5H-PYRROLO[3,2-d]PYRIMIDINE-2,4-DIAMINO COMPOUNDS AND ANTIBODY CONJUGATES THEREOF

Abstract

To provide compounds useful in methods of treatment and prevention of cell proliferation and cancer, methods of detection of cell proliferation and cancer, and methods of diagnosis of cell proliferation and cancer, and also useful in methods of treatment, prevention, detection and diagnosis of inflammatory diseases or conditions.SOLUTION: The present disclosure provides 5H-pyrrolo[3,2-d]pyrimidine-2,4-diamino compounds represented by the formula in the figure.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 859,638, filed on June 10, 2019, the content of which is incorporated herein by reference in its entirety.

[0002]

[0001] Provided herein are 5H - pyrrolo[3,2 - d]pyrimidine - 2,4 - diamino compounds and / or their antibody conjugates, pharmaceutical compositions comprising the compounds and / or conjugates, methods of making the compounds and / or conjugates, and methods of using the compounds, conjugates, and compositions for therapy. The compounds, conjugates, and compositions are useful in methods for treating and preventing cell proliferation and cancer, methods for detecting cell proliferation and cancer, and methods for diagnosing cell proliferation and cancer. The compounds, conjugates, and compositions are also useful in methods for treating, preventing, detecting, and diagnosing inflammatory diseases or conditions.

Background Art

[0003]

[0002] The innate immune system recognizes structurally conserved pathogen - associated molecular patterns via Toll - like receptors (TLRs), which are normally expressed in immune cells such as macrophages and dendritic cells. It is recognized. Activation of TLRs induces innate immune (rapid, non-specific) and / or adaptive immune (slower, more specific) responses such as induction of cytokines and / or co-stimulation of phagocytes and / or activation of T cell responses. Among TLRs, TLR3, 7, 8, and 9 are expressed in intracellular endosomes, and the other TLRs (TLR1, 2, 4, 5, 6, 10, and 11) are localized in the plasma membrane. Each TLR induces a cell response specific to pathogens through various uses of intracellular adapter proteins. TLR7 is an intracellular receptor expressed in the endosomal membrane and is closely related to TLR8. TLR7 recognizes nucleosides and nucleotides derived from intracellular pathogens. Activation of TLR7 can induce type I interferons and an inflammatory response. Saitoh, S-I et al., Nature Communications 2017,8, Article number:1592.

[0004]

[0003] Malignant cells utilize the natural immune regulatory function of TLRs to promote their survival, invasion, and avoidance of the anti-tumor immune response. Current research has demonstrated the context-specific role of TLR activation in various malignancies, where in certain cases, disease progression is promoted while in other cases, cancer growth is restricted. Braunstein M. J. et al., Target Oncol.2018,13(5),583-598.

[0005]

[0004] Some TLR agonists have been found to induce antitumor activity by indirectly activating the tolerant host immune system to destroy cancer cells. When TLR7 agonists such as imiquimod, loxoribine, CL264 (a 9-benzyl-8-hydroxyadenine derivative containing glycine in the benzyl group), ssRNA40, R848, and SM-276001 are used alone or as a vaccine adjuvant, a strong immune response leading to antitumor therapeutic effects is induced in some mouse models. Injection of TLR7 agonists reduces tumor progression and modulates systemic and intratumoral immune responses in colorectal cancer, renal cancer, and breast cancer. Antitumor effects associated with TLR7 stimulation have been demonstrated in human skin cancer and cervical intraepithelial neoplasia. Dajon, M. et al., Oncoimmunology. 2015, 4(3), e991615.

Summary of the Invention

Problems to be Solved by the Invention

[0006]

[0005] Targeting TLR7 can provide new treatment options for both anti-inflammatory therapy and / or anti-cancer therapy. There is a need in the art for new therapies for inflammatory diseases and / or immunomodulatory diseases, particularly cancer. Antibody conjugates to TLR7 agonists can be used to deliver a therapeutic or diagnostic payload portion to target cells expressing tumor antigens for the treatment and / or diagnosis of such diseases.

Means for Solving the Problems

[0007]

[0006] This specification provides 5H-pyrrolo[3,2-d]pyrimidine-2,4-diamino compounds of formula (I-P), formula (I), and their sub-formulas, compositions containing the compounds, methods for manufacturing the compounds, and methods for using the compounds, conjugates, and compositions for the treatment of cell proliferation and / or cancer and / or inflammation. The conjugates are useful in methods for treating and preventing cell proliferation and cancer, methods for detecting cell proliferation and cancer, and methods for diagnosing cell proliferation and cancer. The conjugates are useful in methods for treating and preventing inflammatory diseases and conditions.

[0008]

[0007] In one aspect, formula (I)

[0009]

Chemical formula

[0010]

[0008] This specification also provides antibody conjugates containing residues of compounds of formula (I-P), formula (I), and their sub-formulas. In some or any embodiments, the conjugate is of formula (V)

[0011]

Chemical formula

[0012]

[0009] In another aspect, a composition comprising a compound of formula (I-P), (I), (II) or (III) or an embodiment thereof or an antibody conjugate comprising a residue of a compound of formula (I-P), formula (I) and its sub-formulas and embodiments is provided. In some or any embodiments, the conjugate is of formula (V)

[0013]

Chemical formula

[0014]

[0010] In another aspect, provided herein is formula (I-P), formula (I), (II) or (III) 、or a compound of an embodiment thereof, or a method of using an antibody-drug conjugate described herein is provided. In some embodiments, the method is a method of delivering one or more payload moieties to a target cell or tissue. In some embodiments, the method is a therapeutic method. In some embodiments, the method is a diagnostic method. In some embodiments, the method is an analytical method. In some embodiments, the compound and / or antibody-drug conjugate is used to treat a disease or condition. In some aspects, the disease or condition is selected from cancer and / or an inflammatory disease or condition.

[0015]

[0011] Also provided herein is the use of the compounds described herein and their antibody conjugates for the treatment of cancer and / or an inflammatory disease or condition.

[0016]

[0012] In a further aspect, herein is provided Formula (IV):

[0017]

Chemical Formula

Brief Description of the Drawings

[0018]

Figure 1

[0013] A graph showing in vitro data demonstrating the ability of compound 10 to stimulate the activation of several immune cell types - monocytes (Figure 1A), B cells (Figure 1B), cDCs (Figure 1C), and pDCs (Figure 1D) in human PBMC (peripheral blood mononuclear cells).

Figure 2

[0014] A graph showing in vitro data demonstrating the ability of compound 10 to stimulate the activation of several immune cell types - monocytes (Figure 2A), B cells (Figure 2B), and cDCs (Figure 2C) from cynomolgus (cyno) PBMC.

Figure 3

[0015] Graph showing in vitro data demonstrating the ability of Compound 10 to stimulate the activation of several immune cell types - monocytes (Figure 3A), macrophages (Figure 3B), cDCs (Figure 3C), and pDCs (Figure 3D) from mouse splenocytes.

