins Modified by linking to (see Section 5.1 for consideration of antibody conjugates), etc. One of numerous chemical modifications, but not limited to, a specific chemical cleavage, acetyl This can be done using known techniques, including chemical synthesis, formylation, and metabolic synthesis of tunicamycin. In addition, derivatives can be made using, for example, AMBRX technology, one or more non-natural ingredients. It can contain amino acids (for example, Wolfson, 2006, Chem. Biol. 13(10):1011-2). reference).

[0035] In yet another embodiment of this disclosure, an antibody or fragment thereof corresponds to a wild-type sequence. In contrast, in order to modify the function of at least one steady-state region-mediated biological effector This may be an antibody or antibody fragment with a modified sequence. For example, in some embodiments... The antibody of this disclosure is modified to exhibit at least one constant region-mediated relationship with respect to the unmodified antibody. It is possible to reduce the function of biological effectors, for example, the Fc receptor (FcγR) Alternatively, the binding to C1q can be reduced. The binding of FcγR and C1q is F In a specific region required for cγR or C1q interaction, the immunoglobulin constant region segment of the antibody It 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 ., 2017, J Biol Chem 292: 3900-08, and Wang et al., 2018, Protein Cell 9:63-73. ).

[0036] The decrease in antibody FcγR binding ability is also another effector that depends on FcγR interaction. - It also has low function, such as opsonization, phagocytosis, and antibody-dependent cytotoxicity ("ADCC"). While it may decrease in some cases, a reduction in C1q binding reduces complement-dependent cytotoxicity ("CDCC"). Therefore, by reducing or eliminating the effector function, the ADC of this disclosure may target This may prevent the T cells being transformed from being destroyed via ADCC or CDC. In some embodiments, the effector function of the antibody is the selective function of the Fc portion of the antibody. Modified by mutation, which consequently maintains antigen specificity and internalization ability, but ADC The C / CDC function will be removed.

[0037] Numerous mutations that reduce FcγR and C1q binding have been described in the art. Such mutations may be included in the ADCs of this disclosure. For example, U.S. Act No. 6, In Specification No. 737,056, positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, Single-position Fc region at 376, 414, 416, 419, 435, 438, or 439 Regional amino acid modifications result in decreased binding to FcγRII and a reduction in FcγRII levels. This has been disclosed. In U.S. Patent No. 9,790,268, amino acid position 29 The asparagine residue at position 8, and the serine or threonine residue at amino acid position 300 It has been disclosed that it reduces FcγR binding. PCT Publication International Publication No. 2014 / In pamphlet No. 190441, 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 / E333K sudden change Modified Fc domains with reduced FcγR binding are described, and here, The set of mutations preceding the semicolon is located in the first Fc polypeptide, and the semicolon The subsequent mutation is located in the second Fc polypeptide of the Fc dimer. FcγR receptor binding. Furthermore, mutations that can reduce C1q binding include N297A, N297Q, N297G, D265A / N297A, D265A / N297G, L235E, L234 Examples include 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).

[0038] To reduce the effector function, the steady-state region is altered, for example, the Fc domain mentioned above. Instead of mutating the antibody, the effector function is to use an antibody fragment (e.g., Fab, Fab'). Alternatively, it can be eliminated by using the F(ab')2 fragment.

[0039] In another embodiment of the present disclosure, an antibody or a fragment thereof is modified against an unmodified antibody. To acquire or improve the function of at least one steady-state region-mediated biological effector This can be achieved, for example, by improving FcγR interaction (for example, US patent application See Publication No. 2006 / 0134709). For example, the antibody of this disclosure is FcγR IIA, FcγRIIB, and / or FcγRIIIA correspond to the wild-type constant region. It can have a constant region that binds with higher affinity.

[0040] Therefore, the antibodies of this disclosure result in opsonization, phagocytosis, or reduction of ADCC. It may undergo changes in physical activity. Such changes are known in the art. For example, A Modifications in antibodies that reduce DCC activity are described in U.S. Patent No. 5,834,597. It is listed.

[0041] In yet another embodiment, the antibody or a fragment thereof is involved in, for example, FcRn interaction. By modifying the immunoglobulin constant region segment in a specific area, Antibodies that increase or decrease the binding affinity of fetal Fc receptors, FcRn. This could be a fragment of it (for example, the pamphlet International Publication No. 2005 / 123780). (See reference). Such mutations can increase antibody binding to FcRn, thus dividing the antibody. It protects against solutions and extends their half-life.

[0042] In yet another embodiment, antibodies are, for example, Jung and Pluckthun, 1997, Protein Engineering. neering 10(9):959-966, Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9, And as described in U.S. Patent Application Publication No. 2007 / 0280931, its super It has one or more amino acids inserted into one or more of the variable regions.

[0043] The antibody target depends on the desired therapeutic application of the ADC. Typically, the target is ALK5 inhibitors. Antibodies are molecules present on the surface of cells that are desirable for delivering harmful agents, such as T cells. Preferably, the antibody is internalized upon binding to the target. Examples of internalized antibodies are found 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 there.

[0044] Applications in which ADCs are intended to stimulate the immune system by reducing TGF-β activity. Regarding this, it is desirable to produce antibodies that bind to T cell surface molecules. This is constrained by theory. Without any intervention, the delivery of ALK5 inhibitors to T cells, particularly CD4 + and / or CD 8 + It can activate T cell activity and inhibit regulatory T cell activity, and both can lead to tumors. It is thought to contribute to immune tolerance of the tumor. Therefore, T cell surface molecules in ADCs of this disclosure The use of antibodies that bind to [specific cells] is useful, for example, in the treatment of various cancers as described in Section 5.7 below. It is used. In various embodiments, the antibody is CD4 + T cells, CD8 + T cells, T RE G It binds to cells, or any combination thereof. In some embodiments, the antibody It binds to pan-T cell surface molecules. Examples of T cell surface molecules suitable for targeting include limited... CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD2 5, CD28, CD70, CD71, CD103, CD184, Tim3, LAG3, C Examples include TLA4 and PD1. They are thought to bind to T cell surface molecules and be internalized. Examples of antibodies include OKT6 (anti-CD1, ATCC accession number 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 number 55662) 0), UCHT1 (anti-CD3, BioXCell catalog number BE0231), M290 (Anti-CD103, BioXCell catalog number BE0026), FR70 (Anti-CD70 BioXCell catalog number BE0022), pembrolizumab (anti-PD1, Mer ck), nivolumab (anti-PD1, Bristol-Myers Squibb), semip Limab (anti-PD1, Regeneron), and dostallimab (anti-PD1, Glax (SmithKline) is one example.

[0045] In some embodiments, targeted T cell surface molecules are transmitted through endosomes. It is a T cell surface molecule that can be recirculated to the cell surface after internalization (Goldenring, (See 2015 Curr. Opin. Cell Biol., 35:117-22). Recirculation via endosomes Examples of T cell surface molecules that are thought to be capable of this include CD5, CD7, and CD5. Examples include 71 and CD2. Without being constrained by theory, recirculation through endosomes. Targeting T cell surface molecules that can be cyclically linked means that ALK5 can also target endosomes. Because it can be recirculated through this process, it promotes the delivery of ALK5 inhibitors to ALK5. It is thought that this is possible. Therefore, T cells can be recycled through endosomes. Targeting cell surface molecules may help bring ALK5 inhibitors closer to ALK5. .

[0046] 5.3. ALK5 inhibitors The ALK5 inhibitors of this disclosure preferably competitively and reversibly inhibit the cytoplasm of the ALK5 receptor. The kinase domain binds to the ATP binding site and prevents downstream R-Smad phosphorylation, He is a child.

[0047] ALK5 inhibitors are used to counteract ALK5 against other TGF-β family receptors, such as ALK4. Specifically or for ALK7 and / or TGF-β receptor II It may be selective, but not necessarily. In some embodiments, ALK5 The inhibitor is active against both ALK5 and TGF-β receptor II. While it is preferable that the inhibitor has limited inhibitory activity against the BMP II receptor, Therefore, the ADCs of this disclosure target T cells with minimal or no BMP II activity. It is not necessary because it is done.

[0048] From at least 3 subjects, at least 5 subjects, or at least 10 subjects When measured in an in vitro cell assay using T cells, the ALK5 inhibitory function of this disclosure is measured in an in vitro cell assay. IC for harmful agents 50 Preferably, the concentration is 100 nM or less, more preferably 50 nM or less, most preferably Or it is 20 nM or less. An exemplary cell assay is described in Section 6.6 below. A If a DC targets humans instead of mouse T cell surface molecules, then instead of mouse cells, This uses st cells and antibodies that recognize humans instead of mouse CD28 and CD3. It is possible.

[0049] Specific examples of ALK5 inhibitors suitable for use in the antibody-drug conjugates of this disclosure include: Imidazole-benzodioxole compounds, imidazole-quinoxaline compounds, pyraz Examples include 3-pyrrolo compounds and thiazole compounds.

[0050] According to one aspect of this disclosure, imidazole-benzodioxole ALK5 inhibitors are , has the following formula.

[0051] [ka]

[0052] In the formula, R 1 R is a lower alkyl group having hydrogen or 1 to about 5 carbon atoms. 2 R is a lower alkyl group having hydrogen or 1 to about 5 carbon atoms. 3 is amide, Nitriles, alkynyls with 1 to about 3 carbon atoms, carboxyls, or 1 to about 5 carbon atoms It is an alkanol having carbon atoms, where A is directly bonded, or has 1 to about 5 carbon atoms. The alkyl group has a direct bond, or an alkyl group having 1 to about 5 carbon atoms. In another preferred embodiment of this disclosure, R 2 is hydrogen or methyl A has one carbon atom, B is directly bonded to a benzyl group, and R 3 is, amide In a preferred combined embodiment of the present disclosure, R 2 is hydrogen or methyl Yes, A has one carbon atom, and B is directly bonded to a benzyl group.

[0053] In accordance with another aspect of this disclosure, imidazole-quinoxaline ALK5 inhibitors are below It has the following formula.

[0054] [ka]

[0055] In the formula, R 1 R is a lower alkyl group having hydrogen or 1 to about 5 carbon atoms. 2 R is a lower alkyl group having hydrogen, halogen, or 1 to about 5 carbon atoms. 3 teeth amides, nitriles, alkynyls having 1 to about 3 carbon atoms, carboxyls, or Alkanols having 1 to approximately 5 carbon atoms, where A is directly bonded, or 1 to approximately 5 It is an alkyl group having carbon atoms, and B is directly bonded or has 1 to about 5 carbon atoms. It is an alkyl group. In another preferred embodiment of the present disclosure, R 2 is hydrogen or me It is chill, the halogen contains fluorine or chlorine, A has one carbon atom, and B is , direct bonding to the benzyl group, R 3 is an amide. The preferred combination of the present disclosure In one embodiment, R 2 is hydrogen or methyl, and A has one carbon atom. B is a direct bond to the benzyl group.

[0056] In accordance with another aspect of this disclosure, a pyrazole-based ALK5 inhibitor has the following formula:

[0057] [ka]

[0058] In the formula, R 2 These are hydrogen, halogens, or lower alkyl groups having 1 to about 5 carbon atoms. Yes, R 4 It consists of hydrogen, halogens, lower alkyl groups having 1 to about 5 carbon atoms, and 1 to about 5 Alkoxy, haloalkyl, carboxyl, carboxyalkylated atoms having carbon atoms It is a ster, nitrile, alkylamine, or a group having the following formula.

[0059] [ka]

[0060] In the formula, R 5 These are lower alkyl groups, halogens, or morphogenetic compounds having 1 to approximately 5 carbon atoms. It is Rino, 6 These include pyrrole, cyclohexyl, morpholino, pyrazole, pyran, and i Midazole, oxane, pyrrolidinyl, or alkylamine, where A is directly bonded. Alternatively, it is an alkyl group having 1 to approximately 5 carbon atoms.

[0061] In accordance with another aspect of this disclosure, a pyrazole-pyrrolo ALK5 inhibitor is defined by the following formula: To possess.

[0062] [ka]

[0063] In the formula, R 7 These are hydrogen, halogens, lower alkyl groups having 1 to about 5 carbon atoms, and alkaline compounds. It is an ol, morpholino, or alkylamine, and R 2 is hydrogen, halogen, or 1 It is a lower alkyl having approximately 5 carbon atoms, R 8 is hydrogen, hydroxyl, amino It is a halogen or a group having the following formula.

[0064] [ka]

[0065] In the formula, R 5 It is piperazinyl, and R 6 These are morpholino, piperidinil, piperadini alkoxy, hydroxyl, oxane, halogen, thioalkyl, or alkyl It is a mine, and A is a lower alkyl having 1 to about 5 carbon atoms.

[0066] In accordance with another aspect of this disclosure, a thiazole ALK5 inhibitor has the following formula:

[0067] [ka]

[0068] In the formula, R 9 These are hydrogen, halogens, or lower alkyl groups having 1 to about 5 carbon atoms. Yes, R 10 These are hydrogen atoms or lower alkyl groups having 1 to about 5 carbon atoms.

[0069] In a particular embodiment, the ALK5 inhibitor is a compound designated A to N in Table 1 below. Select from one of the following combinations.

[0070] [Table 1-1]

[0071] [Table 1-2]

[0072] [Table 1-3]

[0073] In a more specific embodiment, the ALK5 inhibitor is selected from any of the compounds designated 1 to 283 in Table 2 below.

[0074]

Table 2-1

[0075]

Table 2-2

[0076]

Table 2-3

[0077]

Table 2-4

[0078]

Table 2-5

[0079]

Table 2-6

[0080]

Table 2-7

[0081]

Table 2-8

[0082]

Table 2-9

[0083]

Table 2-10

[0084]

Table 2-11

[0085]

Table 2-12

[0086]

Table 2-13

[0087]

Table 2-14

[0088]

Table 2-15

[0089]

Table 2-16

[0090]

Table 2-17

[0091]

Table 2-18

[0092] The preparation and use of ALK5 inhibitors are well-known in the scientific and patent literature, This has been well demonstrated. PCT Publication International Publication No. 2000 / 61576, And in U.S. Patent Application Publication No. 2003 / 0149277, the triarylimida Its use as a zole derivative and as an ALK5 inhibitor is disclosed. PCT published International Publication No. 2001 / 62756 describes pyridinylimidazole derivatives, Its use as an ALK5 inhibitor has been disclosed. PCT Publication No. 200 The 2 / 055077 pamphlet lists imidazolyl cyclic acetate as an ALK5 inhibitor. The use of derivatives is disclosed. PCT Publication No. 2003 / 087304 Frets's approach involves tri-substituted heteroaryls, as well as ALK5 and / or ALK4 inhibitors. Its use as a pharmaceutical agent is disclosed. International Publication No. 2005 / 103028 pamphlet. U.S. Patent Application Publication No. 2008 / 0319012, and U.S. Patent Application Publication No. 7,40 Specification No. 7,958 describes 2-pyridyl as an ALK5 and / or ALK4 inhibitor. Substitutive imidazoles are disclosed. One representative compound, IN-1130, has several... In that animal model, it exhibits ALK5 and / or ALK4 inhibitory activity. Xu and the patent publication provide additional examples of ALK5 inhibitors, exemplary synthetic schemes, and A method for using ALK5 inhibitors is provided: U.S. Patent No. 6,465,493, U.S. Japanese Patent No. 6,906,089, U.S. Patent No. 7,365,066, U.S. Patent U.S. Patent No. 7,087,626, U.S. Patent No. 7,368,445, U.S. U.S. Patent No. 7,265,225, U.S. Patent No. 7,405,299, U.S. Patent No. 7, U.S. Patent No. 407,958, U.S. Patent No. 7,511,056, U.S. Patent No. 7,61 U.S. Patent No. 2,094, U.S. Patent No. 7,691,865, U.S. Patent No. 7,863, U.S. Patent No. 288, U.S. Patent No. 8,410,146, U.S. Patent No. 8,410,14 U.S. Patent No. 6, U.S. Patent No. 8,420,685, U.S. Patent No. 8,513,222 Specification, U.S. Patent No. 8,614,226, U.S. Patent No. 8,791,113 The document, U.S. Patent No. 8,815,893, U.S. Patent No. 8,846,931, U.S. Patent No. 8,912,216, U.S. Patent No. 8,987,301, United States Japanese Patent No. 9,051,307, U.S. Patent No. 9,051,318, U.S. Patent Specification No. 9,073,918, and PCT Publication No. 2004 / 06539 Pamphlet No. 2, International Publication No. 2009 / 050183, International Publication No. 20 Pamphlet No. 09 / 133070, International Publication No. 2011 / 146287 , and International Publication No. 2013 / 009140 brochure. The aforementioned patent and patent publication. The entire thing is incorporated by reference.

[0093] 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) Examples include aA, Darmstadt, Germany.

[0094] The structures and names of ALK5 inhibitors described herein are those of antibodies and / or linkers. This refers to the molecule before it is bonded to the target.

[0095] Preferred ALK5 inhibitors have a free NH or NH2 group, preferably alkyl or hetero NH or NH2 groups bonded to a reel or aryl group, or alkyl or hetero Bonded to the linker via the aryl group, or the NH or NH2 portion of the aryl group. This is possible (for example, compounds 1-23, 26-29, 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). Derivatization of ALK5 inhibitors to free NH or NH2 groups It can be added. The design of the derivatized ALK5 inhibitor is preferably such that the activity is transmissible. It can also be determined experimentally, but adding an NH or NH2 group will invalidate the inhibitory activity. To reduce the likelihood of this happening, the structure-activity relationship (SAR) of the inhibitor should be taken into consideration. Exemplary derivatized counterparts of some of the compounds shown in Table 1 are shown in Table 3 below. It will be done.

[0096] [Table 3]

[0097] 5.4. Linker Typically, an ADC contains a linker between the ALK5 inhibitor and the antibody. The linker is, A portion containing covalent bonds, or a chain of atoms that covalently bond the antibody to the drug portion. Various implementations Morphologically, the linker is alkyldiyl, aryldiyl, heteroaryldiyl A divalent group like -(CR2) n O(CR2) n - Alkyloxy (e.g., polyethylene (Lenoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethylene) Amino, a repeating unit-like part of Jeffamine (trademark), and succinate Diglycerides containing succinamide, diglycolate, malonate, and caproamide Examples include tellums and amides. For example, linkers containing various PEGs are considered in this technology. Known in the field, (for example, BroadPharm (broadpharm.com)) It is commercially available from [location]. An example of a PEG-containing linker is Mal-PEG2- Val-Cit-PAB-OH (BroadPharm catalog number BP-23203) Mal-PEG4-Val-Cit-PAB-OH (BroadPharm catalog number) No. BP-23204), Mal-PEG4-Val-Cit-PAB-PNP (Broa dPharm catalog number BP-23668), Mal-amido-PEG2-Val -Cit-PAB-PNP (BroadPharm catalog number BP-23675), A ZID-PEG3-Val-Cit-PAB-OH (BroadPharm catalog number B) P-23206), Azido-PEG4-Val-Cit-PAB-OH(BroadPh ARM catalog number BP-23207), Azide-PEG3-Val-Cit-PAB- PNP (BroadPharm catalog number BP-23368), Fmoc-PEG4- Ala-Ala-Asn-PAB (BP-23328), Azide-PEG5-Ala-A la-Asn-PAB (BroadPharm catalog number BP-23329), Fmo c-PEG3-Ala-Ala-Asn(Trt)-PAB (BroadPharm catalog) Log number BP-23285), Azide-PEG4-Ala-Ala-Asn(Trt)- PAB (BroadPharm catalog number BP-23284), and Fmoc-PE G3-Ala-Ala-Asn(Trt)-PAB-PNP(BroadPharm Catalog) Log number BP-23297 is one example.