Figure 4

[0016] Graph showing in vitro data demonstrating the ability of Compound 10 to produce cytokines - IL-6 (Figure 4A), MCP-1 (Figure 4B), and IL1Ra (Figure 4C) from human PBMCs.

Figure 5

[0017] Graph showing in vitro data demonstrating the ability of Compound 10 to produce cytokines - IL-6 (Figure 5A) and MCP-1 (Figure 5B) from cynomolgus monkey PBMCs.

Figure 6

[0018] Graph showing in vitro data demonstrating the ability of Compound 10 to produce cytokines - IL-6 (Figure 6A), MCP-1 (Figure 6B), TNFa (Figure 6C), and IP-10 (Figure 6D) from mouse splenocytes.

Figure 7

[0019] Graph showing in vivo data regarding the antitumor activity of a specific compound in mice bearing established MC38-hFolRα tumors. Figure 7A shows the dose-related minimal body weight loss (less than 10% before administration). The antitumor effect of the treatment of Compound 2 against MC38-hFolRα regarding tumor growth is shown in Figure 7B.

Mode for Carrying Out the Invention

[0019]

[0020] This specification describes Toll-like receptor 7 (TLR7) agonists and their antibody conjugates for the treatment of cancer and / or inflammatory conditions. In some examples, the compounds described herein are selective for TLR7 and do not affect TLR8.

[0020] 1. Definitions

[0021] Unless otherwise defined, all technical, notation, and other scientific and technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or to facilitate reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a departure from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood, and are widely used by those of ordinary skill in the art using conventional methodologies such as, for example, the widely used molecular cloning methodologies described in Green & Sambrook., Molecular Cloning: A Laboratory Manual 4 th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Son. Procedures, including the use of commercially available kits and reagents, are generally carried out according to the protocols and conditions specified by the manufacturer, unless otherwise noted.

[0021]

[0022] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0022]

[0023] The term "about" indicates a range of values above and below the stated value, including those values. In certain embodiments, the term "about" indicates ±10%, ±5%, or ±1% of the specified value. In certain embodiments, the term "about" indicates ± one standard deviation of the value from the specified value. In certain embodiments, for example, for a logarithmic scale (e.g., pH), the term "about" indicates ±0.3, ±0.2, or ±0.1 of the specified value.

[0023]

[0024] The term "immunoglobulin" generally refers to a class of structurally related proteins that contain two pairs of polypeptide chains: one pair of light (L ) chains and one pair of heavy (H) chains. In "intact immunoglobulins", all four of these chains are interconnected by disulfide bonds . The structure of immunoglobulins is well-characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically contains a heavy chain variable region (V or VH) and a heavy chain constant region (C H or CH). The heavy chain constant region typically contains three domains abbreviated as C H (or CH1), C H1 (or CH2), and C H2 (or CH3). Each light chain typically contains a light chain variable region (V H3 or VL) and a light chain constant region. The light chain constant region typically contains one domain abbreviated as C L or CL.

[0024]

[0025] The term "antibody" is used herein in its broadest sense. Antibodies include intact antibodies (e.g., intact immunoglobulins) and antibody fragments (e.g., antigen-binding fragments of antibodies). Antibodies contain at least one antigen-binding domain. An example of an antigen-binding domain is an antigen-binding domain formed by a V H -V L dimer.

[0025]

[0026] The V H region and the V L region can be further subdivided into regions of hypervariability (the "hypervariable region (HVR)"; also called the "complementary determining region" (CDR)) that are dispersed among more conserved regions. The more conserved regions are called framework regions (FR). Each V H and V​L Generally includes three CDRs and four FRs arranged in the following order (from the N-terminus to the C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and affect the antigen specificity and binding affinity of the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD. This document is incorporated by reference in its entirety.

[0026]

[0027] Light chains from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domain.

[0027]

[0028] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes) IgA, IgD, IgE, IgG, and IgM. These classes are also denoted as α, δ, ε, γ, and μ, respectively. The IgG class and IgA class are further subdivided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0028]

[0029] The amino acid sequence boundaries of the CDRs are known to those skilled in the art, Kabat et al., supra (the "Kab at" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCall Determined using any of several known numbering schemes, including those described by Um et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme). Each of these references is incorporated herein by reference in its entirety. It can be determined using any of these known numbering schemes. Each of these references is incorporated herein by reference in its entirety.

[0029]

[0030] The CDRs can be obtained, for example, from www.bioinf.org.uk / abs / abnum / and assigned using antibody numbering software such as Abnum described by Abhinandan and Martin, Immunology, 2008, 45:3832-3839. This reference is incorporated herein by reference in its entirety.

[0030]

[0031] The "EU numbering scheme" is generally used when referring to residues of the constant region of the antibody heavy chain (e.g., as reported by Kabat et al. , as described above). Unless otherwise indicated, the EU numbering scheme is used to refer to the residues of the antibody heavy chain constant region described herein.

[0031]

[0032] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fv fragments, Fab fragments, F(ab') 2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0032]

[0033] An "Fv" fragment includes a dimer in which one heavy chain variable domain and one light chain variable domain are non-covalently linked.

[0033]

[0034] The "Fab" fragment includes the constant domain of the light chain and the first constant domain (C H1 ) of the heavy chain in addition to the variable domains of the heavy and light chains. The Fab fragment can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody.

[0034]

[0035] The "F(ab')2" fragment contains two Fab' fragments joined by a disulfide bond near the hinge region . The F(ab')2 fragment can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab') fragment can be dissociated, for example, by treatment with β-mercaptoethanol.

[0035]

[0036] The "single-chain Fv" or "sFv" or "scFv" antibody fragment contains the V H domain and the V L domain in a single polypeptide chain. V H and V L are generally linked by a peptide linker. See Pluckthun A. (1994). Antibodies from Escherichia coli. Rosenberg M. & Moore G.P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, which is incorporated herein by reference in its entirety.

[0036]

[0037] The "scFv-Fc" fragment contains an scFv attached to the Fc domain. For example, the Fc domain may be attached to the C-terminus of the scFv. The Fc domain depends on the orientation of the variable domains of the scFv (i.e., V H -V L or V L -V H ), and V H or V Lmay subsequently exist. Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG1 Fc domain. can be used. In some cases, the Fc domain comprises an IgG1 Fc domain.

[0037]

[0038] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies is substantially similar and comprises antibodies that bind to the same epitope, excluding variants that may typically arise during the production of monoclonal antibodies. Such variants generally exist in only trace amounts. Monoclonal antibodies are typically obtained by a process that involves selecting a single antibody from a plurality of antibodies. For example, the selection process may be to select a unique clone from a pool of multiple clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further modified, for example, to improve its affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0038]

[0039] The term "chimeric antibody" refers to an antibody in which portions of the heavy and / or light chains are derived from a particular source or species, while the remaining heavy and / or light chains are derived from a different source or species.