[0098] A linker is one or more linkers such as stretcher and spacer parts It may contain components. For example, peptidyl linker may contain two or more amino acids. Furthermore, the peptidyl component in one or more stretcher and / or spacer portions. It may contain various linker components, some of which are known in the art. The details are described below.

[0099] Linkers can be "cleavable linkers" that facilitate the release of drugs within cells. Acid-unstable linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptiates) (Ze-sensitive) linker, photosensitive linker, dimethyl linker, or disulfide-containing phosphorus Carr (Chari et al., 1992, Cancer Research 52:127-131, U.S. 5,208,02) You may use Specification No. 0.

[0100] Examples of linkers and linker components known in the art include alleimidocaproil. (mc); maleimidocaproyl-p-aminobenzylcarbamate; maleimidocapro Il-peptide-aminobenzylcarbamate linkers, e.g., maleimidocaproyl -L-phenylalanine-L-lysine-p-aminobenzylcarbamate, and Murray Midcaproyl-L-valine-L-citrulline-p-aminobenzylcarbamate (vc );N-succinimidyl 3-(2-pyridyldithio)propionate (N-succinimidyl 3-(2-pyridyldithio)propionate 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 (SPDP); N-succinimidyl 4-(2-pyridyldithio)butyrate (SPD B); 2-Iminothiolane; S-Acetyl succinic anhydride; Disulfide benzylcarbame t;carbonate;hydrazone linker;N-(α-maleimideacetoxy)sucrine Imide ester; N-[4-(p-azidosalicylamide)butyl]-3'-(2'-p Lysyl dithio)propionamide (AMAS); N[β-maleimidopropyl oxy] Cucineimide ester (BMPS); [N-ε-maleimidocaproyloxy]sucine Imide ester (EMCS); N-[γ-maleimidobutyryloxy]succinimide Stel(GMBS); succinimidyl-4-[N-maleimidomethyl]cyclohexane -1-carboxy-[6-amidecaproate](LC-SMCC); succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoate (LC-SPD P); m-maleimidobenzoyl-N-hydroxysuccinimide (MBS); N-succinimidyl[4-iodoacetyl]aminobenzoate (SIAB); succin Imidyl 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); sulfosuccinate Midyl 6-(3'-[2-pyridyldithio]-propionamide)hexanoate (sul H-LC-SPDP); m-maleimidobenzoyl-N-hydroxysulfosuccinimi Doester (sulfo-MBS); N-sulfosuccinimidyl[4-iodoacetyl] Minobenzoate (sulfo-SIAB); sulfosuccinimidyl 4-[N-maleimide] Methyl cyclohexane-1-carboxylate (sulfo-SMCC); sulfosuccinate Imidyl 4-[p-maleimidophenyl]butyrate(sulfo-SMPB); ethylene Recall-bis(N-hydroxysuccinimide succinate)(EGS); Disk Synimidyl tartrate (DST); 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA); diethylenetriamine-pentaacetic acid (DTPA); Examples include thiourea linkers and oxime-containing linkers.

[0101] In some embodiments, the linker is cleavable under intracellular or extracellular conditions. Therefore, cleavage of the linker releases the ALK5 inhibitor from the antibody in a suitable environment. In further embodiments, the linker is not severable, and the drug is, for example, Released by the degradation of antibodies in sosomes (U.S. Patent Application Publication No. 2005 / 023864) See Specification No. 9, which is incorporated herein by reference in its entirety for all purposes. ).

[0102] An example of a non-disconnectable linker that can be used in the ADC of this disclosure is the N-Malaymi. Methylcyclohexane 1-carboxylate, maleimidocaproyl, or mercap Toacetamide caproyl linker is one example.

[0103] In some embodiments, the linker is located in the intracellular environment (e.g., lysosome or It can be cleaved by cleavage agents present in endosomes or caveolae. The linker is For example, including, but not limited to, lysosomes or endosomal proteases, intracellular It may be a peptidyl linker that is cleaved by a peptidase or protease enzyme. In some embodiments, the peptidyl linker has a length of at least 2 amino acids, or It contains a peptidyl component that is at least 3 amino acids long or longer.

[0104] The cleavage agent may include, but is not limited to, cathepsin B and D, and plasmin. All of these processes involve hydrolyzing dipeptide drug derivatives that release active drugs within target cells. It is known that (for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:6) (See 7-123). For example, peptidyl linkers are thiol-dependent protease catepsis. By linker B (for example, Phe-Leu or Gly-Phe-Leu-Gly linker) It is possible to cut it. Another example of such a linker is, for example, U.S. Patent No. 6,214,3 It is described in Specification No. 45 and is incorporated herein by reference in its entirety for all purposes. To be absorbed.

[0105] In some embodiments, peptidyl can be cleaved by intracellular proteases. The linker is either the Val-Cit linker or the Phe-Lys linker (for example, va The synthesis of doxorubicin using an L-CIT linker is described in U.S. Patent No. 6,214,34 (See Item No. 5).

[0106] In other embodiments, the cleavable linker is pH-sensitive, i.e., at a certain pH It is susceptible to hydrolysis at certain values. Typically, pH-sensitive linkers are susceptible to hydrolysis under acidic conditions. It is degradable. For example, acid-unstable linkers that are hydrolyzable in lysosomes. For example, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitate ammonium (For example, o-esters, acetals, ketals, etc.) can be used. U.S. Patent No. 5,122,368, U.S. Patent No. 5,824,805, United States Japanese Patent No. 5,622,929, Dubowchik and Walker, 1999, Pharm. Therapeutic See s 83:67-123, Neville et al., 1989, Biol. Chem. 264:14653-14661. Linkers are relatively stable under neutral pH conditions such as those in blood, but under lysosome pH conditions It is unstable at a pH of 5.5 or less than 5.0, close to 5.0. In certain embodiments, water is added. Degradable linkers are thioether linkers (e.g., therapeutic via acylhydrazone binding). It is a thioether that binds to the agent (for example, U.S. Patent No. 5,622,929). reference).

[0107] In yet another embodiment, the linker is cleavable under reducing conditions (for example, Sulfide linkers). Various disulfide linkers are known in the field of technology. 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- It can be formed using dithio)-toluene), SPDB, and SMPT. This includes things like (e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931, Wawrzynczak). et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (See CW Vogel ed., Oxford University Press, 1987. Also see US 4,880 (See also Specification No. 935)

[0108] In other embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anti cancer Res. 15:1387-93), maleimide benzoyl linker (Lau et al., 1995, Bioorg) -Med-Chem. 3(10):1299-1304), or 3'-N-amide analog (Lau et al., 1995, Bi (org-Med-Chem. 3(10):1305-12).

[0109] In some embodiments, the linker links multiple drug molecules to a single antibody molecule. It is a multivalent linker that can be used for that purpose. For example, developed by Mersana The Fleximer linker technology solubilizes drug molecules through the arrangement of ester bonds. The method is based on incorporating it into a polyacetal backbone. While maintaining scientific characteristics, it incorporates a high-performance ADC (for example, up to 20 drug-antibody ratios (DAR)). It enables (having). An example of a multivalent linker is, for example, International Publication No. 2009 / 073. Pamphlet No. 445, International Publication No. 2010 / 068795, International Publication No. Pamphlet No. 2010 / 138719, International Publication No. 2011 / 120053 International Publication No. 2011 / 171020, International Publication No. 2013 / 096 Pamphlet No. 901, International Publication No. 2014 / 008375, International Publication No. Pamphlet No. 2014 / 093379, International Publication No. 2014 / 093394 This is described in the pamphlet, International Publication No. 2014 / 093640, and The contents are incorporated herein by reference in their entirety.

[0110] In many cases, linkers are substantially insensitive to the extracellular environment. In this case, "substantially insensitive to the extracellular environment" in the context of linkers means ADC In the sample, approximately 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, Approximately 1% or less of the linker is cleaved when the ADC is present in an extracellular environment (e.g., plasma). It means to be.

[0111] Whether linkers are substantially insensitive to the extracellular environment can be determined, for example, by using ADCs in plasma. Incubate for a specified time (e.g., 2, 4, 8, 16, or 24 hours), then incubate with plasma. This can be determined by quantifying the amount of free drug present.

[0112] In other non-exclusive embodiments, the linker facilitates intracellular relocation. This is possible. In a particular embodiment, the linker is conjugated with the therapeutic agent. In combination (i.e., in the environment of the linker-therapeutic portion of the ADC described herein), It promotes intracellular integration. In another embodiment, the linker is an ALK5 inhibitor and It promotes intracellular relocation when conjugated with both antibodies.

[0113] In many embodiments, the linker is of the self-sacrificing type. The term "self-sacrifice" refers to the covalent bonding of two spaced-apart chemical parts to form a stable triple molecule. It refers to a bifunctional chemical moiety that can be formed when its bond to the first part is broken. In such cases, it spontaneously separates from the second chemical part. For example, PCT Publication No. 200 Pamphlet No. 7 / 059404, International Publication No. 2006 / 110476, International Publication No. 2005 / 112919, International Publication No. 2010 / 062171 Pamphlet No. 2009 / 017394, International Publication No. 200 Pamphlet No. 7 / 089149, International Publication No. 2007 / 018431, International Publication No. 2004 / 043493 and International Publication No. 2002 / 083 Refer to pamphlet No. 180, which states that drugs and cleavageable substances can be used at will as self-sacrificial liabilities. This concerns drug-cleavable substance conjugates linked through a linker, and all of them are clearly defined. It is incorporated by reference into white. It can be used to generate a self-sacrificing linker. An example of a self-sacrificing spacer unit is shown in the following formula.

[0114] Various exemplary linkers that can be used in this composition and method are PCT published. International Publication No. 2004 / 010957 brochure, U.S. Patent Application Publication No. 2006 / 00 Specification No. 74008, U.S. Patent Application Publication No. 2005 / 0238649, and U.S. As described in National Patent Application Publication No. 2006 / 0024317 (each of them is all (For the purposes of this document, the entire text is incorporated herein by reference.)

[0115] The ADC of this disclosure may be of the following formula I, where antibody (Ab) is any linker (L It is conjugated with the drug portion (D) of one or more ALK5 inhibitors via ). Ab-(LD) p I

[0116] Therefore, antibodies can interact with drugs and condyloma, either directly or via a linker. It can be galvanized. In formula I, p is the amount of drug per antibody (i.e., ALK This is the average number of inhibitory parts, which is, for example, about 1 to 20 drug parts per antibody. This can range from minutes, and in certain embodiments, there may be 2 to about 8 drug moieties per antibody. This is possible. Further details regarding drug loading are described in Section 5.5 below.

[0117] In some embodiments, the linker component is, for example, via a cysteine ​​residue, another It may include a “stretcher” that links an antibody to the linker component or drug portion. (Example) The stretcher is shown below (in the formula, the wavy line on the left indicates the site of covalent binding to the antibody). The wavy line on the right indicates the site of covalent bonding to another linker component or drug moiety.

[0118] [ka] U.S. Patent No. 9,109,035, Ducry et al., 2010, Bioconjugate Chem. 21: See 5-13.

[0119] In some embodiments, the linker component may include amino acid units. In one embodiment, the amino acid unit allows the linker to be cleaved by a protease, Therefore, when exposed to intracellular proteases such as lysosomal enzymes, drugs from ADCs The release of substances is promoted. For example, Doronina et al., 2003, Nat. Biotechnol. 21:778-784 See also. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, and tetrapeptides. Examples include trapeptides and pentapeptides. A representative dipeptide is valine. -Citrulline (VC or val-cit), alanine-phenylalanine (AF) (also known as ala-phe), phenylalanine-lysine (FK or phe-lys), and One example is N-methyl-valine-citrulline (Me-val-cit). Examples of glycine include glycine-valine-citrulline (gly-val-cit) and glycine. One example is syn-glycine-glycine (gly-gly-gly). The amino acid unit is, It contains naturally occurring amino acid residues, as well as a small number of amino acids and unnatural amino acid analogs. For example, the citrulline amino acid unit is found in certain enzymes, such as cathepsin B, C, and Designed and optimized for selection of enzyme cleavage by D or plasmin protease. It can be transformed.

[0120] In some embodiments, the linker component is directly or stretcher and A spacer that links the antibody to the drug portion by either a pi or an amino acid unit. It may have units. Spacer units can be "self-sacrificing" or "non-self-sacrificing." "Non-self-sacrificing" spacer units are those in which some or all spacer units are enzymes of ADC (e.g.) For example, a non-self-sacrificing spacer that remains bound to the drug portion during protease cleavage. Examples of units include, but are not limited to, glycine spacer units and glycine-glycine units. Pacer units are an example. "Self-sacrificing" spacer units perform a separate hydrolysis step. The drug portion can be released without performing this action. In a particular embodiment, the linker's spec The ser unit contains a p-aminobenzyl unit. In such one embodiment, p-A Minobenzyl alcohol binds to amino acid units via amide bonds, forming carbamate salts. Methylcarbamate, or carbonate, is used in the interaction between benzyl alcohol and cytotoxic agents. It is manufactured. For example, Hamann et al., 2005, Expert Opin. Ther. Patents 15:1087-1103 See reference. In one embodiment, the spacer unit is p-aminobenzyloxycarbonyl ( In a particular embodiment, the phenylene portion of the p-aminobenzyl unit The minutes are Q m Substituted with, where Q is -C1~C8 alkyl, -O-(C1~C8 alkyl m is an integer in the range of 0 to 4, and is a halogen, nitro, or cyano(L) element. Examples of self-sacrificing spacer units include, but are not limited to, p-aminobenzyl alcohol. Aromatic compounds that are electronically similar to (for example, U.S. Patent Application Publication No. 2005 / 025603) See Specification No. 0), for example, 2-aminoimidazole-5-methanol derivatives (Hay et al. (1., 1999, Bioorg. Med. Chem. Lett. 9:2237), and ortho- or para-aminobutyric acid Further examples include 4-aminobutyric acid acetals. Substituted and unsubstituted 4-aminobutyric acid amides (Rodrig Ues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1 ] and bicyclo[2.2.2] ring systems (Storm et al., 1972, Amer. Chem. Soc. 94:5815 ), and 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Use a spacer that forms a ring during the hydrolysis of the amide bond, such as in Chem. 55:5867. This can be achieved. Elimination of amine-containing drugs substituted at the α position of glycine (Kingsbury et al. (l., 1984, J. Med. Chem. 27:1447) also provides an example of a self-sacrificing spacer useful for ADCs. be.

[0121] In one embodiment, the spacer unit is a branched screw (hydroxymethyl (L) Styrene (BHMS) units are used to incorporate and release multiple drugs. It is possible.

[0122] [ka] (In the formula, Ab and D are defined above for formula I, and A is on the stretcher) Yes, a is an integer in the range of 0 to 1, W is an amino acid unit, and w is 0 to 1 Q is an integer in the range of 2, and Q is -C1~C8 alkyl, -O-(C1~C8 alkyl), - is a halogen, - nitro, or - cyano, where m is an integer in the range of 0 to 4, and n is (where p is 0 or 1, and p is in the range of 1 to approximately 20)

[0123] The linker may include any one or more of the linker components described above. In a particular implementation In its form, the linker is shown in parentheses in the following ADC formula: Ab-(-[Aa-Ww-Yy]-D) p II (In the formula, Ab, A, a, W, w, D, and p are defined in the previous paragraph, and Y is S Pacer units (where y is 0, 1, or 2). An example of such a linker. The embodiment is described in U.S. Patent Application Publication No. 2005 / 0238649, which is , incorporated herein by reference.

[0124] Exemplary linker components and their combinations are shown below in the context of the ADC of Equation II:

[0125] [ka]

[0126] Linker components, including stretchers, spacers, and amino acid units, are part of the technology. Methods known in the field, for example, as described in U.S. Patent Application Publication No. 2005 / 0238649. It can be synthesized by combining the elements.

[0127] 5.5. Drug Carrying Drug loading is represented by p, which is the average number of ALK5 inhibitor moieties per antibody in the molecule. The average number is often a fraction or decimal, but the drug load ("p") is 1 per antibody. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20 or more parts (D). Generally, ALK5 inhibitors On average, there are 2 to 8 drug molecules per antibody, more preferably 2 to 4 per antibody. This results in one drug portion, or 5 to 7 drug portions per antibody.

[0128] As will be understood by those skilled in the art, in many cases, references to ADCs are (sometimes pharmaceutical associations) (In the context of products) This is an abbreviated expression for a group or collection of ADC molecules, where each molecule is one Alternatively, it is composed of antibodies covalently bound to multiple ALK5 inhibitor moieties, and the ratio at each molecular base is While it may vary for each ADC molecule within a population, the drug loading ratio is the average drug load for the population or set. This indicates the presence of a group or assembly of 1 to 30 drug portions. In some embodiments, there are 1 to 20, 1 to 15, 2 to 12, Antibody covalently binds to one of two drug moieties: 2-8, 4-15, or 6-12. It contains ADC molecules, including the body. Preferably, the mean in the population is as described in the previous paragraph. For example, 2 to 8 drug molecules per antibody, more preferably 4 to 8 molecules per antibody. This consists of the drug portion, or 5 to 7 drug portions per antibody.

[0129] Some ADC populations are ADCs described herein, and antibodies lacking the drug portion. This may take the form of a composition containing a somatic molecule, for example, an antibody that failed to bind to an ALK5 antibody.

[0130] In the preparation of ADCs from conjugate reactions, the ALK5 inhibitor portion per antibody The average number is characterized by conventional methods such as mass spectrometry and ELISA assays. It can be done.

[0131] The quantitative distribution of the ADC can also be determined with respect to p. In some examples, the average The isolation, purification, and characterization of one ADC is performed when p is accompanied by other ALK5 inhibitors in the ADC. This is a specific value obtained from a certain point, but it can be achieved by means such as electrophoresis.

[0132] For some antibody-drug conjugates, p is limited by the number of binding sites on the antibody. It can be determined. For example, if the bond is a cysteinethiol, then the above exemplary embodiment In this context, the antibody may have only one or more cysteinethiol groups, or , one or more thiol groups with sufficient reactivity to bond a linker It may have a higher drug load, for example p>5. Aggregation, insolubility, toxicity, or decreased cell permeability of certain antibody-drug conjugates may occur. This can happen. In certain embodiments, drug loading onto the ADC of this disclosure is 1 to about 8 Approximately 2-6, approximately 3-5, approximately 3-4, approximately 3.1-3.9, approximately 3.2-3.8, approximately 3.2 to approximately 3.7, approximately 3.2 to approximately 3.6, approximately 3.3 to approximately 3.8, or approximately 3.3 to approximately 3. It is in the range of 7. In fact, for a particular ADC, the optimal ratio of drug portion per antibody is It has been shown that it can be less than 8 and can be approximately 2 to approximately 5. U.S. Patent Application Publication No. 20 See Specification No. 05 / 0238649 (which is incorporated herein by reference in its entirety). .

[0133] In certain embodiments, the drug portion below the theoretical maximum value is conjugated. During the response, it is conjugated into an antibody. The antibody is, for example, a drug-phosphorus compound, as discussed below. It may contain lysine residues that do not react with the Kerr intermediate or linker reagent. Generally, antibodies are It does not contain many free reactive cysteinethiol groups that can be linked to the drug portion. In fact, many cysteine ​​thiol residues within antibodies exist as disulfide crosslinks. In certain embodiments, the antibody is subjected to dithiothrelation under partial or total reduction conditions. Reduction such as itol (DTT) or tricarbonylethylphosphine (TCEP) It can be reduced with an agent to produce a reactive cysteinethiol group. In this state, the antibody is exposed to denaturing conditions that allow reactive nucleophiles such as lysine or cysteine ​​to react. To reveal the basis.

[0134] ADC loading (drug / antibody ratio) can be done using various methods, for example, (i) drug-to-antibody ratio (ii) Limiting the molar excess of the linker intermediate or linker reagent, and conjugate (iii) Limiting the time or temperature of response (iv) Reducing conditions or restricting the reducing conditions, Linker-drug binding (e.g., PCT Publication International Publication No. 2006 / 034488 pamphlet) Thiomab as disclosed in Let (the whole of which is incorporated herein by reference) Recombinant techniques to modify the number and / or position of (or thioFab) This can be controlled by manipulating the amino acid sequence of the antibody through surgery.

[0135] Two or more nucleophilic groups react with a drug-linker intermediate or linker reagent, and then the drug When reacted with a partial reagent, the resulting product is one or more antibodies bound to the antibody. It should be understood that it is a mixture of ADC compounds having the distribution of the drug portion. The average number of drugs per person is calculated from the mixture using a dual ELISA antibody assay. This can be achieved, and it is specific to the antibody and specific to the drug. Individual ADC molecules are mass Identified within the mixture by spectroscopy, and then by HPLC, e.g., hydrophobic interaction chromatography. It can be separated by [this method].

[0136] In some embodiments, a uniform ADC having a single mounting value is used for electrophoresis or It can be isolated from the complex mixture by chromatography.

[0137] 5.6. Formulation and Administration Preferred routes of administration for ADCs include, but are not limited to, oral, parenteral, rectal, transmucosal, and intestinal. Intravenous administration, intramedullary, intrascalable, intravenous, intravitreous, intracavitary, intraperitoneal, or intratumor injection Injection is one possible method of administration. The preferred route of administration is parenteral, and more preferably intravenous. Alternatively, one could be localized rather than systemic, for example, solid or hematological malignancies. The compound can be administered by direct injection of the compound into the body.

[0138] Immunoconjugates are formulated according to known methods for preparing pharmaceutically useful compositions. It can be converted, thereby allowing ADC to combine the mixture with pharmaceutically useful additives. Sterile phosphate-buffered saline is one example of a pharmaceutically useful additive. Other useful The additives are well known to those skilled in the art. For example, Ansel et al., Pharmaceutical Dosage Fo RMS and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), and Gennaro ( ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company See 1990) and its revised edition.

[0139] In a preferred embodiment, the ADC is N-(2-acetamide)-2-aminoethane Sulfonic acid (ACES); N-(2-acetamide)iminodiacetic acid (ADA); N,N- Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 4-(2-H Droxyethyl)piperazine-1-ethanesulfonic acid (HEPES); 2-(N-morpho (Lino) 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] Formulate with Good's biological buffer (pH 6-7) using a buffer selected from the group. A more preferable buffer solution is preferably in the range of 20 to 100 mM, more preferably about MES or MOPS at a concentration of 25 mM. Most preferably at pH 6.5. It is a 5 mM MES. The formulation contains 25 mM trehalose and 0.01 as additives. The final buffer solution further contains %v / v polysorbate 80, and as a result of the added additives, The concentration is adjusted to 22.25 mM. The preferred method of storage is freeze-drying of the conjugate. It is a pharmaceutical product and is most preferably stored at 2°C to 8°C, and most preferably at -20°C to 2°C. It is stored at temperatures within the range of °C.

[0140] ADCs are administered intravenously, for example, by bolus injection, slow infusion, or continuous infusion. It can be formulated for use. Preferably, the ADC should be administered for less than about 4 hours, more preferably about It is administered over a period of less than 3 hours. For example, the first 25-50 mg is preferably administered within 30 minutes. Either inject it in exactly 15 minutes, and inject the remainder over the next 2-3 hours. The formulation is provided in a unit dosage form with added preservatives, for example, in an ampoule or a multi-dose container. The composition may be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. It can take on a state and contains formulation agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be stored in a suitable vehicle, such as a sterile pipe, before use. It may be in powder form, composed of water without rhogen.

[0141] Further pharmaceutical methods can be used to control the duration of action of ADCs. The release preparations are prepared by using polymers that either composite or adsorb ADCs. For example, as a biocompatible polymer, poly(ethylene-co-vinyl acetate) Matrix of stearate dimer and polyanhydride copolymer of sebaciate - A matrix can be cited. Sherwood et al., 1992, Bio / Technology 10:1446. The release rate of ADCs from a matrix like this depends on the molecular weight of the ADC and the amount of A in the matrix. It depends on the amount of DCs and the size of the dispersed particles. Saltzman et al., 1989, Biophys. J. 5 5:163, Sherwood et al., see above. Other solid dosage forms are available from Ansel et al., Pharmaceutical D. Osage Forms and Drug Delivery Systems, 5th Edition (Lea & Febiger 1990), and G ennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing This is described in Company 1990, and its revised editions.

[0142] Generally, the dosage of ADCs administered to humans depends on the patient's age, weight, height, sex, and overall health. The dosage varies depending on factors such as health status and medical history. Approximately 0.3 mg / kg to 5 mg. The ADC can be administered to the recipient in doses within the range of / kg as a single intravenous infusion. While desirable in some cases, lower or higher doses may be administered depending on the situation. In some cases, the dosage of 0.3-5 mg / kg for a 70 kg patient is 21 This is approximately 350 mg, which corresponds to 12-206 mg / m² for a patient with 1.7 m² of saturation. 2 With this dosage There are also other options. The dosage may vary as needed, for example, once a week for 2 to 10 weeks, or once a week for 8 weeks. It can be repeated once a week or once a week for four weeks as needed for maintenance therapy. Depending on the frequency, for example, every week for several months, or for several months It can also be given once a month or once every three months. The preferred dosage is limited The dosage is not fixed, but it is 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1.0 mg / kg, 1.2mg / kg, 1.5mg / kg, 2.0mg / kg, 2.5mg / kg, 3 0.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5. A possible dose is 0 mg / kg. A more preferred dose is 0.6 mg per week. The dosage is / kg, and for lower-frequency administration, it is 1.2 mg / kg. 0.3~5m Any amount within the range of g / kg can be used. The dosage is preferably 1 per week It is administered once or multiple times. For 4 weeks, more preferably 8 weeks, more preferably 16 weeks, A minimum dose schedule can be used for a longer period, and the frequency of administration is The administration schedule depends on adverse side effects, most commonly associated with hematological toxicity, and recovery from them. Joules' regimen consists of (i) once a week; (ii) every other week; and (iii) a week of therapy followed by two weeks. (iv) Two weeks of therapy followed by one, two, or three weeks Interval or 4-week break; (v) 3 weeks of therapy followed by 1, 2, 3, or 4 weeks (vi) 4 weeks of therapy followed by 1, 2, 3, and 4 weeks (vii) 5 weeks of therapy followed by 1, 2, 3, and 4 weeks (viii) a break of 5 weeks or in between; and a cycle selected from the group consisting of once a month This may include administration once or twice a week. Cycles may consist of 2, 4, 6, or 8 cycles. It can be repeated 10, 12, or more times.

[0143] Alternatively, ADCs may 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 a week for 4-6 weeks. The dosage can be administered once every week, or even less frequently, so patients It can recover from any drug-related toxicity. Or, the dosage schedule This can be shortened, meaning every two or three weeks over a period of two to three months. The administration schedule may be repeated at other intervals, and the dose may be appropriately adjusted. And according to the schedule, it is administered via various parenteral routes.

[0144] 5.7. Treatment Methods The ADCs of this disclosure can be used for the treatment of various cancers. For example, the ADCs can be used for the treatment of various cancers. Sub-care medications or regimens, used as monotherapy or as part of a combination therapy regimen. It can be used. In some embodiments, the combination therapy is immunotherapy, for example, Checkpoint inhibitor therapy, chimeric antigen receptor (CAR) therapy, adoptive T cell therapy (for example) (autologous T-cell therapy), oncolytic virus therapy, dendritic cell vaccine therapy, interfero agonist therapy using agonist genes (STING), and Toll-like receptor (TLR) agonists. Streptococcal therapy, intratumor CpG therapy, cytokine therapy (e.g., IL2, IL12, IFN-) ADCs are administered in combination with α- or INF-γ therapy, or a combination thereof. This includes, in some embodiments, combination therapy is immunoconservative chemotherapy (e.g., An antimetabolite such as 5-fluorouracil, gemcitabine, or methotrexate Cyclophosphamide, dacarbazine, mechloretamine, diazicon, or temozolo Alkylating agents like MIDE, anthraxes like doxorubicin or epirubicin Antimicrotubule agents such as Ikurin and vinblastine, cisplatin or oxaliplatin Platinum compounds such as paclitaxel or docetaxel, taxanes, or Topoisomerase inhibitors such as etoposide or mitoxantrone, or vinca This includes administering ADCs in combination with vinca alkaloids such as ristine. Suitable antibodies to include in ADCs for cancer treatment are those that target T cell surface antigens. Examples of antibodies are described in Section 5.2.

[0145] Examples of cancers that can be treated using the ADCs of this disclosure include, but are not limited to, pancreatic cancer. Cancer, glioblastoma, myelodysplastic syndrome, prostate cancer (e.g., castration-resistant prostate cancer) Liver cancer (e.g., hepatocellular carcinoma), melanoma, breast cancer, urothelial carcinoma (e.g., bladder cancer, urethral cancer) (and ureteral cancer), kidney cancer (e.g., renal cell carcinoma and urothelial carcinoma), lung cancer (e.g., Non-small cell lung cancers (NSCLCs) such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, as well as This includes small cell lung cancer, as well as colorectal cancer (e.g., adenocarcinoma, carcinoid tumor, gastrointestinal stromal tumor). Examples include uterine tumors and colorectal lymphoma.

[0146] The ADCs of this disclosure are checkpoint inhibitors, such as PD1, PDL1, CTLA4 Check to target TIGIT, LAG3, OX40, CD40, or VISTA. It can be used in combination with checkpoint inhibitors. Checkpoint inhibitors are antibodies and small molecules. As an example of a checkpoint inhibitor that targets PD1, Pem Examples include brolizumab, nivolumab, semiprimab, and dostallimab. PDL Examples of checkpoint inhibitors that target 1 include atezolizumab, avelumab, Examples include durvalumab, BMS-1001, and BMS-1166. CTLA4 An exemplary checkpoint inhibitor that targets TIGIT is ipilimumab. Examples of targeted checkpoint inhibitors include etigilimab and tyragone. Examples include tiragolumab and AB154, which target LAG3. Examples of checkpoint inhibitors include LAG525, Sym022, and relatrimab (relatl). Examples include imab) and TSR-033. Exemplary checkpoints targeting OX40 As inhibitors, MEDI6469, PF-04518600, and BMS 986 178 is listed. As an exemplary checkpoint inhibitor that targets CD40, Examples include selicrelumab, CP-870, CP-893, and APX005M. An exemplary checkpoint inhibitor that targets VISTA is HMBD-002. be.

[0147] For the treatment of melanoma carrying BRAF mutations, the ADC of this disclosure is venuraphenic. BRAF mutations such as venurafenibm, dabrafenib, and trametinib It can be used in combination with drugs that specifically target the target.

[0148] For the treatment of malignant melanoma, the ADCs of this disclosure include ipilimumab, nivolumab, and pembrow. Combined with checkpoint inhibitors such as lizumab, semiprimab, or avelumab. It can be used in this way.

[0149] For the treatment of non-small cell lung cancer (NSCLC), the ADC of this disclosure includes systolic acid Chin, carboplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, Used in combination with standard care chemotherapy treatments such as etoposide or vinblastine. It can be used. In addition, ADCs can be used with a standard drug such as bevacizumab or Erbitux. It can be used in combination with other therapies. In addition, ADCs include pembrolizumab and ni. Volumab, semiprimab, dostallumab, atezolizumab, avelumab, durvalum It can be used in combination with checkpoint inhibitors such as ipilimumab. Cut.

[0150] For the treatment of bladder cancer, the ADC of this disclosure contains cisplatin, mitomycin C, and carboxymethyl phosphate. Voplatin, docetaxel, paclitaxel, doxorubicin, 5-FU, methotrexate This includes, but is not limited to, vitblastine, ifosfamide, and pemetrexed. It can be used in combination with standard care procedures. In addition, ADCs contain ipilimumab. It can be used in combination with checkpoint inhibitors such as [specific checkpoint inhibitors].

[0151] For the treatment of renal cancer, the ADC of this disclosure is used for standard care procedures, such as angiogenesis and / or drugs that block specific tyrosine kinases, e.g., sorafenib, sunitinib Used in combination with temsirolimus, everolimus, pazopanib, and axitinib. It is possible. In addition, ADCs can be combined with checkpoint inhibitors such as nivolumab. They can be used together.

[0152] For the treatment of breast cancer, the ADCs of this disclosure are anthracyclines (doxorubicin or (epirubicin), and taxanes (paclitaxel or docetaxel), as well as f Standard care chemistry such as ruoruracil, cyclophosphamide, and carboplatin. It can be used in combination with therapeutic agents. In addition, the ADC of this disclosure can be used in combination with targeted therapy. They can be used in combination. As a targeted therapy for HER2 / neu-positive tumors, Trastuzumab and pertuzumab are examples, and they are used for estrogen receptor (ER) positive tumors. Targeted therapies for this include tamoxifen, toremifene, and fulvestrant. In addition, ADCs can be combined with checkpoint inhibitors such as atezolizumab. It can be used in this way.

[0153] For pancreatic cancer, the ADCs of this disclosure are gemcitabine, 5-fluorouracil, and irinotecanthin. Can, oxaliplatin, paclitaxel, capecitabine, cisplatin, or doceta It can be used in combination with standard care chemotherapy agents, such as Kiseru. In addition, ADCs are used in combination with targeted therapies, such as erlotinib, which inhibits EGFR. It is possible.

[0154] For glioblastoma, the ADC of this disclosure contains carboplatin, cyclophosphamide, Standard care medications such as etoposide, lomustine, methotrexate, or procarbazine. It can be used in combination with chemotherapeutic agents.

[0155] For prostate cancer, the ADC of this disclosure is a standard care chemotherapy agent containing docetaxel. It can be used in combination with the steroid prednisone or cabazitax Cell therapy may be used in combination. In addition, ADCs can be used as checkpoint inhibitors like ipilimumab. It can be used in combination with other agents.

[0156] The use of the ADCs of this disclosure in combination with one or more therapies may affect the order in which the therapies are administered. No limitations. For example, the ADCs of this disclosure are used before the subject is treated with one or more therapies. It can be administered during or after the procedure. In some embodiments, The ADCs of this disclosure are used before treatment of the patient with another therapy (e.g., the second therapeutic agent described above). 5 minutes ago, 15 minutes ago, 30 minutes ago, 45 minutes ago, 1 hour ago, 2 hours ago, 4 hours ago, 6 hours ago 12 hours ago, 24 hours ago, 48 hours ago, 72 hours ago, 96 hours ago, 1 week ago, 2 weeks ago (3 weeks prior, 4 weeks prior, 5 weeks prior, 6 weeks prior, 8 weeks prior, or 12 weeks prior), treatment and Simultaneously, or following the procedure (for example, 5 minutes later, 15 minutes later, 30 minutes later, 45 minutes later, 1 hour later) Later, 2 hours later, 4 hours later, 6 hours later, 12 hours later, 24 hours later, 48 hours later, 72 hours later 96 hours later, 1 week later, 2 weeks later, 3 weeks later, 4 weeks later, 5 weeks later, 6 weeks later, 8 weeks later It is administered later (or 12 weeks later). In some embodiments, the ADC of the present disclosure It is incorporated into the same regimen as a second treatment. [Examples]

[0157] The following abbreviations will be seen throughout the examples. Boc-tert-butyloxycarbonyl DCM - Dichloromethane DMA - Dimethylamine DMF - Dimethylformamide DIPEA - N,N-diisopropylethylamine HCl - ethyl acetate EtOH - Ethanol Fmoc - Fluorenylmethyloxycarbonyl HOBt - Hydroxybenzotriazole MeOH - methanol NaHMDS - Sodium hexamethyldisylazide RT - Room temperature, approximately 21℃ TBTU - O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl Tyluronium tetrafluoroborate TEA - Triethylamine THF - Tetrahydrofuran TFA - Trifluoroacetic acid TMS-imidazole-1-(trimethylsilyl)imidazole

[0158] 6.1. [Example 1] 4-(6-methylpyridine-2-yl)-5-(1,5-naphthyridine-2-yl)-1 Synthesis of 3-thiazole-2-amine (compound A) Compound A was prepared according to the general method in Scheme 1 below.

[0159] [ka]

[0160] 6.1.1. 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 the substance (167 mg, 0.60 mmol) was stirred at room temperature. Glycerol (1. 5 ml) followed by 5-amino-2-methylpyridine (A-SM) (500 mg, 4.6 Add 2 mmol) of the reaction mixture and 2.5 ml of water, and heat at 135°C for 18 hours. The reaction was measured by TLC. After the reaction was complete, the reaction mixture was cooled to approximately 21°C, and 4N N was added. The solution was based using aOH and extracted with ethyl acetate (2 × 100 ml). The organic extract was then combined. Then, wash with water (200 ml), dry with Na2SO4, evaporate under reduced pressure, and remove the raw material. Compound A1 was obtained. The crude compound was collected in silica gel using (2% MeOH / CH2Cl2). The compound A1 (200m) was purified by Lamb chromatography and obtained as a light brown crystalline solid. g, 30%) was obtained.