[0039]

[0040] The "humanized" form of a non-human antibody is a chimeric antibody that contains a minimal sequence derived from a non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which the residue(s) of one or more CDRs have been replaced by the residue(s) of one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues of the donor antibody. Also, a humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596. Each of these references is incorporated herein by reference in its entirety.

[0040]

[0041] A "human antibody" corresponds to the amino acid sequence of an antibody produced by a human or human cell, or has an amino acid sequence derived from a non-human source in which a human antibody repertoire or human antibody coding sequence (e.g., obtained from a human source or designed de novo) is used. Human antibodies specifically exclude humanized antibodies.

[0041]

[0042] An "isolated antibody" is one that has been separated and / or recovered from the components of its natural environment. Components of the natural environment can include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, an isolated antibody is, for example, by using a spinning cup sequenator and is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence. In some embodiments, the isolated antibody is purified to greater homogeneity than by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and is detected by Coomassie blue staining or silver staining. The isolated antibody is free of at least one component of the antibody's natural environment and thus includes an in situ antibody within a recombinant cell. In some aspects, the isolated antibody is prepared by at least one purification step. Since it is absent, it includes an in situ antibody within a recombinant cell. In some embodiments, the isolated antibody is prepared by at least one purification step.

[0042]

[0043] In some embodiments, the isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, the isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight. In some embodiments, the isolated antibody is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by volume.

[0043]

[0044] "Affinity" refers to the overall strength of non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can be represented by the dissociation constant (K D ). Affinity can be measured by conventional methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR ) technology such as a Biacore® instrument. In some embodiments, the affinity is determined at 25°C.

[0044]

[0045] With respect to the binding of an antibody to a target molecule, the terms "specific binding", "specifically binds to", "specific for", "selectively binds to", and "selective for" with respect to a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean that the binding is a measurable binding that is distinct from non-specific or non-selective interactions. Specific binding can be measured, for example, by determining the binding of a molecule as compared to the binding of a control molecule. Specific binding can also be determined by competition with a control molecule that mimics the antibody binding site on the target. In that case, if the binding of the antibody to the target is competitively inhibited by the control molecule, it is shown to be specific binding.

[0045]

[0046] An "affinity matured" antibody has one or more changes in one or more CDRs or FRs that result in an improvement in the affinity of the antibody for its antigen as compared to the parent antibody that does not possess the change. In one embodiment, the affinity matured antibody has a nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies can be produced using a variety of methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, which is incorporated herein by reference in its entirety) describe affinity maturation by V H and V L domain shuffling. Random mutagenesis of CDR residues and / or framework residues is described, for example, in Barbas et al. (Proc. Nat. Acad. Sci. U.S.A., 1994, 91:3809-3813); Schier et al., Gene, 1995, 169:147-155; Yelton et al., J. Immunol., 1995, 155:1994-2004; Jackson et al., J. Immunol., 1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992, 226:889-896. Each of these references is incorporated herein by reference in its entirety.

[0046]

[0047] The term "amino acid" refers to the 20 common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V), as well as the rare pyrrolysine and selenocysteine. Natural amino acids also include citrulline. Natural coded amino acids include 22 naturally occurring post-translational variants of amino acids such as prenylated amino acids, isoprenylated amino acids, myrisoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids. The term "amino acid" includes non-naturally occurring (or unnatural) or synthetic α, β, γ or δ amino acids, including but not limited to the amino acids found in proteins, namely glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine, and histidine. In certain embodiments, the amino acid is in the L-form. Alternatively, the amino acid may be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolynyl, phenylalaninyl, tryptophanyl, methioninyl, glycyl, seryl, threonyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaryl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolynyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycyl, β-seryl, β-threonyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaryl, β-lysinyl, β-argininyl, or β-histidinyl. Unnatural amino acids are not proteinogenic amino acids or their post-translational modified variants. In particular, the term "unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolidine or selenocysteine, or a post-translational modified variant thereof.

[0047]

[0048] The term "conjugate" or "antibody conjugate" refers to an antibody linked to one or more payload moieties. The antibody may be any of the antibodies described herein. The payload may be any of the payloads described herein. The antibody may be directly linked to the payload via a covalent bond, or the antibody may be indirectly linked to the payload via a linker. Typically, the linker is covalently bound to the antibody and also covalently bound to the payload. The term "antibody-drug conjugate" or "ADC" refers to a conjugate in which at least one payload is a therapeutic moiety such as a drug.

[0048]

[0049] The "pAMF" mutation refers to a variant of the phenylalanine residue, i.e., para-azidomethyl-L-phenylalanine, added or substituted to a polypeptide.

[0049]

[0050] The term "payload" refers to a molecular moiety that can be conjugated to an antibody. In certain embodiments, the payload is selected from the group consisting of a therapeutic moiety and / or a labeling moiety described herein.

[0050]

[0051] The term "linker" refers to a molecular moiety capable of forming at least two covalent bonds. Typically, a linker is capable of forming at least one covalent bond to an antibody and at least another covalent bond to a payload. In certain embodiments, the linker can form more than one covalent bond to an antibody. In certain embodiments, the linker may form more than one covalent bond to a payload or form covalent bonds with more than one payload. The remaining structure of the linker after forming a bond with the antibody or payload or both, i.e., the residues of the linker after one or more covalent bonds are formed, may still be referred to herein as the "linker". The term "linker precursor" refers to a linker having one or more reactive groups capable of forming a covalent bond with an antibody or payload or both. In some embodiments, the linker is a cleavable linker. For example, the cleavable linker may be a linker released by a biodegradation function, which may or may not be genetically engineered. In some embodiments, the linker is a non-cleavable linker. For example, the non-cleavable linker may be a linker released upon degradation of the antibody. ー. For example, the cleavable linker may be a linker released by a biodegradation function, which may or may not be genetically engineered. In some embodiments, the linker is a non-cleavable linker. For example, the non-cleavable linker may be a linker released upon degradation of the antibody.

[0051]

[0052] When referring to the compounds provided herein, the following terms have the following meanings unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Where there are multiple definitions of a term in this specification, this section shall prevail unless otherwise specified.

[0052]

[0053] As used herein, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon, unless otherwise specified. In certain embodiments, the alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, the alkyl group contains 1 to 10 carbon atoms, i.e., C1-C 10It is alkyl. In certain embodiments, the alkyl group includes a saturated straight-chain or branched hydrocarbon having 1 to 6 carbon atoms, that is, C1-C6 alkyl or lower alkyl. This term includes both substituted and unsubstituted moieties. This term includes both substituted alkyl groups and unsubstituted alkyl groups, including, for example, halogenated alkyl groups. In some or any embodiments, the alkyl is unsubstituted. In some or any embodiments, the alkyl is substituted. In certain embodiments, the alkyl group is a fluorinated alkyl group. Non-limiting examples of moieties where the alkyl group can be substituted are known to those skilled in the art and are taught, for example, in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, which is incorporated herein by reference, and are selected from the group consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected or protected as necessary. In certain embodiments, the alkyl group is selected from the group consisting of methyl, CF3, CCl3, CFCl2, CF2Cl, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0053]

[0054] As used herein, unless otherwise specified, the term "alkylene" refers to a divalent alkyl group as defined herein. In some or any embodiments, the alkylene is unsubstituted.

[0054]

[0055] "Alkenyl", in certain embodiments, refers to an olefinically unsaturated hydrocarbon group having up to about 11 carbon atoms or 2 to 6 carbon atoms which may be linear or branched and having at least 1 or 1 to 2 alkenyl unsaturation sites.

[0055]

[0056] "Alkenylene" refers to a divalent alkenyl as defined herein. Lower alkenylene is C2-C6-alkenylene.

[0056]

[0057] "Alkynyl", in certain embodiments, refers to an acetylenically unsaturated hydrocarbon group having up to about 11 carbon atoms or 2 to 6 carbon atoms which may be linear or branched and having at least 1 or 1 to 2 alkynyl unsaturation sites. Non-limiting examples of alkynyl groups include an acetylene group, ethynyl (-C≡CH), propargyl (- CH2C≡CH), and the like.

[0057]

[0058] "Alkynylene" refers to a divalent alkynyl as defined herein. Lower alkynylene is C2-C6-alkynylene.

[0058]

[0059] As used herein, unless otherwise specified, the term "aryl" refers to phenyl, biphenyl, or naphthyl. This term includes both substituted and unsubstituted moieties. An aryl group may be substituted with one or more moieties selected from the group consisting of, but not limited to, halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected or protected as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, where the aryl of the arylamino substituent and the aryloxy substituent is further unsubstituted.

[0059]

[0060] As used herein, unless otherwise specified, the term "arylene" refers to a divalent aryl group as defined herein.

[0060]

[0061] "Alkarylene" refers to an arylene group as defined herein, where the aryl ring is substituted with one or two alkyl groups. "Substituted alkarylene" refers to an alkarylene as defined herein, where the arylene group is further substituted as defined for aryl.

[0061]

[0062] "Aralkylen" refers to a -CH2-arylene-, -arylene-CH2-, or -CH2-arylene-CH2- group, where arylene is as defined herein. "Substituted aralkylen" refers to an aralkylen as defined herein, where the aralkylen group is substituted as defined for aryl.

[0062]

[0063] "Alkoxy" and "alkoxyl" refer to an -OR'' group where R'' is alkyl or cycloalkyl. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0063]

[0064] "Alkoxycarbonyl" refers to a -C(O)-alkoxy radical where alkoxy is as defined herein.

[0064]

[0065] "Amino" refers to the radical -NH2.

[0065]

[0066] As used herein, unless otherwise specified, the term "alkylamino" refers to an -NHR'' group where R'' is C 1~10 alkyl as defined herein. In some or any embodiments, alkylamino is C 1~6 alkylamino.

[0066]

[0067] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon. In certain embodiments, the cycloalkyl group can be saturated, and / or bridged, and / or unbridged, and / or fused bicyclic groups. In certain embodiments, the cycloalkyl group contains from 3 to 10 carbon atoms, i.e., C3 - C 10 cycloalkyl. In some embodiments, cycloalkyl is from 3 to 15 (C 3~15 ) or from 3 to 10 (C 3~10 ) or from 3 to 7 (C 3~7) has carbon atoms. In certain embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl, or adamantyl. In some or any embodiments, the cycloalkyl is substituted with 1, 2, or 3 groups independently selected from halogen (fluoro, chloro, bromo, or iodo), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.

[0067]

[0068] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group as defined herein. Lower cycloalkylene refers to C3-C6-cycloalkylene.

[0068]

[0069] As used herein, unless otherwise specified, the term "dialkylamino" refers to an -NR"R" group where each R" is independently C 1~10 alkyl as defined herein. In some or any embodiments, the dialkylamino is di-C 1~6 alkylamino.

[0069]

[0070] "Carboxyl" or "carboxy" refers to the -C(O)OH radical.

[0070]

[0071] As used herein, "fused bicyclic aryl" is naphthyl.

[0071]

[0072] As used herein, "lower heteroalkylene" refers to a lower alkylene group in which 1, 2, or 3 carbon atoms are replaced with heteroatoms independently selected from N, O, and S(O) 0~2 and.

[0072]

[0073] The terms "heterocyclyl" and "heterocyclic" refer to monocyclic non-aromatic ring systems and / or polycyclic ring systems containing at least one non-aromatic ring, where one or more of the non-aromatic ring atoms are heteroatoms independently selected from O, S, and N, the remaining ring atoms of the non-aromatic ring are carbon atoms, any aromatic ring atoms are optionally heteroatoms independently selected from O, S, and N, and the remaining ring atoms of the non-aromatic ring are carbon atoms. In certain embodiments, a heterocyclyl or heterocyclic group has 3 to 20, 3 to 15, 3 to 10, 3 to 8, 4 to 7, 4 to 11, or 5 to 6 ring atoms. The heterocyclyl group is attached to the remainder of the molecule through the non-aromatic ring. In certain embodiments, the heterocyclyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused ring system or a bridged ring system, the nitrogen or sulfur atoms may optionally be oxidized, the nitrogen atoms may optionally be quaternized, some of the rings may be partially or fully saturated, or may be aromatic. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom of its non-aromatic ring to form a stable compound. Heterocycloalkyl refers to a heterocycle that is a monocyclic or polycyclic non-aromatic ring system. In some or any embodiments, the heterocycloalkyl is a monovalent, monocyclic or polycyclic, fully saturated ring system. Such heterocyclic and / or heterocycloalkyl radicals include 2,5-diazabicyclo[2.2.2]octanyl, 3,9-diazabicyclo[3.3.2]decanyl), azepinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, β-carbolinyl, chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl , such as dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiomorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl, but not limited thereto. In certain embodiments, the heterocyclic ring may be optionally substituted as described herein. In some or any embodiments, the heterocyclic ring and heterocycloalkyl are substituted with 1, 2, or 3 groups independently selected from halogen (fluoro, chloro, bromo, or iodo), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, the heterocycloalkyl group may contain 1, 2, 3, or 4 heteroatoms. One of ordinary skill in the art will recognize that a 4-membered heterocycloalkyl may generally contain 1 or 2 heteroatoms, a 5- to 6-membered heterocycloalkyl may generally contain 1, 2, or 3 heteroatoms, and a 7- to 10-membered heterocycloalkyl may generally contain 1, 2, 3, or 4 heteroatoms.

[0073]

[0074] "Heterocycloalkylene" refers to a divalent heterocycloalkyl as defined herein.