[0161] 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, 1st hour), 2.8 (s, 3rd hour)

[0162] LC-MS(ESI): m / z 145[M+H] +

[0163] 6.1.2. 1-(6-methylpyridine-2-yl)-2-(1,5-naphthyridine- 2-yl)ethane-1-one (A2) A1 (200 mg, 1.38 mmol) and methyl 6-methyl picolinate (209 The solution of mg (1.38 mmol) in anhydrous THF (10 ml) is placed under an N2 atmosphere. It was cooled to -78°C. Potassium bis(trimethylsilyl)amide (0.5M in toluene) 6.9 ml (3.47 mmol) was added dropwise over 5 minutes. The reaction mixture was The mixture was stirred at -78°C for 1 hour, then heated to approximately 21°C and maintained at that temperature for 20 hours. After the reaction was complete... (Measured by TLC), reaction mixture, citrate with saturated ammonium chloride solution (20 ml) The aqueous layer was extracted with ELISA (2 x 20 ml). The combined organic extract was then mixed with water ( Washed with 100 ml, dried with Na2SO4, and evaporated to obtain crude compound A2. The substance was purified by column chromatography (1% MeOH / CH2Cl2), Compound A2 (110 mg, 30.5%) was obtained as an orange-yellow solid.

[0164] 1 ¹H 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)

[0165] LC-MS(ESI): m / z 264[M+H] +

[0166] 6.1.3. 4-(6-methylpyridine-2-yl)-5-(1,5-naphthyridine- 2-yl)-1,3-thiazole-2-amine (compound A) Solution of A2 (110 mg, 0.418 mmol) in 1,4-dioxane (10 ml) This was treated with bromine (0.025 ml, 0.501 mmol). The resulting reaction mixture was The mixture was stirred at approximately 21°C for 1 hour, then concentrated under reduced pressure to obtain crude A3 (120 mg). This was used in the next step without further purification. Crude A3 (120 mg) was used as ethanol. It was dissolved in 15 ml of water. Then, thiourea (3.5 mg, 0.046 mmol) was added. Add the starting material and continue the reaction mixture for 78 minutes (until complete consumption of the starting material is observed by TLC). The mixture was heated at °C for 4 hours. The reaction mixture was cooled to approximately 21°C and then mixed with ammonia solution (25%). 1.5 ml was added while gently stirring. The solvent was evaporated, and the residue was then collected in CH2 It was dissolved in Cl2 (2 × 20 ml) and washed with water (50.0 ml). Then, the separated... The organic layer was washed with 1N HCl (30 ml x 2). The combined aqueous layer was then treated with 35% hydroxide The solution was based with a sodium aqueous solution (20 ml) and extracted with CH2Cl2 (2 × 20 ml). The organic layer was dried over sodium sulfate and evaporated to obtain crude compound A. Crude compound A was then acetone. Compound A (35) was purified as a yellow crystalline solid by recrystallization from tonitrile (2 ml). mg was obtained in 49% yield over two steps.

[0167] 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)

[0168] LC-MS(ESI): m / z 320[M+H] +

[0169] UPLC purity: 97.6%

[0170] 6.2. [Example 2] N-methyl-2-(4-{4-[3-(pyridine-2-yl)-1H-pyrazole-4- Synthesis of ylpyridine-2-ylphenoxy)ethane-1-amine (compound B) Compound B was prepared according to the general method in Scheme 2 below.

[0171] [ka]

[0172] 6.2.1. tert-butyl(2-chloroethyl)(methyl)carbamate (B7) A stirred solution of Boc-anhydrous (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 in 4 ml of THF (7.69 mmol) was added over 1 hour. The resulting mixture was stirred at approximately 21°C for 16 hours. The reaction mixture was then prepared using a saturated NaCl solution. Diluted with (20 ml) and extracted with DCM (3 x 50 ml). The combined organic extract was then mixed with Na The compound was dried with 2SO4 and concentrated under vacuum to obtain the crude compound, which was then mixed with 10% siRNA / H2O4. The liquid was purified using xan by silica gel column chromatography to obtain a pale yellow liquid. Compound B7 (1 g, 5.18 mmol, 71%) was obtained.

[0173] 1 H NMR (400 MHz, CDCl3): δ 3.58-3.52 (m, 4H), 2.93 (s, 3H), 1.46 (s, 9H)

[0174] 6.2.2. tert-butylmethyl(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) In a stirred solution in 13 ml of DMF, add B7 (900 mg, 4.66 mmol) and KI. (18 mg, 0.10 mmol), and Cs2CO3 (2.57 g, 7.88 mmol) The mixture was added under an argon atmosphere. The reaction mixture was heated to 65°C and stirred for 16 hours. The reaction mixture was poured into water (20 ml) and extracted with phenyl (3 x 20 ml). The combined organic layers were concentrated under reduced pressure to obtain a crude product, which was then mixed with 7% siRNA / hexane. It was used and purified by column chromatography to obtain Int-B(58) as a pale yellow solid. 0 mg, 1.53 mmol, and 43% were obtained.

[0175] 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) )

[0176] 6.2.3. 2-(2-bromopyridine-4-yl)-1-(pyridine-2-yl) Tan-1-on (B2) 2-Bromo-4-methylpyridine (B1) (2g, 11.62 mmol) THF (3 In the stirred solution in 0 ml, NaHMDS (2 M in THF, 1) was added at -78°C under argon. A 2.7 ml (25.58 mmol) solution was added dropwise. The yellow solution was incubated at -78°C for 30 minutes. Stirring for 1 minute. Then, ethyl picolinate (1.72 ml, 12.79 mmol) was added to the TH. Add the solution in F (10 ml), warm the reaction mixture to approximately 21°C, and stir for 16 hours. The solvent was evaporated under reduced pressure, the solid residue was ground with diethyl ether, filtered, and then... Washed with chill ether. Then the solid was diluted with saturated NH4Cl solution (30 ml) and water. The organic layer was extracted with SiO(2 × 200 ml). The organic layer was dried with Na2SO4 and concentrated. It shrunk. The crude product was subjected to silica gel column chromatography using 10% sorbate / hexane. The compound B2 (2.06 g, 7.46 mmol) was purified by tography and obtained as a yellow solid. l, 64.3%) was obtained.

[0177] 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)

[0178] LC-MS(ESI): m / z 277[M] +

[0179] 6.2.4. 2-Bromo-4-[3-(pyridine-2-yl)-1H-pyrazole-4 -ylpyridine (B3) A solution of B2 (850 mg, 3.07 mmol) in dry DMF (3.4 ml) is prepared by A Under Gon's conditions, it was treated with glacial acetic acid (0.45 ml, 7.39 mmol) in DMF. DMA( Add 0.6 ml (4.61 mmol) dropwise, and incubate the mixture at approximately 21°C for 2 hours using an Algebra. The mixture was stirred under a humid atmosphere. Hydrazine monohydrate (1.15 ml, 23.09 mmol) was added dropwise. The mixture was then added and heated at 50°C for 3 hours, followed by heating 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 obtain the crude compound. The crude product was obtained by silica gel column chromatography using 30% sorbate / hexane. The compound B3 (560 mg, 1.86 mmol) was purified by fluoroscopy and obtained as a yellow solid. , 60.6% was obtained.

[0180] 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)

[0181] LC-MS(ESI): m / z 301[M] +

[0182] 6.2.5. 2-bromo-4-(3-(pyridine-2-yl)-1-trityl-1H- Pyrazole-4-yl)pyridine (B4) In a stirred solution of B3 (500 mg, 1.66 mmol) in acetone (10 ml), K2 CO3 (1.37g, 9.99mmol) and trityl chloride (464mg, 2.4 9 mmol) was added. The reaction mixture was then heated under reflux and stirred for 24 hours. The mixture is filtered, the filtrate is concentrated, and then CH2Cl2 (20 ml) and water (10 ml) The mixture was distributed between the two parts. The organic phase was dried with Na2SO4 and concentrated. The crude solid was mixed with 2% MeO2. Purified by silica gel column chromatography using H / CH2Cl2, resulting in a pale yellow color. Compound B4 (402 mg, 0.74 mmol, 44%) was obtained as a colored solid.

[0183] 1 1H 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)

[0184] 6.2.6. tert-butylmethyl(2-(4-(4-(3-(pyridin-2-yl )-1-trityl-1H-pyrazole-4-yl)pyridine-2-yl)phenoxy) Chilled carbamate (B5) A stirred solution of B4 (100 mg, 0.18 mmol) in toluene (2 ml) is mixed with EtO Int-B (185 mg, 0.49 mmol) in H (0.75 ml), followed by 2M 0.45 ml of Na2CO3 solution was added under an argon atmosphere. The reaction mixture was then... Degass with argon for 20 minutes, then add Pd(PPh3)4 (16 mg, 0.01 mmol) The mixture was added and refluxed for 3 hours. After complete consumption of the starting material (monitored by TLC), the reaction proceeded. The mixture was poured into water and extracted with toluene (3 x 15 ml). The organic layer was Na2SO4. The product was dried and concentrated under reduced pressure to obtain the crude product, which was then mixed with 30% Â / hexane. The compound was purified by silica gel column chromatography using [the specified method] and obtained as a colorless solid. B5 (70 mg, 0.09 mmol, 53%) was obtained.

[0185] 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).

[0186] 6.2.7. N-methyl-2-(4-(4-(3-(pyridine-2-yl)-1H-py Razole-4-yl)pyridine-2-yl)phenoxy)ethane-1-amine hydrochloride (chemical Compound B) In a stirred solution of B5 (70 mg, 0.09 mmol) in CH2Cl2 (6 ml), add 1, 4N HCl was added to 4-dioxane (0.5 ml) at 0°C. The reaction mixture was A The mixture was stirred for 1 hour under a GON atmosphere. After complete consumption of the starting material (monitored by TLC), The solvent was evaporated under reduced pressure to obtain the crude compound, which was then powdered with n-pentane (2 × 1 ml). The material was crushed and dried to obtain the HCl salt of compound B as a colorless solid (25 mg, 0.06 mm). ol, 69%).

[0187] 1H 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)

[0188] LC-MS(ESI): m / z 372[M+H] +

[0189] 6.3. [Example 3] N-methyl-2-(4-{4-[3-(6-methylpyridine-2-yl)-1H-pyrazo [4-yl]pyridine-2-yl}phenoxy)ethane-1-amine (compound C) synthesis Compound C was prepared according to the general method in Scheme 3 below.

[0190] [ka]

[0191] 6.3.1. 2-(2-bromopyridine-4-yl)-1-(6-methylpyridine-2) -Il)ethane-1-one (C2) 2-Bromo-4-methylpyridine (B1) (1g, 5.81 mmol) THF (15 In a stirred solution in (ml), NaHMDS (2M in THF, 6.0 ml) was added at -78°C under argon. A 39 ml (12.8 mmol) solution was added dropwise. The yellow solution was incubated at -78°C for 30 minutes. Stirring. Then, methyl 6-methyl picolinate (1.19 ml, 8.72 ml) Add the solution of (ol) in THF (7 ml), and heat the reaction mixture to a maximum of approximately 21°C. The mixture was then stirred for 16 hours. The solvent was evaporated under reduced pressure, and the solid residue was ground with diethyl ether. The solid was then filtered and washed with diethyl ether. Next, the solid was sterilized in a saturated NH4Cl solution (20 ml). The aqueous phase was diluted with 1 / 100 ml of thiosulfate and extracted with SiO2SO4. The organic layer was then extracted with Na2SO4. The product was dried and concentrated in 4. The crude product was treated with silica using 10% Depositphotos / hexane. The compound C2 (1.1g) was purified by gel column chromatography and obtained as a yellow solid. 3.79 mmol, 65.4%, was obtained.

[0192] 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)

[0193] LC-MS(ESI): m / z 291[M] +

[0194] 6.3.2. 2-Bromo-4-[3-(6-methylpyridine-2-yl)-1H-pyra Zole-4-yl]pyridine (C3) A solution of C2 (300 mg, 1.03 mmol) in dry DMF (1 ml) is heated to argon Below, it was treated with glacial acetic acid (0.14 ml, 2.48 mmol) in DMF. DMA(0. Add 2 ml (1.55 mmol) dropwise, and steep the mixture at approximately 21°C for 1 hour under an argon atmosphere. The mixture was stirred under air pressure. Hydrazine monohydrate (0.37 ml, 7.75 mmol) was added dropwise. The resulting mixture was then heated at 50°C for 3 hours, and then at approximately 21°C for 16 hours. The mixture was poured into water (20 ml) and extracted with CH2Cl2 (3 × 20 ml). Organic The layer was dried with Na2SO4 and filtered. The solvent was evaporated under reduced pressure to obtain crude C3. Crude C3 was analyzed by silica gel column chromatography using 2% MeOH / DCM. After purification, purified C3 (172 mg, 0.54 mmol, 53%) was obtained as a yellow solid. Ta.

[0195] 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)

[0196] LC-MS(ESI): m / z 315[M+H] +

[0197] 6.3.3. 2-Bromo-4-(3-(6-methylpyridine-2-yl)-1-trithio (Lu-1H-pyrazole-4-yl)pyridine (C4) A stirring solution of C3 (40 mg, 0.12 mmol) in acetone (2 ml) is mixed with K2CO2. 3 (53 mg, 0.38 mmol) and trityl chloride (53 mg, 0.19 mmol) l) was added. The reaction mixture was then heated under reflux and stirred for 24 hours. Filter, concentrate the filtrate, then partition it between CH2Cl2 (5 ml) and water (5 ml). The organic phase was dried with Na2SO4 and concentrated. The crude solid was mixed with 2% MeOH / CH2C Purified by silica gel column chromatography using L2, as a pale yellow solid. Compound C4 (30 mg, 0.05 mmol, 41%) was obtained.

[0198] 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)

[0199] LC-MS(ESI): m / z 558[M+H] +

[0200] 6.3.4. tert-butylmethyl(2-(4-(4-(3-(6-methylpyridine) (-2-yl)-1-trityl-1H-pyrazole-4-yl)pyridine-2-yl) C5 oxyethyl carbamate A stirred solution of C4 (150 mg, 0.26 mmol) in toluene (5 ml) is mixed with EtO Int-B (152 mg, 0.40 mmol) in H (1 ml), followed by 2 M Na2 CO3 solution (0.7 ml) was added under an argon atmosphere. The reaction mixture was then halved with argon. Degass for 20 minutes, then add Pd(PPh3)4 (25 mg, 0.02 mmol), The mixture was refluxed for 6 hours. After complete consumption of the starting material (monitored by TLC), the reaction mixture was then prepared. The mixture was poured into water and extracted with toluene (3 x 10 ml). The organic layer was dried with Na2SO4. Then, concentrate under reduced pressure to obtain crude C5, which is then used with 30% siRNA / hexane. Purified C5(5) as a brown solid by silica gel column chromatography. 1 mg, 0.07 mmol, and 26% were obtained.

[0201] 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)

[0202] 6.3.5. N-methyl-2-(4-{4-[3-(6-methylpyridine-2-yl) -1H-pyrazole-4-yl]pyridine-2-yl}phenoxy)ethane-1-amine (Compound C) In a stirred solution of C5 (51 mg, 0.07 mmol) in CH2Cl2 (5 ml), add 1, 4N HCl was added to 4-dioxane (0.3 ml) at 0°C. Then the reaction mixture was prepared. The mixture was stirred under an argon atmosphere for 1 hour. After complete consumption of the starting material (monitored by TLC), (ring), the solvent was evaporated under reduced pressure to obtain crude compound C. Then, crude compound C was n-pe Grind the compound (2 x 1 ml) with a granule (2 x 1 ml), dry it, and obtain a brown solid. Compound C is then converted to an HCl salt. Obtained (20 mg, 0.05 mmol, 74%)

[0203] 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)

[0204] LC-MS(ESI): m / z 386[M+H] +

[0205] 6.4. [Example 4] (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfur (Phenamide)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.

[0206] [ka]

[0207] 6.4.1. Methyl 1-acetyl-2-oxoindoline-6-carboxylate (D 2) Methyl 2-oxoindoline-6-carboxylate (D1) (2.0g, 10.47 A stirred solution of mmol of acetic anhydride (16 ml) was heated to 130°C under an inert atmosphere for 6 hours. The mixture was heated for a while. After the complete consumption of the starting material (monitored by TLC), the reaction mixture was then heated. The solution was cooled to approximately 21°C. The precipitate was filtered, washed with n-hexane (2 x 50 ml), and then vacuum-sealed. The mixture was dried to obtain compound D2 (1.5 g, 61.5%) as a yellow solid.

[0208] 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)

[0209] 6.4.2. Methyl(Z)-1-acetyl-3-(hydroxy(phenyl)methylene) -2-oxoindoline-6-carboxylate (D3) To the 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) The reaction mixture was then mixed with triethylamine (2.2 ml) at 0°C under an inert atmosphere. The mixture was heated to approximately 21°C and stirred for 16 hours. After complete consumption of the starting materials (TLC) (For further monitoring), the reaction mixture was quenched with ice-cold water (30 ml) and toluene (2 Extraction was performed using 40 ml of water. The combined organic extract was dried with Na2SO4, filtered, and vacuum filtered. It was concentrated to obtain crude product D3, which was then converted using 80% toluene / hexane. The compound D3 (900m) was purified by Kagel column chromatography and obtained as a yellow solid. g, 42%) was obtained.

[0210] 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)

[0211] LC-MS(ESI): m / z 338.3[M+H] +

[0212] 6.4.3. (Z)-3-(hydroxy(phenyl)methylene)-2-oxoindri n-6-carboxylic acid (D4) Mixed solution of compound D3 (900 mg, 2.67 mmol) in MeOH (15 ml) Then, 15 ml of 1N NaOH aqueous solution was added at approximately 21°C. The reaction mixture was 100 The mixture was heated to °C and stirred for 6 hours. After complete consumption of the starting material (monitored by TLC), The reaction mixture was cooled to approximately 21°C and quenched with 13 ml of 1N HCl aqueous solution. Stirred for 30 minutes. Filter the precipitated solid and wash with 20% pharmaceutically / hexane. Compound D4 (580 mg, 77%) was obtained as an off-white solid, and this was further purified. It was used in the next step without needing to be done.

[0213] 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)

[0214] 6.4.4. (Z)-N-ethyl-3-(hydroxy(phenyl)methylene)-2-O Xoindoline-6-carboxamidelate (fragment A) Mixed 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) It was added at approximately 21°C under an inert atmosphere. After 30 minutes, THF (2.1 ml, 4.12 2N ethylamine in mmol was added at 0°C and stirred for 1 hour. Then the reaction mixture was added. The mixture was heated to approximately 21°C and stirred for a further 16 hours. After complete consumption of the starting materials (T (Monitoring by LC), volatile matter was removed under vacuum. The residue was diluted with water (15 ml). The mixture was filtered, washed with 20% phenylhexane (2 × 10 ml), and the crude product was obtained. This was then analyzed using silica gel column chromatography with 10% MeOH / CH2Cl2. By purification, fragment A (410 mg, 64.5%) was obtained as an off-white solid. .