[0074]

[0075] "N-linked heterocycloalkyl" or "N-linked heterocyclyl" refers to a heterocycloalkyl as defined above that contains at least one nitrogen, and the heterocycloalkyl is attached to the main structure through a nitrogen atom in the non-aromatic ring. In some or any embodiments, the N-linked heterocycloalkyl and / or N-linked heterocyclyl is fully saturated.

[0075]

[0076] The term "heteroaryl" refers to a monovalent monocyclic aromatic group and / or polycyclic aromatic group, where at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N within the ring. Each ring of the heteroaryl group can contain one or two O atoms, one or two S atoms, and / or one to four N atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, heteroaryl has 5 to 20, 5 to 15, or 5 to 10 ring atoms. Heteroaryl may be attached to the rest of the molecule via a nitrogen atom or a carbon atom. In some embodiments, monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, imidazolyl, triazolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl, and triazolyl. Bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thienopyridyl. It is not possible. Examples of the tricyclic heteroaryl group include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In certain embodiments, the heteroaryl may optionally be substituted as described herein. "Substituted heteroaryl" is heteroaryl substituted as defined for aryl.

[0076]

[0077] The term "heteroarylene" refers to a divalent heteroaryl group as defined herein. "Substituted heteroarylene" is heteroarylene substituted as defined for aryl.

[0077]

[0078] "Partially saturated heteroaryl" refers to a polycyclic (e.g., bicyclic, tricyclic) fused ring system containing at least one non-aromatic ring and at least one aromatic ring, where one or more of the non-aromatic ring atoms and / or one or more of the aromatic ring atoms are independently heteroatoms selected from O, S, and N, and the remaining ring atoms are carbon atoms. The partially saturated heteroaryl group is attached to the rest of the molecule via the aromatic ring. In certain embodiments, the partially saturated heteroaryl group has 6 to 20, 6 to 15, 6 to 10, 6 to 8, or 8 to 11 ring atoms. In certain embodiments, the partially saturated heteroaryl group has 8, 9, 10, or 11 (in some embodiments, 9 or 10) ring atoms. The partially saturated heteroaryl may be attached to the main structure at any heteroatom or carbon atom of its aromatic ring to form a stable compound. In some or any embodiments, an oxo group may be present as a substituent on one of the ring atoms.Partially saturated heteroaryl radicals consist of, or include one or more of, benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, chromanyl, chromonyl, cinnolinyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, tetrahydropyrazinyl, dihydropyrazinonyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, furanonyl, imidazolinyl, indolinyl, tetrahydroindolyl, isoindolinyl, tetrahydroisoindolyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, dihydroisoxazolyl, oxazinyl, dihydrooxazinyl, oxo-oxazolyl, dihydrooxazolyl, dihydropiperidonyl, dihydro-4-piperidonyl, dihydropyrazolyl, dihydropyrazolinyl, dihydropyrrolyl, azabicyclo[2.2.2]oct-2-enyl, dihydrofuryl, tetrahydroisoquinolinyl, dihydropyranyl, pyranyl, dihydrothienyl, oxathiazinyl, dihydrothiazolyl, tetrahydroquinolinyl, and 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl.In certain embodiments, the partially saturated heteroaryl radical is benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, chromanyl, chromonyl, coumarinyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydroisoindolyl, indolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, or 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl. In certain embodiments, the partially saturated heteroaryl may optionally be substituted as described herein.

[0078]

[0079] "Spiroheterocyclic" or "spiroheterocycle" or "spiroheterocycloalkyl" refers to a heterocycle as defined herein that includes two rings linked to each other through a common atom. Non-limiting examples of spiroheterocycles include an azetidinyl ring, a morpholinyl ring, and / or a piperidinyl ring attached through a common atom to another ring (e.g., ring B shown below):

[0079]

Chemical Formula

[0080] As used herein and unless otherwise specified, the term "protecting group" refers to a group attached to an oxygen atom, a nitrogen atom, or a phosphorus atom to prevent further reaction thereof or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.

[0081]

[0081] "Pharmaceutically acceptable salts" refer to any salts of the compounds provided herein that retain their biological properties and are either non-toxic or otherwise not undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions well known in the art. Such salts include, but are not limited to, (1) acid addition salts formed with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylsulfamic acid, quinic acid, muconic acid, etc.; or (2) the acidic protons present in the parent compound are replaced by (a) metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions, or alkali metal or alkaline earth metal hydroxides such as the hydroxides of sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium, or ammonia, or (b) organic bases such as aliphatic, alicyclic, or aromatic organic amines such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, Examples of salts formed when coordinating with chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, etc.

[0082]

[0082] Pharmaceutically acceptable salts further include, but are not limited to, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc. When the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as hydrohalic acid salts, for example, hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate, etc.

[0083] For a composition, the terms "substantially free of" or "substantially absent" refer to a composition that contains at least 85 wt%, 90 wt%, in certain embodiments, 95 wt%, 98 wt%, 99 wt% or 100 wt% of the designated enantiomer of the compound. In certain embodiments, in the methods and compounds provided by the present invention, the compound is substantially free of enantiomers.

[0084] Similarly, for a composition, the term "isolated" refers to a composition that contains at least 85 wt%, 90 wt%, 95 wt%, 98 wt%, 99 wt% to 100 wt% of the compound, with the balance containing other chemical species or enantiomers.

[0085] "Solvate" refers to a compound or a salt thereof provided herein that further contains a solvent bonded by non-covalent intermolecular forces in a stoichiometric or non-stoichiometric amount. When the solvent is water, the solvate is a hydrate.

[0086] "Isotopic composition" refers to the amount of each isotope present for a given atom, and "natural isotopic composition" refers to the composition or abundance of isotopes that are naturally present for a given atom. Atoms containing these natural isotopic compositions may also be referred to herein as "non-enriched" atoms. Unless otherwise specified, atoms of the compounds described herein are meant to represent any stable isotope of that atom. For example, unless otherwise specified, if a position is specifically designated as "H" or "hydrogen", that position is understood to have hydrogen in its natural isotopic composition.

[0087]

[0087] "Isotope enrichment" refers to the percentage by which the amount of a particular isotope in a given atom is incorporated into a molecule instead of the natural isotope abundance of that atom. For example, 1% deuterium enrichment at a given position means that 1% of the molecules in a given sample contain deuterium at the designated position. Since the naturally occurring distribution of deuterium is approximately 0.0156%, the deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is approximately 0.0156%. The isotope enrichment of the compounds provided herein can be determined using conventional analytical methods known to those of skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy. and can be determined using conventional analytical methods known to those of skill in the art.

[0088]

[0088] "Isotopically enriched" refers to an atom having an isotope composition other than the natural isotope composition of that atom. "Isotopically enriched" may also refer to a compound containing at least one atom having an isotope composition other than the natural isotope composition of that atom.

[0089]

[0089] As used herein, the "alkyl", "alkylene", "alkylamino", "dialkylamino", "cycloalkyl", "aryl", "arylene", "alkoxy", "alkoxycarbonyl", "amino", "carboxyl", "heterocyclyl", "heterocycloalkyl", "heteroaryl", "heteroarylene", "partially saturated heteroaryl", "spiroheterocyclyl", "carboxyl", and "amino acid" groups optionally contain deuterium at one or more positions where a hydrogen atom is present, and the deuterium composition of one or more atoms is other than the natural isotope composition.

[0090]

[0090] Also, as used herein, the terms “alkyl,” “alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “arylene,” “alkoxy,” “alkoxycarbonyl,” “amino,” “carboxyl,” “heterocyclyl,” “heterocycloalkyl,” “heteroaryl,” “heteroarylene,” “partially saturated heteroaryl,” “spiroheterocyclyl,” “carboxyl,” and “amino acid” groups optionally contain carbon-13 in amounts other than the natural isotopic composition.

[0091]

[0091] As used herein, EC 50 refers to the dose, concentration, or amount of a particular test compound that induces a dose-dependent response at 50% of the maximal manifestation of a particular response induced, elicited, or enhanced by that particular test compound.

[0092]

[0092] As used herein, IC 50 refers to the amount, concentration, or dose of a particular test compound that achieves 50% inhibition of the maximal response in an assay that measures such a response.

[0093]

[0093] As used herein, the terms “subject” and “patient” are used interchangeably herein. The term “subject” (singular and plural) refers to a mammal, e.g., a human, including non-primates (e.g., cows, pigs, horses, cats, dogs, rats, and mice) as well as primates (e.g., monkeys such as cynomolgus monkeys, chimpanzees, etc., and humans). In certain embodiments, the subject is resistant or non-responsive to current treatments for hepatitis C infection. In another embodiment, the subject is a farm animal (e.g., a horse, cow, pig, etc.) or a pet (e.g., a dog or a cat). In certain embodiments, the subject is a human.

[0094]

[0094] As used herein, the term "therapeutic agent" (singular and plural) refers to any agent that can be used in the treatment or prevention of a disorder or one or more of its symptoms. In certain embodiments, the term "therapeutic agent" includes the compounds and / or antibody conjugates provided herein. In certain embodiments, a therapeutic agent is a drug that is known to be useful, or has been used, or is currently used for the treatment or prevention of a disorder or one or more of its symptoms.

[0095]

[0095] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an antibody or composition that is effective for the treatment of a disease or disorder when administered to a subject. In some embodiments, a therapeutically effective amount or effective amount refers to the amount of an antibody or composition that, when administered to a subject, is effective for the prevention or amelioration of a disease or the progression of a disease, or results in an improvement in symptoms. A "therapeutically effective amount" will vary, inter alia, depending on the compound, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0096] As used herein, "treating" or "treatment" of any disease or disorder, in certain embodiments, refers to ameliorating a disease or disorder present in a subject. In another embodiment, "treating" or "treatment" includes ameliorating at least one physical parameter that may be less perceptible to the subject. In yet another embodiment, "treating" or "treatment" includes modulating a disease or disorder, either physically (e.g., stabilization of a perceivable symptom) or physiologically (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treating" or "treatment" includes delaying or preventing the onset of a disease or disorder, or delaying or preventing the recurrence of a disease or disorder. In yet another embodiment, "treating" or "treatment" includes reducing or eliminating any of a disease or disorder, or delaying the progression of a disease or disorder or one or more symptoms thereof, or reducing the severity of a disease or disorder or one or more symptoms thereof.

[0097]

[0097] As used herein, the term "inhibiting growth" (e.g., with reference to cells such as tumor cells) is intended to include measurably decreasing cell growth (e.g., tumor cell growth) upon contact with an antibody or antibody conjugate as compared to the growth of the same cells not in contact with the antibody or antibody conjugate. In some embodiments, growth can be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. The decrease in cell growth can occur by a variety of mechanisms including, but not limited to, antibody internalization, apoptosis, necrosis, and / or effector function-mediated activity.

[0098] As used herein, the term "preventive agent" (singular and plural) refers to any agent(s) that can be used in the prevention of a disorder or one or more symptoms thereof. In certain embodiments, the term "preventive agent" includes the compounds provided herein. In certain other embodiments, the term "preventive agent" does not refer to the compounds provided herein. For example, a preventive agent is a drug known or used or currently used to be useful for preventing or inhibiting the onset, development, progression, and / or severity of a disorder.

[0099]

[0099] As used herein, the phrase "preventive effective amount" refers to an amount of a treatment (e.g., a preventive agent) sufficient to effect the prevention or reduction of the development, recurrence, or onset of one or more symptoms associated with a disorder (or to enhance or improve the preventive effect of another treatment (e.g., another preventive agent)).

[0100]

[0100] In some of the chemical structures shown herein, certain substituents, chemical groups, and and atoms are shown with a curve / wavy line (e.g.,

[0101]

Chem.

[0102]

Chem.

[0103]

Chem.

[0104]

[0101] The term "site-specific" refers to the modification of a polypeptide at a predetermined sequence position of the polypeptide. The modification is at a single predictable residue of the polypeptide with little or no mutation. In certain embodiments, the modified amino acid is introduced at its sequence position, for example, recombinantly or synthetically. Similarly, a moiety may be "site-specifically" linked to a residue at a specific sequence position of the polypeptide. In certain embodiments, the polypeptide may contain more than one site-specific modification.

[0105] 2. Payload - Compounds of Formula (I-P) and (I) and their sub-formulas

[0102] Provided herein are compounds capable of modulating the activity of a disease or disorder associated with Toll-like receptor 7 / 8. Pyrazoloquinolines can be formed as described herein and can be used for the treatment of diseases or disorders associated with diseases or disorders associated with Toll-like receptor 7 / 8. In certain embodiments, the disease or disorder is cancer or an inflammatory disease or condition.

[0106]

[0103] Embodiments described herein include the compounds recited, as well as their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, or mixtures thereof.

[0107]

[0104] In one aspect, provided herein is a compound of formula (I-P)

[0108]

Chem.

[0109]

Chemical formula

[0110]

[0105] In another aspect, provided herein is a compound of formula (I)

[0111]

Chemical formula

[0112]

Chemical formula

[0113]

[0106] In a group of embodiments, the compound of formula (I) has the formula (II):

[0114]

Chemical formula

[0115]

Chemical formula

[0116]

[0107] In some embodiments of the compounds of formula (I-P), (I) and / or formula (II), ring A is a phenyl ring. In some embodiments of the compounds of formula (I-P), (I) and / or formula (II), ring A is a monocyclic heteroaryl ring. In some embodiments of the compounds of formula (I-P), (I) and / or formula (II), ring A is pyridinyl. In some embodiments of the compounds of formula (I-P), (I) and / or (II), ring A is a fused bicyclic heteroaryl ring. In some embodiments of the compounds of formula (I-P) and (I), ring A is a cycloalkyl ring. In some embodiments of the compounds of formula (I-P) and (I), ring A is a heterocycloalkyl ring.