[0215] 1 H 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)

[0216] LC-MS(ESI): m / z 307.1 (MH) + )

[0217] 6.4.5. N-(2-(dimethylamino)ethyl)-N-(4-nitrophenyl)methyl Tansulfonamide (D8) Mixed solution of compound D7 (800 mg, 3.70 mmol) in acetone (15 ml) Potassium carbonate (1.32g, 9.62mmol), sodium iodide (110mg, 0 Compound B6 (799 mg, 5.55 mmol) and compound B6 (799 mg, 5.55 mmol) were tested in an inert atmosphere. The mixture was added at 0°C under gaseous conditions. The reaction mixture was heated to 50°C and stirred for 20 hours. After complete consumption (monitored by TLC), volatile matter was removed under vacuum. The residue was then removed with water. Diluted with (20ml) and extracted with ELISA (2 x 40ml). The combined organic extract was then... The product was dried with Na2SO4, filtered, and concentrated under vacuum to obtain the crude product, which was then mixed with 5% Me Purified by silica gel column chromatography using OH / CH2Cl2, then lightly Compound D8 (460 mg, 43%) was obtained as a yellow solid.

[0218] 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) )

[0219] LC-MS(ESI): m / z 288.3[M+H] +

[0220] 6.4.6. N-(4-aminophenyl)-N-(2-(dimethylamino)ethyl) Tansulfonamide (Fragment B) Mixed solution of compound D8 (460 mg, 1.60 mmol) in MeOH (10 ml) , 10% Pd / C (40 mg) was added and stirred at approximately 21 °C under a hydrogen atmosphere (balloon pressure) for 3 hours. After complete consumption of the starting material (monitored by TLC), the reaction mixture was filtered through a pad of Celite® and washed with MeOH (10 ml). The filtrate was concentrated in vacuo to afford the crude product, which was purified by silica gel column chromatography using 10% MeOH / CH2Cl2 to give fragment B as a pale yellow solid (300 mg, 73%).

[0221] 1 1H 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).

[0222] LC-MS (ESI): m / z 258.2 [M+H]. +

[0223] 6.4.7. (Z)-3-(((4-(N-(2-(dimethylamino)ethyl)methyl sulfonamido)phenyl)amino)(phenyl)methylene)-N-ethyl-2-oxo indoline-6-carboxamide (D5) A solution of fragment A (200 mg, 0.64 mmol), fragment B (500 mg, 1.94 mmol) and TMS-imidazole (455 mg, 3.24 mmol) in THF (5 ml) was heated at 170 °C for 1 hour under microwave irradiation. After consumption of the starting materials (monitored by TLC and LC-MS), the volatiles were removed in vacuo. The residue was partitioned between water (10 ml) and the residue was partitioned between water (10 ml) Dilute with HCl, extract with HCl (3 × 25 ml) to obtain the crude product, and separate it with preparative HPL. The compound D5 (150 mg, 42%) was purified with C to obtain a pale yellow solid.

[0224] 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)

[0225] LC-MS(ESI): m / z 548.6[M+H] +

[0226] 6.4.8. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl (Phenyl) Methylsulfonamide (Phenyl) Amino (Phenyl) Methylene)-2-oxo Ndrin-6-carboxamide (compound D) Mixed 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) l) was added under an inert atmosphere at approximately 21°C. The reaction mixture was then heated to reflux temperature (120°C). The material was heated 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 HCl (30 ml), and then diluted with 1N HCl. The organic layer was washed with 15 ml of aqueous solution. The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The compound (40 mg) was reduced, mono-demethylated, and di-troc protected.

[0227] 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 then heated to 50°C. The mixture was heated and stirred for 8 hours. After complete consumption of the starting materials (monitored by TLC), the reaction mixture was mixed. The mixture was cooled to approximately 21°C, and volatile substances were removed under vacuum. The residue was then mixed with water (20 ml). The mixture was diluted and extracted with ۶ (2 x 25 ml). The combined organic extract was then saturated with NaHCl. Wash with O3 solution (20 ml), dry with Na2SO4, filter, and concentrate under reduced pressure. Crude compound D was obtained and then dissolved in silica gel using 5-6% MeOH / CH2Cl2. The compound D was purified by chromatography to obtain 12 mg of compound D with 83% HPLC purity. Ta.

[0228] The reaction was repeated on a 60 mg scale, and the resulting crude product was combined with the batch described above. The compound D (8.0 mg, 6.3%) was purified by preparative HPLC and obtained as a pale yellow solid. I got it.

[0229] 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)

[0230] LC-MS(ESI): m / z 534.6[M+H] +

[0231] UPLC purity: 99.18%

[0232] 6.5. [Example 5] (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl)methylsulfur (Phenamide)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.

[0233] [ka]

[0234] 6.5.1. N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfone Amido (D9) Mixing solution of compound D7 (1.0 g, 4.65 mmol) in DMF (10 ml) with water Sodium oxide (60%, 320 mg, 7.99 mmol in mineral oil) under an inert atmosphere The mixture was added at 0°C and stirred at approximately 21°C for 30 minutes. To this mixture, 1,2-dibromoate Thanh (2.18 g, 11.60 mmol) was added at approximately 21°C. The mixture was then heated to 90°C. The mixture was heated to °C and stirred for 24 hours. The reaction mixture was monitored by TLC. Cool to approximately 21°C, quench with ice water (30 ml), and add phenylalanine (2 x 40 ml). Extracted using [method]. The combined organic extracts were dried with Na2SO4, filtered, and concentrated under vacuum. The crude product was obtained and then subjected to silica gel column chromatography using 5% MeOH / CH2Cl2. Purified by matrixing, the mixture containing 40% unreacted starting material yields 1.2 g of D9 was obtained. The resulting mixture was used directly in the next reaction without further purification.

[0235] 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)

[0236] 6.5.2. N-(2-(methylamino)ethyl)-N-(4-nitrophenyl)meth Sulfonamide (D10) A stirring solution of compound D9 (1.2g, impure) in THF (10ml) is prepared by adding triethylamine. N (1.6 ml) and methylamine (2M 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 then heated to 80°C. The mixture was heated and maintained for 16 hours. After complete consumption of the starting material (monitored by TLC), the reaction was performed. The reaction mixture was cooled to approximately 21°C and concentrated under reduced pressure to obtain crude D10. Crude D10 was then used. Silica gel column chromatography using 15% MeOH / CH2Cl2 Purified to give compound D10 (500 mg, 39% overall yield over 2 steps) as a yellow solid. in 2 steps) as a yellow solid.

[0237] 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)

[0238] 6.5.3. tert-Butylmethyl(2-(N-(4-nitrophenyl)methylsulfonamide)ethyl)carbamate (D11) A stirred solution of D10 (500 mg, 1.83 mmol) in CH2Cl2 (10 ml) was treated with triethylamine (0.4 ml, 2.61 mmol) and anhydrous Boc (659 mg, 3.02 mmol) at approximately 21 °C under an inert atmosphere and maintained for 5 h. After complete consumption of the starting material (monitored by TLC), the volatiles were removed in vacuo to give a crude product, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 to give D11 (320 mg, 47%) as a colorless thick syrup. in vacuo to give a crude product, which was purified by silica gel column chromatography using 5% MeOH / CH2Cl2 to give D11 (320 mg, 47%) as a colorless thick syrup.

[0239] 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)

[0240] LC-MS(ESI): m / z 274.2(M + -B℃)

[0241] 6.5.4. tert-butyl(2-(N-(4-aminophenyl)methylsulfonate Mido(ethyl)(methyl)carbamate (Boc variant of fragment B) In a solution of compound D11 (250 mg, 0.67 mmol) in EtOH (10 ml), Add Laney-Ni (40 mg) and incubate at approximately 21°C under a hydrogen atmosphere (balloon pressure) for 1 hour. The mixture was stirred. After complete consumption of the starting material (monitored by TLC), the reaction mixture was converted to C. The sample was filtered through an elite® pad and washed with EtOH (10 ml). The combined filtrate is concentrated under vacuum to obtain the crude product, which is then mixed with 10% MeOH / CH2Cl2 The fragments were purified by silica gel column chromatography using the following method, and obtained as a pale yellow solid. We obtained a Boc variant of B (180 mg, 77%).

[0242] 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)

[0243] LC-MS(ESI): m / z 244.2(M + -B℃)

[0244] 6.5.5. tert-butyl(Z)-(2-(N-(4-(((6-(ethylcarb Moyl)-2-oxoindoline-3-ylidene)(phenyl)methyl)amino)pheni (Methyl sulfonamide) ethyl (methyl)carbamate (D10) Fragment A (70 mg, 0.22 mmol), Boc variant of Fragment B (155 mg, 0.4 THF (5 mmol) and TMS-imidazole (159 mg, 1.13 mmol) The solution in 3 ml was heated under microwaves at 170°C for 160 minutes. Consumption of the starting material. (Monitoring by TLC and LC-MS) Afterwards, volatile matter is removed under vacuum, and the residue is This was obtained and purified by preparative HPLC to obtain compound D10 (50 mg) as a pale yellow solid. , 36% was obtained.

[0245] 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)

[0246] LC-MS(ESI): m / z 634.6[M+H] +

[0247] 6.5.6. (Z)-N-ethyl-3-(((4-(N-(2-(methylamino)ethyl (Phenyl) Methylsulfonamide (Phenyl) Amino (Phenyl) Methylene)-2-oxo Ndolin-6-carboxamide hydrochloride (compound D as an HCl salt) Stirring of compound D10 (20 mg, 0.03 mmol) in diethyl ether (3 ml) Add 4N HCl from 1,4-dioxane (0.3 ml) to the solution and heat under an inert atmosphere at 0°C. The mixture was added. The reaction mixture was stirred at approximately 21°C for 1 hour. Complete consumption of the starting material (T After monitoring by LC, volatile matter is removed under vacuum to obtain the crude product, which is then subjected to n- Grind with pentane (2 x 4 ml) to obtain compound D(1) as a pale yellow solid and its HCl salt. 2 mg was obtained, with a 71% response rate.

[0248] 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).

[0249] LC-MS(ESI): m / z 534.7[M+H] +

[0250] UPLC purity: 96.26%

[0251] 6.6. [Example 6] In vitro assay for testing the activity of compounds A-D 6.6.1. N-(2-bromoethyl)-N-(4-nitrophenyl)methanesulfone Amido (2) Compounds A-D were tested, and these were found to be the TGF of HEK293T cells in vitro. We determined whether it could inhibit β-inducible luciferase activity.

[0252] 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 per well was added. The substance was transfected into cells for 24 hours using lipofectamine. The next day The cells were treated with compounds A-D and 100 pM TGFβ for 24 hours. Lucifer Luciferase activity is measured using the Dual-Glo(registered trademark) luciferase assay kit (Promeg a) was used for measurement. The assay was performed twice for compounds A, B, and D. The experiment was performed three times on compound C. The results are shown in Table 4.

[0253] [Table 4]

[0254] The activity data from Experiment 1 is shown in Figure 5.

[0255] Compounds A through C showed the greatest inhibitory activity.

[0256] 6.6.2. MTS proliferation assay Compounds A-D were tested, and these were found to be the first generation mouse CD4 + T cell TGF-β signaling We determined whether or not transmission could be inhibited.

[0257] Original Mouse CD4 + T cells are separated using the RoboSep® cell isolation system (Stemc Using ell Technologies, isolated from the spleen of C57 / B6 mice 0.5 μg / ml hamster anti-mouse CD3e antibody (145-2C11, eBio (science) was coated overnight onto a 96-well flat-bottom plate. 1 × 10 5 Individually refined CD4 + T cells were treated with 1 μg / ml of soluble hamster anti-mouse CD28 anti- (37.51, BD Biosciences), 1 nM TGF-β1, and compound The cells were incubated with 8-fold sequential dilutions of substances A-D. After 72 hours, cell proliferation was observed by the manufacturer. The measurements were taken using the MTS assay (Promega) according to the instructions. The results are shown in the table. This is shown in 5.

[0258] [Table 5]

[0259] The data from Experiment 1 is shown in Figure 6.

[0260] In two different experiments, IC 50 The value was not obtained for compound D. The same was true for compound A. Also, Mouse CD4 + It did not show consistent effects in T cells. However, compound B C reversed the TGF-β-mediated inhibition of T cell proliferation.

[0261] Based on the two assays, compound C was selected for conjugation to ADC. Ta.

[0262] 6.7. [Example 7] 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2H-Pyrrole-1(5 H)-Il)hexaneamide)-3-methylbutanamide)-5-ureidopentanamide (d)benzylmethyl(2-(4-(4-(3-(6-methylpyridine-2-yl)-1H -Pyrazole-4-yl)pyridine-2-yl)phenoxy)ethyl)carbamate compound Growth Compound C is prepared according to the general method in Scheme 6 below, using valine-citrulline It was connected to the linker.

[0263] [ka]

[0264] L1 (122 mg, 0.165 mmol, 1.1 equivalents) and TEA (52 μl, 0. Compound C (375 mmol, 2.5 equivalents) (58 mg, 0.150 mmol, 1.0 equivalent) Add the solution (in 2 ml) of DMF at 0°C, and allow the reaction mixture to ferment at approximately 21°C for 2 hours. The mixture was stirred to obtain crude ADC-1. Crude ADC-1 was purified by preparative HPLC to obtain a white solid. A purified form of ADC-1 (34 mg, 24% yield) was obtained.

[0265] 6.8. [Example 8] Generation of antibody-drug conjugate 1 (ADC1) Anti-mouse transferrin receptor antibody R17217, and rat anti-mouse IgG2A Isotype control antibody (BioXCell) is mixed with a composite buffer (25 mM borate). (Sodium / 25 mM NaCl and 0.3 mM EDTA, final pH 7.4) I underwent dialysis overnight. Tris(2-carboxyethyl)phosphine (TCEP) was used as the antibody. Then, reduction was performed for 2 hours at a reduction rate of 10-30. ADC-1 was DM-treated to a final concentration of 10 mM. The solution is dissolved in SO, and then conjugated to the antibody in the presence of 15% DMSO at a conjugation ratio of 5-30. The reaction was gated. All reactions were carried out at approximately 21°C. Several drug-antibody ratios (DARs) were observed. In contrast, 50% propylene glycol is added during the conjugation step, in an organic solution. Used as a medium. The final ADC was dialyzed overnight in PBS and filtered through a 0.22 μm filter. The sample was used, filtered, and analyzed via HPLC-HIC to determine the DAR, and then HPLC-SEC was performed. The level of aggregation was determined by analysis via HPLC-HIC. For HPLC-HIC, the sample was flowed through The procedure was performed at a rate of 0.5 ml / min on a TSKgel® butyl-NPR column. A is 25 mM sodium phosphate and 1.5 M ammonium sulfate at pH 6.95. Phase B, on the other hand, was 75% 25 mM sodium phosphate at pH 6.95, and It was 25% isopropyl alcohol. HPLC-SEC analysis was performed using TSKg el(registered trademark) G3000SW column (Tosoh Bioscience), flow rate It was used at 0.25 ml / min, 280 nM, for 25 minutes.

[0266] 6.9. [Example 9] Synthesis of compound C(ADC-2) linked to a disulfide linker Compound C is disulfide according to the general method in Scheme 7A-B below. It was connected to the linker.

[0267] [ka]

[0268] [ka]

[0269] 6.9.1. Synthesis of Intermediate A 2-Chlorotrityl chloride resin (L2) (4g, 4mmol) is used in DCM (2×40 Wash in ml, swell in 50 ml of DCM for 10 minutes, then drain. Fmoc -Cys(Trt)-OH(L3) (7.03g, 12 mmol) in 40 ml of DCM Dissolve in and add to a container containing 2-chlorotrityl chloride resin. 8.7 ml of D Add IPEA (6.8 ml, 40 mmol) to the container and incubate the mixture at approximately 21°C for 2 hours. Stir. Then add 10 ml of methanol to the mixture and stir for 30 minutes. Next Next, the obtained resin (L4) is discharged and washed five times with DMF. Then, the resin L4 is removed Protect the mixture and add approximately 40 ml of 20% piperidine in DMF to resin L4. The resin L5 is produced by shaking the mixture and then draining the liquid from the resin. Add another 40 ml of 20% piperidine to the resin and shake for 15 minutes. Then, Remove resin L5 from the liquid and wash it with DMF (6 x 40 ml).

[0270] The solution of Fmoc-amino acids is Fmoc-Asp(OtBu)-OH(4.93g, 1 2mmol), Fmoc-Asp(OtBu)-OH (4.93g, 12mmol), F moc-Arg(Pbf)-OH(7.79g, 12mmol), Fmoc-Asp(O tBu)-OH (4.93g, 12mmol), and Fmoc-Glu-OtBu(5 0.1g, 12 mmol) to HBTU / HOBT (4.55g, 12 mmol / 1.62 (g, 12 mmol) and DIPEA (2 ml, 12 mmol) are prepared separately in combination. To manufacture.

[0271] Add the Fmoc-Asp(OtBu)-OH solution to resin L5 and shake for 60 minutes. Next, resin L6 is produced. Resin L6 is washed with DMF (6 x 40 ml), and then as described above. Then, deprotect with 20% piperidine in DMF. Next, resins L7, L8, L9, and L10 was produced by performing a series of couplings using an Fmoc-amino acid solution. Using the same procedure, resin L5 is used to produce resin L6.

[0272] In the exemplary synthesis, 8g of dry resin L10 is added to the flask, and 80ml is cut. The solution was added (TFA:TES:EDT:H2O=90:5:3:2, v / v / v / v The reaction was allowed to proceed for 1.5 hours. Then, the resin was filtered under pressure to remove the reaction mixture. They were then separated. Next, the resin was washed twice with TFA. The filtrates were combined and cooled to 10 times their volume. TBE was added dropwise. Then, the precipitated peptide (intermediate A) was centrifuged and chilled MTB was used. It was washed four times with E. Then, intermediate A was dried under reduced pressure and purified by preparative HPLC. 1.1 g of intermediate A (37% yield) was obtained as a white solid. LC-MS(ESI)m / z:752[M+H]+.

[0273] 6.9.2. 2-(pyridine-2-yldisulfanyl)ethylmethyl(2-(4-( 4-(4-(6-methylpyridine-2-yl)-1H-pyrazole-3-yl)pyridine -2-yl(phenoxy)ethyl(carbamate)(L12) Compound C (40 mg, 0.1038 mmol) and 4-nitrophenyl 2-(pyridinyl (n-2-yldisulfanil)ethylcarbamate (L11) (80mg, 0.2272 To the solution of mmol) in DMF (5 ml), add DIPEA (0.5 ml) and HOBt ( 14 mg (0.1038 mmol) was added. The mixture was left at approximately 21°C under N2 conditions for 16 hours. The mixture was stirred to produce L12. Crude L12 was purified by preparative HPLC to obtain a white solid. This yielded 35 mg of purified L12 (56% yield).