[0117]

[0108] In some embodiments of the compounds of formula (I-P), (I) and / or formula (II), ring A has at least one -OR 4 is ortho to the group

[0118]

Chem.

[0119]

Chem.

[0120]

[0109] In a group of embodiments, the compounds of formula (I-P), (I) and / or formula (II) have the structure of formula (III):

[0121]

Chem.

[0122]

Chemical formula

[0123]

[0110] In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R 1a and R 1b are each hydrogen. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R 2a and R 2b are each hydrogen. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R , R 1a , R 1b , R 2a and R 2b are each hydrogen.

[0124]

[0111] In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R 4 is methyl, ethyl, propyl, or isopropyl. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R 4 is methyl. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R , R 4is ethyl. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III ), R 4 is propyl. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R 4 is isopropyl . In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III it is R 4 is butyl, isobutyl, pentyl, neopentyl, or hexyl.

[0125]

[0112] In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), R 5 is one, two, or three R 1~6 alkylamino, C 1~6 dialkylamino, C 3~6 cycloalkyl, aryl, and heteroaryl independently selected from halo, hydroxy, alkoxy, amino, C 5a alkyl optionally substituted with 1~6 groups, wherein heteroaryl contains one, two, three, or four heteroatoms independently selected from N, S, and O, and wherein R any of the C 5a cycloalkyl group, aryl group, and heteroaryl group of 3~6 is optionally substituted with halo, hydroxy, alkyl, or haloalkyl.

[0126]

[0113] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is one, two, or three R 1~6 alkylamino, and C 1~6 dialkylamino independently selected from halo, hydroxy, alkoxy, amino, C 5a alkyl optionally substituted with 1~6is alkyl. In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is C alkyl optionally substituted with one or two 1~6 hydroxy groups. In some such examples, R 5 is branched C 1~6 alkyl optionally substituted with one or two hydroxy groups.

[0127]

[0114] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is C 1~6 alkyl optionally substituted with hydroxy or alkoxy.

[0128]

[0115] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is

[0129]

Chemical formula

[0130]

Chemical formula

[0131]

[0116] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is C 3~6 alkyl optionally substituted with C 3~6 cycloalkyl. In some such examples, R 5 is -CH2-cyclopropyl or -CH2-cyclobutyl.

[0132]

[0117] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R5 is C optionally substituted with aryl or heteroaryl 1~6 alkyl, wherein heteroaryl is selected independently from N, S, and O and contains 1, 2, 3, or 4 heteroatoms, wherein aryl and heteroaryl are optionally further substituted with halo, alkyl, or haloalkyl.

[0133]

[0118] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is

[0134]

Chemical formula

[0135]

Chemical formula

[0136]

[0119] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is 1, 2, or 3 R 1~6 groups independently selected from halo, hydroxy, alkoxy, amino, C 1~6 alkylamino, C 3~6 dialkylamino, C 5a cycloalkyl, aryl, and heteroaryl, optionally substituted with C 3~6 alkyl, wherein heteroaryl contains 1, 2, 3, or 4 heteroatoms selected independently from N, S, and O, and any of the C 5a cycloalkyl group, aryl group, and heteroaryl group of R 3~6 is optionally substituted with halo, hydroxy, alkyl, or haloalkyl.

[0137]

[0120] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is unsubstituted C 3~6 cycloalkyl. In some such examples, R 5 is cyclopropyl or cyclobutyl.

[0138]

[0121] In some or any embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 4-membered N-linked heterocycloalkyl substituted with 1 to 2 R 3 , where R 3 is, in each occurrence, independently, -N(R 3a )2, -OR 3b , -C(R 3c )2NH2, C 1~6 alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two R 3 bonded to the same carbon together with the carbon atom to which they are attached form a spiroheterocycloalkyl, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R 3 contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1 to 2 C 1~3 alkyl.

[0139]

[0122] In some or any embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 5- to 6-membered N-linked heterocycloalkyl substituted with 1 to 3 R 3 , where R 3 is, in each occurrence, independently, -N(R 3a )2, -OR 3b , -C(R 3c )2NH2, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two R 3forms a spiroheterocycloalkyl together with the carbon atom to which they are attached, where R 3 The heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of have 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and optionally 1 to 2 C 1~3 alkyl substituted.

[0140]

[0123] In some or any embodiment of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 7- to 10-membered N-linked heterocycloalkyl substituted with 1 to 3 R 3 or a 5- to 10-membered N-linked heteroaryl substituted with 1 to 3 R 3 where R 3 is, in each occurrence, independently, -N(R 3a )2, -OR 3b , -C(R 3c )2NH2, C 1~6 alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two R 3 bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are attached, where R 3 The heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of have 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and optionally 1 to 2 C 1~3 alkyl substituted.

[0141]

[0124] In some or any preceding embodiment of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a fully saturated heterocycloalkyl ring substituted with 1 to 3 R 3 . In some or any embodiment of the compounds of formula (I), formula (II) and / or formula (III), ring B is two R 3 bonded to the same carbon and forming a spiroheterocycloalkyl together with the carbon atom to which they are attacheda fully saturated heterocycloalkyl ring substituted with, where spiroheterocycloalkyl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and optionally 1 to 2 C 1~3 further substituted with alkyl.

[0142]

[0125] In some or any preceding embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), ring B is an N-linked azetidinyl ring substituted with 1 to 2 R 3 , an N-linked piperidinyl ring substituted with 1 to 2 R 3 , an N-linked triazolyl ring substituted with 1 to 2 R 3 , an N-linked morpholinyl ring substituted with 1 to 2 R 3 , or an N-linked piperazinyl ring substituted with 1 to 2 R 3 . In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is an N-linked azetidinyl ring substituted with 2 R 3 , an N-linked piperidinyl ring substituted with 2 R 3 , an N-linked morpholinyl ring substituted with 2 R 3 , or an N-linked piperazinyl ring substituted with 2 R 3 , where the 2 R 3 are attached to the same carbon and together with the attached carbon atom form a spiroheterocycloalkyl optionally substituted with one or two C1-C6 alkyls.