[0274] 6.9.3. (2R,5S,8S,11S,14S,19S)-19-amino-5,8 ,14-tris(carboxymethyl)-11-(3-guanidinopropyl)-2-((( 2-(methyl(2-(4-(4-(4-(6-methylpyridine-2-yl)-1H-pyra Zol-3-yl)pyridine-2-yl)phenoxy)ethyl)carbamoyloxy) (Tyl)disulfanyl)methyl)-4,7,10,13,16-pentaoxo-3,6, 9,12,15-Pentazycosan-1,20-Diacid (L13) Dissolution of L12 (35 mg, 0.058 mmol) in THF / H2O (5 ml / 5 ml) Intermediate A (80 mg, 0.106 mmol) was added to the solution under N2 conditions. The mixture was then mixed. The mixture was stirred at 21°C for 16 hours to produce L13. Crude L13 was purified by preparative HPLC. This yielded 23 mg of purified L13 as a white solid (31% yield).

[0275] 6.9.4. (2R,5S,8S,11S,14S,19S)-19-(2-(ter t-Butoxycarbonylaminooxy)acetamide)-5,8,14-Tris(Carbo Xymethyl)-11-(3-guanidinopropyl)-2-(((2-(methyl(2-(4 -(4-(4-(6-methylpyridine-2-yl)-1H-pyrazole-3-yl)pyri Zin-2-yl(phenoxy(ethyl))carbamoyl(oxy)(ethyl)disulfanyl) Methyl)-4,7,10,13,16-pentaoxo-3,6,9,12,15-penta Azaicosan-1,20-dioxide (L15) A solution of L13 (32 mg, 0.025 mmol) in DMF (3 ml) contains 2,5-di Oxopyrrolidine-1-yl-2-(tert-butoxycarbonylaminooxy)acetate Add L14 (28 mg, 0.097 mmol), followed by TEA (0.5 ml). The reaction mixture was stirred at approximately 21°C under an N2 atmosphere for 16 hours to produce L15. Crude L15 was purified by preparative HPLC, and 12 mg of purified L was obtained as a white solid. 15 (33% yield) was produced.

[0276] 6.9.5. (2R,5S,8S,11S,14S,19S)-19-(2-(amino Oxy(acetamide)-5,8,14-tris(carboxymethyl)-11-(3-g Anidinopropyl)-2-(((2-(methyl(2-(4-(4(4-(6-methylpyryl) (Din-2-yl)-1H-pyrazole-3-yl)pyridine-2-yl)phenoxy) (Cyl)carbamoyloxy)ethyl)disulfanyl)methyl)-4,7,10,13, 16-Pentaoxo-3,6,9,12,15-Pentaazycosan-1,20-Diacitic Acid ADC-2) A mixture of L15 (12 mg, 0.0085 mmol) in DCM (5 ml) contains TFA (1 ml) was added. The mixture was stirred at approximately 21°C for 30 minutes to produce ADC-2. Crude ADC-2 was concentrated and purified by preparative HPLC, yielding 3.5 mg as a white solid. Purified ADC-2 (31% yield) was produced.

[0277] 6.10. [Example 10] Generation of antibody-drug conjugate 2 (ADC2) ADC-2 is subjected to antibody lysine residues according to the general method in Scheme 8 below. It then bound to the anti-TfR antibody.

[0278] [ka]

[0279] I purchased the heterobifunctional polymer linker S-4FB from Solulink. (Rat) Anti-mouse IgG2a and anti-mouse transferrin receptor antibody R17217, pH Dialysis was performed in PBS at pH 7.4. S-4FB was dialyzed in PBS at pH 7.4 in different molar ratios. The antibody was added and incubated at approximately 21°C for 3 hours. S-4FB modified antibody solution. This was prepared using a 2-hydrazinopyridine solution (0.5 mM in 100 mM MES buffer, pH 5). Combined with (0), incubate at 37°C for 30 minutes in various compound ratios ranging from 5 to 50. The S4FB / Ab molar substitution ratio was determined by UV-Vis with A354. The body was subjected to a Zeba® spin desalination column, 50 mM phosphate buffer (pH 6.5, 15 The solution was purified using a buffer solution (0 mM NaCl), and then the linker-SS drug was removed. Mix ADC-2 (10 mM in DMSO) with different molar ratios at 37°C for 24 hours. ADC2 was generated. The following day, the ADC2 sample was dialysis overnight against PBS. The sample was filtered, and then subjected to HPLC-SEC, SDS-PAGE, and LC-MS. The tests were conducted using ADC2 prepared with an S-4FB / Ab ratio of 6 and an ADC-2 / Ab ratio of 20. An exemplary LC-MS data for the tested ADC2 sample is shown in Figure 7. Figure 7 shows the results for the tested ADC2 sample. The average DAR was 4.99, with the heavy chain having a DAR of 1.97 and the light chain having a DAR of 0.53. This indicates that...

[0280] If ADC2 aggregation at 5% is detected by HPLC-SEC, the aggregated components are... This is the SEC column (GE Healthcare Life Sciences, Sup AKTA with erdex 200 increase 10 / 300GL The samples were separated and analyzed again by HPLC-SEC. The samples were purified by SEC to remove aggregates. The chromatogram of ADC2 is shown in Figure 8.

[0281] 6.11. [Example 11] Antibody-inducible receptor internalization assay 96-well flat-bottom plates were coated overnight with anti-mouse CD3e antibody at 4°C. CD4+ T cells are separated using the RoboSep™ cell isolation system (Stemcell Te It was isolated from the mouse spleen using (Chemologies). Approximately 2 × 10 5 individual The cells were plated in each well with soluble anti-CD28 antibody at 37°C for 24-48 hours. It activated. When activated, CD4 + T cells were collected, washed, and then exposed to air at 37°C. Then, plated again with 5 μg / ml primary (anti-transferrin receptor) antibody, Internalization was induced. The reaction was stopped with ice-cold staining buffer and kept on ice to stop internalization. At the end of the assay, the cells were washed twice with ice-cold staining buffer to remove unbound antibodies. The cells were pelleted, then stained with PE, and then treated with a goat anti-rat secondary antibody and conjugate. The cells were sterilized and incubated on ice for 30 minutes. The cells were washed with staining buffer and then FAC. We analyzed the expression mediated by S. As shown in Figure 9, TfR expression was primary CD4 within 1 hour. + Internalization begins in T cells, and within 3 hours, more than 70% of TfRs are anti-transferrin receptors. It is internalized by the antibody R17217.

[0282] 6.12. [Example 12] In vitro assay 6.12.1. Growth Assay Mouse CTLL2 cells were divided into 0.2 ng / ml IL2 cells, 1 × 10⁶ cells. 5 by individual / well The cells were cultured. In each well, indicated by 1 nM TGF-β, 1 μg / ml ADC was added, and / Alternatively, 100 nM of ALK5 inhibitor compound C was added to each well for 24 hours to promote growth. Quantification was performed by adding BrdU reagent (Abcam) to the wells for an additional 12 hours, and then... The analysis was performed using ELISA.

[0283] As shown in Figure 10, treatment of CTLL2 cells with TGF-β resulted in approximately 60% proliferation. This was inhibited. However, by adding ADC1 (DAR2-4, 4-6, or 6-8) Therefore, TGF-β inhibition is almost completely reversed compared to treating cells with ALK5 inhibitors alone. The condition reversed, and CTLL2 proliferation recovered. (Rat anti-mouse IgG2A isotype control) Cells treated with ALK5 ADC did not recover CTLL2 proliferation. In cells treated with ADC1 without it, or with a naked Tfr antibody alone. No inhibition of proliferation occurs, which means that ADC1 does not affect proliferation unless TGF-β is present. It was shown that it does not have an effect (data was not shown).

[0284] 6.12.2. Granzyme B Expression Assay Mouse CD3 + T cells, EasySep(trademark) mouse T cell isolation kit (negative Using Stemcell Technologies, we can select the mouse spleen or Then it was purified. CD3 + T cells are treated with plate-bound anti-CD3e and soluble anti-CD28. The T cells were activated for 48 hours before use. The T cells were washed and treated with 5% serum and 1 nM TGF. The cells were plated again in a medium containing -β- / +ADC.

[0285] Golgistop reagent was added during the last 4 hours, and then the cells were sterilized by CD8(BD) on the surface. Immunostaining was performed against GzmB (eBioscience) in the cells, and flow cytometry was performed. Analysis was performed via Tory. Granzyme B (GzmB) is CD8 + T cells cause tumor cells It is a serine protease released to kill cells. Therefore, GzmB expression The increase in CD8 + It exhibits cytotoxic T cell activity.

[0286] As shown in Figure 11, TGF-β is the first CD8 + Suppresses GzmB expression in T cells. However, treatment at all ADC1s of the three DARs, 2-4, 4-6, and 6-8, is also We were able to restore GzmB expression to the same extent as with ALK5 compounds. In addition, rat anti-mau The IgG2A isotype control ALK5 ADC did not restore GzmB expression. .

[0287] 6.12.3. iTreg Conversion Assay Naive CD4 T cells were isolated from mouse spleen cells using a negative selection kit. They were isolated from the cells. The cell density was 0.4 × 10⁶ cells. 6Adjust to 10 ng / ml Mouse IL-2, 20 ng / ml TGF-β, and 1 μg / ml soluble anti-CD28 This was added to the cell suspension.

[0288] A 10 μg / ml anti-mouse CD3 antibody was coated onto a 24-well plate. The cells were incubated overnight at 4°C. Then, the antibodies were aspirated from the plate. 1 ml of cell suspension was used. The turbidity was added to each well of a 24-well plate at concentrations of 3 μg / ml and 5 μg / ml. ADC1 (DAR4-6), anti-transferrin receptor antibody, rat anti-mouse IgG2 A isotype control ALK5 ADC, and A at 100 nM and 1 μM LK5 inhibitor compound C was added to a separate well of a 24-well plate. Then, cells were added. The cells were cultured for 72 hours. TfR expression was tested at 48 hours (data not shown). The cells were stained with FoxP3 (eBioscience FoxP3 staining buffer) and left for 72 hours. At that point, the selection was performed using FACS.

[0289] As shown in Figure 12, ADC1 (+CD71-ALK5 ADC) at 5 μg / ml is Similar to 100nM free ALK5 inhibitor alone (+ALK5 inh 100nM), The amount of iTreg generated is moderately reduced. In contrast, the control ALK5 ADC (+Iso-ALK5 ADC) and naked anti-TfR antibody (+anti-CD71) are used in iTre There was no effect on g FoxP3 expression.

[0290] 6.13. [Example 13] Synthesis and Characterization of Compound N Compound N was synthesized according to the general method shown in Scheme 9 below.

[0291] [ka]

[0292] Compound N was compared to compound C in several in vitro assays. Its IC50 activity and its K in exchange kinase assays i A summary of the values ​​is shown in Table 6. Table 6 also shows the TGF-β sigma of compound C in human HEK cells and mouse T cells. It also exhibits inhibitory activity against Nal signaling. Compound C showed 10 times greater inhibitory activity than compound N in recombinant assays. It was found to be powerful.

[0293] [Table 6]

[0294] 6.14. [Example 14] Internalization of CD2 and CD5 into T cells Two different internalization studies were conducted, using anti-CD2 antibodies and anti-CD5 antibodies, respectively. Following T cell incubation, we measured the internalization of CD2 and CD5 cells.

[0295] 6.14.1. Study 1: No antibody washout Mouse CD3+ T cells were treated with plate-bound anti-CD3 antibody (1 μg / ml) and soluble anti- The cells were activated with CD28 antibody (2 μg / ml) for 36 hours. The cells were washed and treated with 1 μg / ml LA. Anti-mouse CD2 antibody (clone 12-15, Southern Biotech, Catalogue number 1525), rat anti-mouse CD5 antibody (clone 53-7.3, South (ern Biotech, catalog number 1547), or rat isotype control With the roll antibody, at the indicated time points (0, 15 minutes, or 0.5, 1, 3, or 6 hours), The cells were incubated at 37°C. At each time point, the assay was performed by placing the cells on ice. The process was stopped. Secondary antibodies conjugated with fluorescence were used to express CD2 and CD5. It was detected by doing so.

[0296] At 6 hours, over 60% of CD5s and over 50% of CD2s were detected, indicating that mouse CD3+T was fine. It was internalized within the vesicle (Figures 13A and 13B, respectively).

[0297] 6.14.2. Study 2: Antibody Washout Repeat Study 1, except that the free antibody was incubated in cells at 4°C for 30 minutes. All receptors on the cell surface were saturated. The antibodies remaining in the supernatant were washed away before the time interval began. did.

[0298] At the 6-hour mark, nearly 90% of CD5s and over 50% of CD2s were found to be mouse CD3+T It was internalized within the cell (Figures 13C and 13D, respectively).

[0299] 6.14.3. Discussion In Study 1, the novel recirculated receptors, if present, remained on the cell surface over time. It approached and was able to bind to the free antibody in the culture medium. In Study 2, the time course was started Because it is possible to monitor only the internalization of the receptor that exists at that time, unbound antibodies are used. It was rinsed off before the time elapsed period began. For CD2, the results of Study 1 and Study 2 are similar. They are similar, suggesting that CD2 does not reverse quickly. Compared to CD5, WOT In the Shuout study (Study 2), internalization increased by approximately 20%, indicating that the novel receptor was 6 Over time, does it recycle or increase through de novo synthesis? This indicates that it was one of the following: a large amount of de novo synthesis occurred over a 6-hour period. Since it is unpredictable, it is considered a highly recyclable option. Therefore, The results of Study 1 and Study 2 suggest that CD5 can be recirculated to the cell surface in greater quantities than CD2. This suggests that it is possible.

[0300] 6.15. [Example 15] ADC-targeted CD2 and CD5 generation and characterization 6.15.1. Example 15: Generation of ADC In this embodiment, four A's are referred to as T cell-targeted TGF-β antagonists (T3A). LK5-ADC was used with rat anti-mouse CD2 antibody (clones 12-15, Southern Biotech (catalog number 1525), and rat anti-mouse CD5 antibody (Crow Using (N53-7.3, Southern Biotech, catalog number 1547) The T3A was constructed using two linker-ALK5 inhibitor payloads. However, one of them is a cleavable Val-Cit(VC) bonded to ALK5-compound C. It contains a linker, and the other is a non-cleavable maleimide caproyl (M) bonded to compound N. C) Includes linker

[0301] The combinations of antibodies, linkers, and ALK5 payloads for the four T3A antibodies are shown in Table 7.

[0302] [Table 7]

[0303] T3A #2~#5 were purified by size exclusion chromatography (SEC) and drug The antibody ratio was calculated by hydrophobic interaction chromatography (HIC). T3A #2 The percentage agglutination, percentage unbound antibody, and DAR values ​​for each of the five antibodies are shown in Table 8.

[0304] [Table 8]

[0305] 6.15.2. Characterization of ADCs To determine the efficacy of T3A #2-5 in reversal TGF-β-mediated immunosuppression Mouse CD3+ T cells were purified from the spleen and treated with 1 nM TGF-β and small molecule ALK. 5. Inhibitor compound C (positive control), T3A #2-5, or isotype control T In the presence of 3A (negative control), the cells were activated for 36-72 hours with anti-CD3 and anti-CD28 antibodies. After 36 hours, the level of CD8+ T cells expressing granzyme (GzmB) was reduced to cytotoxicity. Measured as a sex marker (Figure 14), and the level of secreted cytokine IL2 (Figure 15) The levels of IFN-γ (Figure 16) were measured by ELISA. Finally, at 72 hours After a period of time, the amount of T cell proliferation was measured using Cell Titer Glo (Promega). (Figure 17). All of these assays relate to tumor clearance in vivo. There is a connection.

[0306] The amount of function observed in relation to activated T cells (set to 100%) is shown in Figures 14-17. Each is shown below. T3A #5 restored GzmB expression and T cell proliferation, but IFN -γ expression was only partially restored. No effect on IL2 expression was observed.

[0307] 6.15.3. Discussion The data from the above examples show that the level of target expression in T cells corresponds to the efficacy in the primary T cell assay. This shows that it is important for function. Both CD2 and CD5 are present in 20-50% of active T cells. Unlike CD71, which is only highly expressed, it is expressed in both naive T cells and activated T cells. Over 85% are highly expressed. However, both CD2 and CD5 are highly expressed in T cells. However, CD5-targeted ADCs have been observed to have higher efficacy than CD2-targeted ADCs. In Example 14, based on the receptor internalization patterns observed in CD2 and CD5, Subsequently, at 6 hours, approximately 85% of CD5 was internalized in primary mouse T cells, but C Only 53% of D2 was internalized. In addition, CD5 began to be internalized earlier than CD2. This was the conclusion. This data suggests that the amount of internalization also affects efficacy.

[0308] The data also shows that both the linker that binds the ALK5 inhibitor to the antibody and the release mechanism are involved. This also shows that it is important for efficacy. Catheterization in combination with anti-CD5 antibody (T3A #5) Syn B-cleavable VC linker was the most effective T3A. However, anti-CD5 antibody (T3 A #4) Non-cleavable MCs combined with a linker also bind to anti-CD5 antibodies. In combination, it exhibited several activities.

[0309] Based on studies using primary mouse T cells, T3A can be ranked in terms of efficacy as follows: The following can be used: 1) T3A #5, 2) T3A #4, 3) T3A #3, and 4) T3 A #2.

[0310] Without being constrained by theory, in response to high ADC activity, ADCs are naive T cells and It is widely expressed across activated T cells (for example, expressed in more than 70% of cells) and rapidly inhibits intracellular activity. T cells have been established and possess an established intracellular release mechanism (e.g., proteolytic processing). It is considered that the target should be the cellular target.

[0311] 6.16. [Example 16] Internalization of CD7 into T cells We conducted an internalization study to incubate T cells with two different anti-CD7 antibodies. Next, we measured the internalization of CD7.

[0312] Human CD3+ T cells were treated with plate-bound anti-CD3 antibody (1 μg / ml) and soluble anti-CD3 antibody. The cells were activated with D28 antibody (2 μg / ml) for 40 hours. The cells were washed and treated with 1 μg / ml anti-inflammatory agent. CD7 antibody (clones 124-D1 and 4H9, Caprico Biotech) Alternatively, incubate with rat isotype control antibody at 4°C for 30 minutes. All receptors on the cell surface were saturated. The antibodies remaining in the supernatant were washed away, and then the cells were heated to 37°C. Incubated for 0-6 hours at each time point (5, 15, 30, 60, 180, and 360). The assay was stopped after (minutes) by placing the cells on ice. CD7 expression was measured using fireflies. Detection was performed using a photoconjugated secondary antibody.

[0313] At 6 hours, approximately 70-80% of CD7 was internalized (Figure 18). The amount of internalization was C CD7 was demonstrated to be as suitable as D2 and CD5 as an ADC target.

[0314] 6.17. [Example 17] Combination of T3A and immune checkpoint inhibitors Perform a cytokine secretion assay to check for T cells T3A #5 (Example 16). The efficacy of combining this treatment with the point inhibitor pembrolizumab was measured.

[0315] In a 96-well round-bottom plate, 1.5 × 10 5 Individual CMV-responsive human PBMCs (A Starte Biologics) in serum-free medium for 48 hours, alone or (i) (ii) 1 nM TGF-β at 1 ng / ml and T3A #5 (iii) 1 nM TGF-β and pembrolizumab at 1 ng / ml, (iv) 1 1 nM TGF-β, T3A #5 at ng / ml, and pembrol at 1 ng / ml Zumab, or (v) 1nM TGF-β and isotype control antibody (negative). In the presence of a control, 1.5 μg / ml CMV antigen (Astarte Biologics The cells were cultured in ) and the IFN-γ levels were measured by odor in the supernatant using the R&D ELISA kit. It was measured.

[0316] The results are shown in Figure 19. T3A #5, pembrolizumab, and T3A #5 and The combination of pembrolizumab and IFN-γ was observed to increase IFN-γ levels. T3A The combination of #5 and pembrolizumab showed the greatest increase in IFN-γ levels. Furthermore, the therapeutic efficacy of the ADCs disclosed herein is observed when combined with T-cell checkpoint inhibitors. In summary, it demonstrated that improvement is possible.

[0317] 7. Specific Embodiments This disclosure is illustrated by the following specific embodiments. 1. ALK operably linked to an antibody or antigen-binding fragment that binds to T cell surface molecules. Antibody-ALK5 inhibitor conjugate (ADC) containing 5 inhibitors. 2. IC55 inhibitors 50However, the AD described in Embodiment 1 is at least 20 nM. C. 3. ALK5 inhibitors include imidazole compounds, pyrazole compounds, or thiazole compounds. ADC according to Embodiment 1 or Embodiment 2, which is a compound of the system. 4. The ADC according to Embodiment 3, wherein the ALK5 inhibitor is an imidazole compound. 5. The ADC according to Embodiment 3, wherein the ALK5 inhibitor is a pyrazole compound. 6. The ADC according to Embodiment 3, wherein the ALK5 inhibitor is a thiazole compound. 7. ALK5 inhibitors include imidazole-benzodioxole compounds, or imidazole - The ADC described in Embodiment 3, which is a quinoxaline compound or an imidazole compound. . 8. The ALK5 inhibitor is an imidazole-benzodioxole compound, in Embodiment 7. The ADC described. 9. The ALK5 inhibitor is an imidazole-quinoxaline compound, as described in Embodiment 7. ADC. 10. ALK5 inhibitors are pyrazole-pyrrolo compounds, or pyrazole compounds. The ADC described in Embodiment 3. 11. ALK5 inhibitors include imidazole-benzodioxole compounds, imidazole-benzodioxole compounds, and imidazole-benzodioxole compounds. The implementation involves noxaline compounds, pyrazole-pyrrolo compounds, or thiazole compounds. ADC as described in Form 3. 12. ALK5 inhibitors are linked to an antibody or antigen-binding fragment via a linker. an ADC according to any one of Embodiments 1 to 11. 13. The ADC according to Embodiment 12, wherein the linker is a linker containing PEG. 14. The AD described in Embodiment 12 or Embodiment 13, wherein the linker is a polyvalent linker. C. 15. The linker is a non-cuttable linker, according to any one of embodiments 12 to 14. The ADC described. 16. Non-cleavable linker, N-maleimidomethylcyclohexane-1-carboxylate It is either maleimidocaproyl or mercaptoacetamidecaproyl linker. The ADC described in Embodiment 15. 17. The non-cleavable linker is N-maleimidomethylcyclohexane 1-carboxylate ADC as described in Embodiment 16, which is a tlinker. 18. The non-cuttable linker is a maleimidocaproyl linker, as described in Embodiment 16. The ADC on board. 19. The non-cleavable linker is a mercaptoacetamide caproyl linker. ADC as described in form 16. 20. The linker is a severable linker as described in any one of Embodiments 12 to 14. The ADC on board. 21. The cleavable linker is a dipeptide linker, a disulfide linker, or a hydra The ADC described in Embodiment 20 is a Zonlinker. 22. The ADC according to Embodiment 21, wherein the cleavable linker is a dipeptide linker. 23. The ADC according to Embodiment 21, wherein the severable linker is a disulfide linker. . 24. The ADC according to Embodiment 21, wherein the severable linker is a hydrazone linker. 25. The linker is a protease-sensitive valine-citrulline dipeptide linker. The ADC described in Embodiment 21. 26. The linker is a glutathione-sensitive disulfide linker, as described in Embodiment 21. The ADC on board. 27. The ADC according to Embodiment 21, wherein the linker is an acid-sensitive disulfide linker. . 28. ALK5 inhibitors can transmit antigens or antigen-binding properties via site-directed conjugation. An ADC according to any one of Embodiments 1 to 27, which conjugates into fragments. 29. ALK5 inhibitors contain one or more cysteine ​​molecules on an antibody or antigen-binding fragment. A as described in Embodiment 28, which is conjugated via a lysine or glutamine residue. DC. 30. ALK5 inhibitors can remove one or more cysteine ​​residues on antibody or antigen-binding fragments. An ADC according to Embodiment 29, which is conjugated via a base. 31. ALK5 inhibitors can destroy one or more lysine residues on an antibody or antigen-binding fragment. The ADC described in Embodiment 29, which is conjugated via [a certain method]. 32. ALK5 inhibitors can remove one or more glutamine residues on antibody or antigen-binding fragments. An ADC according to Embodiment 29, which is conjugated via a base. 33. ALK5 inhibitors react with one or more unnatural amino acids on an antibody or antigen-binding fragment. The ADC according to Embodiment 28, which is conjugated via an acid residue. 34.1 or more non-natural amino acid residues are p-acetylphenylalanine (pAc The ADC according to Embodiment 33, including F). 35.1 or more non-natural amino acid residues, p-azidomethyl-L-phenylalanine An ADC according to Embodiment 33, including a (pAMF). 36.1 or more non-natural amino acid residues, including selenocysteine ​​(Sec), ADC as described in the application method 33. 37. ALK5 inhibitors act via one or more glycans on an antibody or antigen-binding fragment. The ADC described in Embodiment 28, which is then conjugated. 38. An ADC according to Embodiment 37, wherein one or more glycans include fucose. 39.1 or more glycans, comprising 6-thioffocose, as described in Embodiment 37 DC. 40.1 or more glycans comprising galactose, as described in Embodiment 37 of ADC . 41. One or more glycans contain N-acetylgalactosamine (GalNAc) The ADC described in Embodiment 37. 42. One or more glycans containing N-acetylglucosamine (GlcNAc) The ADC described in Embodiment 37. 43.1 or more glycans containing sialic acid (SA), as described in Embodiment 37. DC. 44. Embodiments 28 to 4 in which the ALK5 inhibitor is conjugated via a linker. The ADC described in any one of item 3. 45. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 1 to 30. An ADC according to any one of Embodiments 1 to 44, which is within the range of an ADC. 46. ​​The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 1 to 20. An ADC according to any one of Embodiments 1 to 44, which is within the range of an ADC. 47. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 1 to 15. An ADC according to any one of Embodiments 1 to 44, which is within the range of an ADC. 48. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 2-12. An ADC according to any one of Embodiments 1 to 44, which is within the range of an ADC. 49. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 4-15. An ADC according to any one of Embodiments 1 to 44, which is within the range of an ADC. 50. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 6-12. An ADC according to any one of Embodiments 1 to 44, which is within the range of an ADC. 51. The average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is 2 to 8. The ADC described in any one of Embodiments 1 to 44, which is within the scope of the ADC. 52. The embodiment according to any one of Embodiments 1 to 51, wherein the antibody is a monoclonal antibody. ADC. 53. The ADC according to Embodiment 52, wherein the antibody is human or humanized. 54. The ADC according to Embodiment 53, wherein the antibody is human. 55. The ADC according to Embodiment 53, wherein the antibody is humanized. 56. The antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. An ADC according to any one of Embodiments 1 to 55. 57. The ADC according to Embodiment 56, wherein the antigen-binding fragment is Fab. 58. The ADC according to Embodiment 56, wherein the antigen-binding fragment is Fab'. 59. The ADC according to Embodiment 56, wherein the antigen-binding fragment is F(ab')2. 60. The ADC according to Embodiment 56, wherein the antigen-binding fragment is an Fv fragment. 61. Embodiment 56, in which the antigen-binding fragment is an antigen-binding fragment of a human or humanized antibody. ADC as described in any one of items 60 to 60. 62. The AD according to Embodiment 61, wherein the antigen-binding fragment is an antigen-binding fragment of a human antibody. C. 63. Embodiment 61, wherein the antigen-binding fragment is an antigen-binding fragment of a humanized antibody. DC. 64. An ADC according to any one of Embodiments 1 to 55, comprising an antibody. 65. An ADC according to any one of Embodiments 1 to 63, comprising an antigen-binding fragment. 66. Any one of Embodiments 1 to 65, wherein the T cell surface molecule is a human T cell surface molecule. The ADC described in section. 67.T cell surface molecules include CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD70, CD71, CD103, CD184, Tim3 , LAG3, CTLA4, or PD1 as described in any one of Embodiments 1 to 66 The ADC on board. 68. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD1. 69. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD2. 70. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD3. 71. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD4. 72. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD5. 73. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD6. 74. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD7. 75. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD8. 76. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD25. 77. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD28. 78. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD70. 79. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD71. 80. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD103. 81. The ADC according to Embodiment 67, wherein the T cell surface molecule is CD184. 82. The ADC according to Embodiment 67, wherein the T cell surface molecule is Tim3. 83. The ADC according to Embodiment 67, wherein the T cell surface molecule is LAG3. 84. The ADC according to Embodiment 67, wherein the T cell surface molecule is CTLA4. 85. The ADC according to Embodiment 67, wherein the T cell surface molecule is PD1. 86. The antibody or antigen-binding fragment is pembrolizumab or its antigen-binding fragment, Volumab or its antigen-binding fragment, semiprimab or its antigen-binding fragment, The ADC according to Embodiment 85, comprising dostallimab or an antigen-binding fragment thereof. 87. The antibody or antigen-binding fragment contains pembrolizumab or its antigen-binding fragment. , the ADC described in Embodiment 86. 88. The antibody or antigen-binding fragment contains nivolumab or its antigen-binding fragment. ADC as described in form 86. 89. The antibody or antigen-binding fragment contains semiprimab or its antigen-binding fragment. ADC as described in the application method 86. 90. The antibody or antigen-binding fragment contains dostallimab or its antigen-binding fragment. The ADC described in Embodiment 86. 91. T cell surface molecules that can be recycled through endosomes The ADC according to any one of Embodiments 1 to 66. 92. Embodiment 9, where the T cell surface molecule is CD2, CD5, CD7, or CD71. The ADC described in 1. 93. The ADC according to Embodiment 91, wherein the T cell surface molecule is CD5 or CD7. 94. The ADC according to Embodiment 92, wherein the T cell surface molecule is CD5. 95. The ADC according to Embodiment 92, wherein the T cell surface molecule is CD7. 96. The ADC according to Embodiment 91, wherein the T cell surface molecule is CD2. 97. The ADC according to Embodiment 91, wherein the T cell surface molecule is CD71. 98. Fc-dominants having one or more amino acid substitutions that reduce effector function. An ADC according to any one of embodiments 1 to 97, including the in. 99. 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 Includes K / L234R / L235R / E269K / D265S / K322E / E333K The ADC described in Embodiment 98. 100. An ADC according to Embodiment 99, wherein one or more substitutions include N297A. 101. An ADC according to Embodiment 99, wherein one or more substitutions include N297Q. 102. An ADC according to Embodiment 99, wherein one or more substitutions include N297G. 103. One or more substitutions include D265A / N297A, as described in Embodiment 99. ADC. 104. One or more substitutions include D265A / N297G, as described in Embodiment 99. ADC. 105. An ADC according to Embodiment 99, wherein one or more substitutions include L235E. 106. One or more substitutions include L234A / L235A, as described in Embodiment 99. ADC. 107. One or more substitutions include L234A / L235A / P329A, in implementation form The ADC described in condition 99. 108. One or more substitutions are L234D / L235E:L234R / L235R / The ADC according to Embodiment 99, including the E233K. 109. One or more substitutions are L234D / L235E / D265S:E233K / The ADC according to Embodiment 99, including L234R / L235R / D265S. 110. One or more substitutions are L234D / L235E / E269K:E233K / ADC according to Embodiment 99, including L234R / L235R / E269K. 111. One or more substitutions are L234D / L235E / K322A:E233K / ADC according to Embodiment 99, including L234R / L235R / K322A. 112. One or more substitutions are L234D / L235E / P329W:E233K / ADC according to Embodiment 99, including L234R / L235R / P329W. 113. One or more substitutions are L234D / L235E / E269K / D265S / K322A:E233K / L234R / L235R / E269K / D265S / K322 The ADC according to Embodiment 99, including A. 114. One or more substitutions are L234D / L235E / E269K / D265S / K322E / E333K:E233K / L234R / L235R / E269K / D265 The ADC according to embodiment 99, including S / K322E / E333K. 115. An ADC according to any one of Embodiments 1 to 114, and a pharmaceutically acceptable A pharmaceutical composition containing a carrier. 116. At least 30% of the ADC molecules in the pharmaceutical composition have a drug-antibody ratio (DAR) of 1 to 30. A pharmaceutical composition according to Embodiment 115, having ) 117. At least 30% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 20. The pharmaceutical composition described in application form 115. 118. At least 30% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 15. The pharmaceutical composition described in application form 115. 119. At least 30% of the ADC molecules in the pharmaceutical composition have 2 to 12 DARs. The pharmaceutical composition described in application form 115. 120. At least 30% of the ADC molecules in the pharmaceutical composition have a DAR of 4 to 15. The pharmaceutical composition described in application form 115. 121. At least 30% of the ADC molecules in the pharmaceutical composition have 6 to 12 DARs. The pharmaceutical composition described in application form 115. 122. At least 30% of the ADC molecules in the pharmaceutical composition have a DAR of 2 to 8. The pharmaceutical composition described in Form 115. 123. At least 40% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 30. The pharmaceutical composition described in application form 115. 124. At least 40% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 20. The pharmaceutical composition described in application form 115. 125. At least 40% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 15. The pharmaceutical composition described in application form 115. 126. At least 40% of the ADC molecules in the pharmaceutical composition have 2 to 12 DARs. The pharmaceutical composition described in application form 115. 127. At least 40% of the ADC molecules in the pharmaceutical composition have 4 to 15 DARs. The pharmaceutical composition described in application form 115. 128. At least 40% of the ADC molecules in the pharmaceutical composition have 6 to 12 DARs. The pharmaceutical composition described in application form 115. 129. At least 40% of the ADC molecules in the pharmaceutical composition have 2 to 8 DARs, The pharmaceutical composition described in Form 115. 130. At least 50% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 30. The pharmaceutical composition described in application form 115. 131. At least 50% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 20. The pharmaceutical composition described in application form 115. 132. At least 50% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 15. The pharmaceutical composition described in application form 115. 133. At least 50% of the ADC molecules in the pharmaceutical composition have 2 to 12 DARs. The pharmaceutical composition described in application form 115. 134. At least 50% of the ADC molecules in the pharmaceutical composition have a DAR of 4 to 15. The pharmaceutical composition described in application form 115. 135. At least 50% of the ADC molecules in the pharmaceutical composition have 6 to 12 DARs. The pharmaceutical composition described in application form 115. 136. At least 50% of the ADC molecules in the pharmaceutical composition have 2 to 8 DARs, The pharmaceutical composition described in Form 115. 137. At least 60% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 30. The pharmaceutical composition described in application form 115. 138. At least 60% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 20. The pharmaceutical composition described in application form 115. 139. At least 60% of the ADC molecules in the pharmaceutical composition have a DAR of 1 to 15. The pharmaceutical composition described in application form 115. 140. At least 60% of the ADC molecules in the pharmaceutical composition have 2 to 12 DARs. The pharmaceutical composition described in application form 115. 141. At least 60% of the ADC molecules in the pharmaceutical composition have 4 to 15 DARs. The pharmaceutical composition described in application form 115. 142. At least 60% of the ADC molecules in the pharmaceutical composition have 6 to 12 DARs. The pharmaceutical composition described in application form 115. 143. At least 60% of the ADC molecules in the pharmaceutical composition have 2 to 8 DARs, The pharmaceutical composition described in Form 115. 144. A method for treating cancer, comprising embodiments 1 to 114, for a subject requiring the treatment. The ADC described in any one of the paragraphs, or the ADC described in any one of the embodiments 115 to 143. A method comprising administering a pharmaceutical composition. 145. The method according to Embodiment 144, wherein the cancer is an immunogenic cancer. 146. The method according to Embodiment 145, wherein the cancer is a solid tumor expressing a tumor antigen. 147. Embodiment 1, in which the tumor antigen is gp100, Melan A, or MAGE A1. Method 46. 148. The method according to Embodiment 147, wherein the tumor antigen is gp100. 149. The method according to Embodiment 147, wherein the tumor antigen is melan A. 150. The method according to Embodiment 147, wherein the tumor antigen is MAGE A1. 151. The method according to Embodiment 144, wherein the cancer is a solid tumor including immune infiltration. 152. Any of embodiments 144 to 151 in which cancer is treatable by immunotherapy. The method described in item 1. 153. Immunotherapy includes cytokine therapy, adoptive T-cell therapy, and chimeric antigen receptor (CAR) therapy. Therapies, T-cell checkpoint inhibitor therapy, oncolytic virus therapy, dendritic cell vaccines Therapy, STING agonist therapy, TLR agonist therapy, or intratumor CpG therapy The method described in Embodiment 152. 154. Immunotherapy includes cytokine therapy, adoptive T-cell therapy, and chimeric antigen receptor (CAR) therapy. The method according to Embodiment 152, which is a therapy or T-cell checkpoint inhibitor therapy. 155. The method according to Embodiment 154, wherein the immunotherapy is cytokine therapy. 156. The method according to Embodiment 155, wherein the cytokine therapy is IL2 therapy. 157. The method according to Embodiment 155, wherein the cytokine therapy is IL12 therapy. 158. The method according to Embodiment 155, wherein the cytokine therapy is IFN-α therapy. 159. The method according to Embodiment 155, wherein the cytokine therapy is IFN-γ therapy. 160. The method according to Embodiment 154, wherein the immunotherapy is adoptive T-cell therapy. 161. The method according to embodiment 160, wherein adoptive T-cell therapy is autologous T-cell therapy. 162. The immunotherapy described in Embodiment 154 is chimeric antigen receptor (CAR) therapy. method. 163. The immunotherapy is T-cell checkpoint inhibitor therapy, as described in Embodiment 154. The method. 164. The method according to Embodiment 163, wherein the T-cell checkpoint inhibitor is an antibody. 165. T-cell checkpoint inhibitors inhibit PD1, PDL1, or CTLA4. The method according to Embodiment 154, Embodiment 163, or Embodiment 164, which is an agent. 166. The T cell checkpoint inhibitor is a PD1 inhibitor, as described in Embodiment 165. Method of loading. 167. The method according to embodiment 166, wherein the PD1 inhibitor is an antibody. 168. PD1 inhibitors include pembrolizumab, nivolumab, semiprimab, or dos The method according to embodiment 167, wherein tarlimab is used. 169. The method according to embodiment 168, wherein the PD1 inhibitor is pembrolizumab. 170. The method according to Embodiment 168, wherein the PD1 inhibitor is nivolumab. 171. The method according to Embodiment 168, wherein the PD1 inhibitor is semiprimab. 172. The method according to Embodiment 168, wherein the PD1 inhibitor is dostallimab. 173. Embodiment 165, in which the T cell checkpoint inhibitor is a PDL1 inhibitor. Method of description. 174. The method according to Embodiment 173, wherein the inhibitor of PDL1 is an antibody. 175. PDL1 inhibitors are atezolizumab, avelumab, or durvalumab. The method described in Embodiment 174. 176. The method according to Embodiment 175, wherein the inhibitor of PDL1 is atezolizumab. 177. The method according to Embodiment 175, wherein the inhibitor of PDL1 is avelumab. 178. The method according to Embodiment 175, wherein the inhibitor of PDL1 is durvalumab. 179. Embodiment 165, where the T cell checkpoint inhibitor is a CTLA4 inhibitor. Methods used. 180. The method according to Embodiment 179, wherein the inhibitor of CTLA4 is an antibody. 181. The method according to Embodiment 180, wherein the inhibitor of CTLA4 is ipilimumab. 182. T cell checkpoint inhibitors target TIGIT, as in Embodiment 163. Or the method described in 164. 183. T cell checkpoint inhibitors target LAG3, Embodiment 163 also This is the method described in 164. 184. T cell checkpoint inhibitors target OX40, Embodiment 163 also This is the method described in 164. 185. T cell checkpoint inhibitors target CD40, Embodiment 163 also This is the method described in 164. 186. T cell checkpoint inhibitors target VISTA, Embodiment 163 Or the method described in 164. 187. Cancers include lung cancer, liver cancer, urothelial carcinoma, kidney cancer, breast cancer, melanoma, pancreatic cancer, and nephroma. Embodiment 1 is transglioblastoma, myelodysplastic syndrome, prostate cancer, or colorectal cancer. The method described in any one of items 44 to 186. 188. Cancers include non-small cell lung cancer (NSCLC), liver cancer, urothelial carcinoma, kidney cancer, and breast cancer. The method according to Embodiment 187, which is a cyst or melanoma. 189. The method according to Embodiment 187, wherein the cancer is lung cancer. 190. The method according to Embodiment 189, wherein the cancer is NSCLC. 191. The method according to Embodiment 190, wherein NSCLC is an adenocarcinoma. 192. The method according to Embodiment 190, wherein NSCLC is squamous cell carcinoma. 193. The method according to Embodiment 190, wherein NSCLC is large cell carcinoma. 194. The method according to Embodiment 189, wherein the cancer is small cell lung cancer. 195. The method according to Embodiment 187, wherein the cancer is liver cancer. 196. The method according to embodiment 195, wherein the liver cancer is hepatocellular carcinoma. 197. The method according to Embodiment 187, wherein the cancer is urothelial carcinoma. 198. The method according to Embodiment 197, wherein the cancer is bladder cancer. 199. The method according to embodiment 197, wherein the cancer is urethral cancer. 200. The method according to Embodiment 197, wherein the cancer is ureteral cancer. 201. The method according to Embodiment 187, wherein the cancer is renal cancer. 202. The method according to Embodiment 201, wherein the renal cancer is renal cell carcinoma. 203. The method according to embodiment 201, wherein the renal cancer is urothelial carcinoma. 204. The method according to Embodiment 187, wherein the cancer is breast cancer. 205. The method according to Embodiment 187, wherein the cancer is melanoma. 206. The method according to Embodiment 187, wherein the cancer is pancreatic cancer. 207. The method according to Embodiment 187, wherein the cancer is glioblastoma. 208. The method according to Embodiment 187, wherein the cancer is myelodysplastic syndrome. 209. The method according to Embodiment 187, wherein the cancer is prostate cancer. 210. The method according to Embodiment 187, wherein the cancer is colorectal cancer. 211. The method according to embodiment 210, wherein the colorectal cancer is adenocarcinoma. 212. The method according to embodiment 210, wherein the colorectal cancer is a carcinoid tumor. 213. The method according to Embodiment 210, wherein the colorectal cancer is a gastrointestinal stromal tumor. 214. The method according to embodiment 210, wherein the colorectal cancer is colorectal lymphoma. 215. Cancer can be treated with ALK5 inhibitors, according to embodiments 144 to 214. The method described in either of the above terms. 216. Any of embodiments 144 to 215 in which the cancer is treatable by chemotherapy. The method described in item 1. 217. Embodiments 144 to 2 in which an ADC or pharmaceutical composition is administered as monotherapy. The method described in any one of paragraphs 16. 218. The ADC or pharmaceutical composition is the AD described in any one of Embodiments 1 to 114. This includes administering one or more drugs other than C (each referred to as the "second treatment agent") It is also administered as part of a good combination therapy regimen, one of embodiments 144 to 216. The method described in item 1. 219. When an ADC or pharmaceutical composition is used in combination with a standard care therapy or treatment regimen. The method according to Embodiment 218, which is administered. 220. The combination therapy includes administering at least one second therapeutic agent. The method according to embodiment 218 or 219. 221. The combination therapy regimen includes immunotherapy, and the immunotherapy is a checkpoint inhibitor therapy. Therapy, chimeric antigen receptor (CAR) therapy, adoptive T-cell therapy, oncolytic virus therapy, dendritic Cell vaccine therapy, STING agonist therapy, TLR agonist therapy, intratumor CpG therapy Any one of Embodiments 218 to 220, which may be a method or cytokine therapy. Methods used. 222. The combination therapy includes checkpoint inhibitor therapy, as in Embodiments 218 to 221. The method described in any one of the items. 223. Checkpoint inhibitor therapy, including T-cell checkpoint inhibitor therapy, The method described in the application method 222. 224. T-cell checkpoint inhibitor therapy, which includes an antibody or its antigen-binding fragment, The method according to Embodiment 223. 225. Checkpoint inhibitor therapy is used for PD1, PDL1, CTLA4, TIGIT, Embodiments targeting LAG3, OX40, CD40, VISTA, or a combination thereof. The method described in any one of paragraphs 222 to 224. 226. The checkpoint inhibitor therapy, as described in Embodiment 225, targets PD1. method. 227. The method according to Embodiment 226, wherein the second therapeutic agent is pembrolizumab. 228. The method according to Embodiment 226, wherein the second therapeutic agent is nivolumab. 229. The method according to Embodiment 226, wherein the second therapeutic agent is semiprimab. 230. The method according to Embodiment 226, wherein the second therapeutic agent is dostallimab. 231. Checkpoint inhibitor therapy targets PDL1, Embodiments 225 to 2 The method described in any one of paragraphs 30. 232. The method according to Embodiment 231, wherein the second therapeutic agent is atezolizumab. 233. The method according to Embodiment 231, wherein the second therapeutic agent is avelumab. 234. The method according to Embodiment 231, wherein the second therapeutic agent is durvalumab. 235. Checkpoint inhibitor therapy targets CTLA4, from Embodiment 225 The method described in any one of paragraphs 234. 236. The method according to Embodiment 235, wherein the second therapeutic agent is ipilimumab. 237. Checkpoint inhibitor therapy targets TIGIT, from Embodiment 225 The method described in any one of paragraphs 236. 238. The method according to Embodiment 237, wherein the second therapeutic agent is etigirimab. 239. The method according to Embodiment 237, wherein the second therapeutic agent is tiragolumab. 240. The method according to Embodiment 237, wherein the second therapeutic agent is AB154. 241. Checkpoint inhibitor therapy targets LAG3, Embodiments 225 to 2 The method described in any one of item 40. 242. The method according to Embodiment 241, wherein the second therapeutic agent is LAG525. 243. The method according to Embodiment 241, wherein the second therapeutic agent is Sym022. 244. The method according to Embodiment 241, wherein the second therapeutic agent is relatrimab. 245. The method according to Embodiment 241, wherein the second therapeutic agent is TSR-033. 246. Checkpoint inhibitor therapy targets OX40, Embodiments 225 to 2 The method described in any one of item 45. 247. The method according to Embodiment 246, wherein the second therapeutic agent is MEDI6469. 248. The method according to Embodiment 246, wherein the second therapeutic agent is PF-04518600. . 249. The method according to Embodiment 246, wherein the second therapeutic agent is BMS 986178. 250. Checkpoint inhibitor therapy targets CD40, Embodiments 225 to 2 The method described in any one of item 49. 251. The method according to Embodiment 250, wherein the second therapeutic agent is sericrelmab. 252. The method according to Embodiment 250, wherein the second therapeutic agent is CP-870,893. 253. The method according to Embodiment 250, wherein the second therapeutic agent is APX005M. 254. Checkpoint inhibitor therapy targets VISTA, from Embodiment 225 The method described in any one of item 253. 255. The method according to Embodiment 254, wherein the second therapeutic agent is HMBD-002. 256. Embodiments 218 to 25 in which the second therapeutic agent is a chimeric antigen receptor (CAR). The method described in any one of item 5. 257. Any one of Embodiments 218 to 256, wherein the combination therapy includes adoptive T cell therapy. Methods used. 258. The method according to Embodiment 257, wherein adoptive T-cell therapy is autologous T-cell therapy. 259. The combination therapy includes oncolytic virus therapy, as in any of embodiments 218 to 258. The method described in any one of the items. 260. The combination therapy includes any of Embodiments 218 to 259, including dendritic cell vaccine therapy. The method described in item 1. 261. The combination therapy includes STING agonist therapy, as in Embodiments 218 to 260. The method described in either of the above terms. 262. The combination therapy includes TLR agonist therapy, as in any of embodiments 218 to 261. The method described in item 1. 263. The combination therapy is described in any one of Embodiments 218 to 262, including chemotherapy. The method. 264. The second therapeutic agent is an antimetabolite, alkylating agent, anthracycline, antimicrotubule These are agents, platinum compounds, taxanes, topoisomerase inhibitors, or vinca alkaloids. The method according to embodiment 263. 265. The method according to Embodiment 264, wherein the second therapeutic agent is an antimetabolite. 266. The method according to Embodiment 265, wherein the antimetabolite is 5-fluorouracil. 267. The method according to Embodiment 265, wherein the antimetabolite is gemcitabine. 268. The method according to Embodiment 265, wherein the antimetabolite is methotrexate. 269. The method according to Embodiment 264, wherein the second therapeutic agent is an alkylating agent. 270. The method according to Embodiment 269, wherein the alkylating agent is cyclophosphamide. 271. The method according to Embodiment 269, wherein the alkylating agent is dacarbazine. 272. The method according to Embodiment 269, wherein the alkylating agent is mechloretamine. 273. The method according to Embodiment 269, wherein the alkylating agent is diazicone. 274. The method according to Embodiment 269, wherein the alkylating agent is temozolomide. 275. The method according to Embodiment 264, wherein the second therapeutic agent is an anthracycline. 276. The method according to embodiment 275, wherein the anthracycline is doxorubicin. 277. The method according to embodiment 275, wherein the anthracycline is epirubicin. 278. The method according to Embodiment 264, wherein the second therapeutic agent is an antimicrotubule agent. 279. The method according to Embodiment 278, wherein the antimicrotubule agent is vinblastine. 280. The method according to Embodiment 264, wherein the second therapeutic agent is a platinum compound. 281. The method according to Embodiment 280, wherein the platinum compound is cisplatin. 282. The method according to Embodiment 280, wherein the platinum compound is oxaliplatin. 283. The method according to Embodiment 264, wherein the second therapeutic agent is a taxane. 284. The method according to Embodiment 283, wherein the taxane is paclitaxel. 285. The method according to Embodiment 283, wherein the taxane is docetaxel. 286. The method according to Embodiment 264, wherein the second therapeutic agent is a topoisomerase inhibitor. . 287. The method according to Embodiment 286, wherein the topoisomerase inhibitor is etoposide. 288. The embodiment described in 286, wherein the topoisomerase inhibitor is mitoxantrone. method. 289. The method according to Embodiment 264, wherein the second therapeutic agent is a vinca alkaloid. 290. The method according to Embodiment 289, wherein the vinca alkaloid is vincristine. 291. The combination therapy includes any one of embodiments 218 to 290, including intratumor CpG therapy. The method described in section [section number]. 292. The second therapeutic agent is a cytokine, one of any one of embodiments 218 to 291. The method described in section [section number]. 293. The method according to Embodiment 292, wherein the cytokine is IL2. 294. The method according to Embodiment 292, wherein the cytokine is IL12. 295. The method according to Embodiment 292, wherein the cytokine is IFN-α. 296. The method according to Embodiment 292, wherein the cytokine is IFN-γ. 297. Any one of embodiments 218 to 296, including treating the subject with combination therapy. The method described in section [section number]. 298. Any of Embodiments 218 to 297, including administering the second therapeutic agent to the target. The method described in item 1.

[0318] Various specific embodiments are illustrated and described, but do not exceed the spirit and scope of this disclosure. It will become clear that various changes can occur without any intervention.

[0319] 8. Citation of References All publications, patents, patent applications, and other documents cited in this application are the respective publications, patents, patent applications, and other documents. Licenses, patent applications, or other documents are incorporated individually, by reference, for all purposes. To the same extent as indicated, the whole is incorporated herein by reference for all purposes. It is incorporated between the teachings of one or more references incorporated herein and in this disclosure. In the event of any discrepancy, the teachings herein are intended.

Claims

1. A pharmaceutical composition for treating cancer in combination with a checkpoint inhibitor, comprising an antibody-ALK5 inhibitor conjugate (ADC) consisting of an ALK5 inhibitor operably linked to an antibody or antigen-binding fragment that binds to a T cell surface molecule, wherein the cancer is a cancer other than malignant melanoma, and the ALK5 inhibitor has the following structure: A pharmaceutical composition having the following characteristics.

2. The aforementioned ALK5 inhibitor has the following structure: A pharmaceutical composition according to claim 1, having the following characteristics.

3. The ALK5 inhibitor has the following structure: A pharmaceutical composition according to claim 1, having the following characteristics.

4. The ALK5 inhibitor has the following structure: A pharmaceutical composition according to claim 1, having the following characteristics.

5. The ALK5 inhibitor has the following structure: A pharmaceutical composition according to claim 1, having the following characteristics.

6. The pharmaceutical composition according to claim 1 or 4, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a non-cleavable linker or a cleavable linker.

7. The pharmaceutical composition according to claim 6, wherein the ALK5 inhibitor linker is linked to the antibody or antigen-binding fragment via a protease-sensitive linker.

8. The pharmaceutical composition according to claim 7, wherein the ALK5 inhibitor is linked to the antibody or antigen-binding fragment via a valine-citrulline dipeptide linker.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the ALK5 inhibitor is conjugated via one or more cysteine ​​residues on the antibody or antigen-binding fragment, or via one or more lysine residues on the antibody or antigen-binding fragment, and the ALK5 inhibitor may also be conjugated via a linker.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the average number of ALK5 inhibitor molecules per antibody or antigen-binding fragment molecule is in the range of 2 to 8.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody.

12. The pharmaceutical composition according to claim 11, wherein the antibody is human or a humanized antibody.

13. The antigen-binding fragment is Fab, Fab', F(ab') ２ The pharmaceutical composition according to any one of claims 1 to 12, wherein the composition is either a fragment of Fv or an Fv fragment.

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

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the T cell surface molecule is CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD25, CD28, CD70, CD71, CD103, CD184, Tim3, LAG3, CTLA4, or PD1.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the T cell surface molecule is a T cell surface molecule that can be recycled through endosomes.

17. The pharmaceutical composition according to claim 16, wherein the T cell surface molecule is CD5 or CD7.

18. The pharmaceutical composition according to claim 16, wherein the T cell surface molecule is CD5.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the checkpoint inhibitor is an antibody or an antibody-conjugated fragment thereof.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the checkpoint inhibitor targets PD1, PDL1, CTLA4, TIGIT, LAG3, OX40, CD40, VISTA, or a combination thereof.

21. The aforementioned checkpoint inhibitors (a) The drug targets PD1, and the checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, or dostallimab; (b) Targeting PDL1, wherein the checkpoint inhibitor is atezolizumab, avelumab, or durvalumab, (c) Targeting CTLA4, the checkpoint inhibitor is ipilimumab, (d) Targeting TIGIT, wherein the checkpoint inhibitor is etigilimab, tiragolumab, or AB154, (e) Targeting LAG3, the checkpoint inhibitor is LAG525, Sym022, relatlimab, or TSR-033. (f) Targeting OX40, the checkpoint inhibitor is MEDI6469, PF-04518600, or BMS 986178, (g) Targeting D40, the checkpoint inhibitor is selicrelumab, CP-870, 893, or APX005M, or (h) The pharmaceutical composition according to claim 20, wherein the checkpoint inhibitor targets VISTA and is HMBD-002.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the checkpoint inhibitor targets PD1.

23. The pharmaceutical composition according to any one of claims 1 to 21, wherein the checkpoint inhibitor targets PDL1.

24. The pharmaceutical composition according to any one of claims 1 to 23, wherein the checkpoint inhibitor is pembrolizumab, nivolumab, semiprimab, or dostallimab.

25. The pharmaceutical composition according to any one of claims 1 to 23, wherein the checkpoint inhibitor is atezolizumab, avelumab, or durvalumab.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is colorectal cancer.

27. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is lung cancer.

28. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is liver cancer.

29. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is urothelial carcinoma.

30. The pharmaceutical composition according to claim 29, wherein the urothelial carcinoma is bladder cancer.

31. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is renal cancer.

32. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is breast cancer.

33. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is pancreatic cancer.

34. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is glioblastoma.

35. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is myelodysplastic syndrome.

36. The pharmaceutical composition according to any one of claims 1 to 25, wherein the cancer is prostate cancer.

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