[0143]

[0126] In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is NH2, -NH(C1-C6 alkyl), -NH(C3-C6 cycloalkyl), heterocycloalkyl, tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, -C(R 3c )2NH2, OH,

[0144]

Chemical formula

[0145]

[0127] In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is an N-linked azetidinyl ring substituted with 1 to 2 Rs 3 In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is unsubstituted 2,5-diazabicyclo[2.2.2]octanyl, or 3,9-diazabicyclo[3.3.2]decanyl. In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a piperidine ring or a morpholinyl ring substituted with 1 to 3 Rs )In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a piperazinyl ring substituted with 1 to 3 Rs 3 In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 5- to 6-membered heteroaryl substituted with 1 to 3 Rs In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 5- to 6-membered heteroaryl substituted with 1 to 3 Rs 3 In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a piperazinyl ring substituted with 1 to 3 Rs In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 5- to 6-membered heteroaryl substituted with 1 to 3 Rs 3 In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is a 5- to 6-membered heteroaryl substituted with 1 to 3 Rs

[0146]

[0128] In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III),

[0147]

Chemical formula

[0148]

Chemical formula

[0149]

[0129] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III),

[0150]

Chemical formula

[0151]

Chemical formula

[0152]

Chemical formula

[0153]

Chemical formula

[0154] Compounds of formula (I-P), formula (I), formula (II) and / or formula (III) In some or any of the preceding embodiments, R 1a , R 1b , R 2a , and R 2b are hydrogen, R 5 is pentyl, and ring B, together with the atoms to which they are attached, is an N-linked azetidinyl ring substituted with two R 3 that forms spiroheterocycloalkyl. In some of such embodiments, the spiroheterocycloalkyl is selected from spiroazetidinyl, spiromorpholinyl, spiro-(gem-dimethyl)morpholinyl, or spiro-piperidinyl, and is optionally substituted as described herein. In some or any of the preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III) R 1a , R 1b , R 2a and R 2b are hydrogen, R 5 is pentyl, and ring B is an azetidine ring substituted with 1 to 2 R

[0155]

Chemical formula

[0156]

Chemical formula

[0157]

[0131] In some or any previous embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 1a , R 1b , R 2a and R 2b are hydrogen, R 5 is pentyl, and ring B is an N-linked morpholinyl or piperidinyl substituted with two R 3 that together with the attached atoms form a spiroheterocycloalkyl. In some of such embodiments, the spiroheterocycloalkyl is an azetidinyl ring or a piperidinyl ring. In some or any previous embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III) , R 1a , R 1b , R 2a and R 2b are hydrogen, R 5 is pentyl, and ring B is an N-linked piperidinyl ring substituted with a partially saturated heteroaryl. In some or any previous embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III) compounds, R 1a , R 1b , R 2a and R 2b are hydrogen, R 5 is pentyl, and ring B is optionally C 1~3It is a piperazinyl ring substituted with a heteroaryl ring substituted with alkyl. In some or any previous embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 1a , R 1b , R 2a and R 2b are hydrogen, R 5 is pentyl, and ring B is an N-linked heteroaryl substituted with one or two R 3 . In some of such embodiments, ring B is an N-linked triazolyl substituted with one or two R 3 . In some or any previous embodiments of the compounds of formula (I-P), formula (I), formula (II), and / or formula (III), R 3 is methyl. In some or any previous embodiments of the compounds of formula (I-P), formula (I), formula (II), and / or formula (III), R 1a , R 1b , R 2a , and R 2b are hydrogen, R 5 is pentyl, and ring B is an N-linked ring substituted with any combination of R 3 described herein and / or in this paragraph.

[0158]

[0132] Compounds of formula (I-P), formula (I), formula (II), and / or formula (III) In some or any previous embodiments, R 1a , R 1b , R 2a , and R 2b are hydrogen, R 5 is pentyl, and ring A is a phenyl ring substituted with one methoxy group at the ortho position with respect to the group

[0159]

Chemical formula

[0160]

Chemical formula

[0161]

Chemical formula

[0162]

Chemical formula

[0163]

[0133] In one aspect, the compound of formula (I-P), formula (I), formula (II), and / or formula (III), or a pharmaceutically acceptable salt, solvate, or N-oxide thereof is

[0164]

Chemical formula

[0165]

Chemical formula

[0166]

Chemical formula

[0167]

[0134] The compounds described above are in the antibody-drug conjugates described herein It is used as a payload. In addition to the payloads described above, the molecular payload can be any molecular entity that one of ordinary skill in the art may desire to conjugate to a polypeptide. In certain embodiments, the payload is a therapeutic moiety (e.g., a compound of Formula (I-P), Formula (I), or a sub-formula thereof described herein). In such embodiments, the antibody conjugate can be used to target a therapeutic moiety (e.g., a TLR7 agonist of Formula (I-P), Formula (I), or a sub-formula thereof described herein) to its molecular target. Other TLR7 agonists are known to those of ordinary skill in the art and include, for example, but are not limited to, 4-amino-2-butoxy-7,8-dihydro-8-[[3-(1-pyrrolidinylmethyl)phenyl]methyl]-6(5H)-pteridinone (Vesatolimod, GS9620, CAS No. 1228585-88-3), 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4 -amine (Imiquimod, CAS No. 99011-02-6), 1-(4-amino-2-(ethoxymeth yl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (Resquimod, CAS No. 144875-48-9), N-[4-(4-amino- 2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide (3M-001), 2-propylthiazolo[4,5-c]quinolin-4-amine (3M-002), 4-amino-2-(ethoxymethyl)-α,α-dimethyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinolin-1-ethanol hydrate (3M-003), N-(1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-yl)methanesulfonamide (CAS No. 642473-62-9, 3M-011, or 854A), and N-(4-(4-amino-2-ethyl-1H-imidazo[4,5-c]quinolin-1-yl)butyl)methanesulfonamide (CAS No. 532959-63-0, 3M-852A, PF-4878691), 2-methyl-1-(2,2,4-trimethylpen ((S)-4-(4-ethylpiperazin-1-yl)-1H-imidazo[4,5-c]quinolin-4-amine (S-34240), Roxolibin, CL264, ssRNA40, R848, and SM-276 including 001.

[0168] 3. Conjugate

[0135] Provided herein are conjugates of antibodies with TLR7 agonists (e.g., any TLR7 agonist described herein). The conjugate comprises an antibody or an antigen-binding fragment thereof against a suitable antigen (e.g., a tumor antigen) covalently linked directly or indirectly via a linker to a payload. In certain embodiments, the antibody is linked to one payload. In further embodiments, the antibody is linked to more than one payload. In certain embodiments, the antibody is linked to one, two, three, four, five, six, seven, eight, or more payloads. Thus, the drug-to-antibody ratio (DAR) can vary from 1 to 30.

[0169]

[0136] The payload may be any payload that one of ordinary skill in the art would consider useful. In certain embodiments, the payload is a therapeutic moiety. In certain embodiments, the payload is a diagnostic moiety, e.g., a label. Useful payloads are described in the following sections and examples.

[0170]

[0137] The linker may be any linker capable of forming at least one bond with the antibody and at least one bond with the payload. Useful linkers are described in the following sections and examples.

[0171]

[0138] An antibody is typically a protein that comprises multiple polypeptide chains. In certain In certain embodiments, the antibody is a heterotetramer comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain may be linked to the heavy chain by one covalent disulfide bond. Each heavy chain may be linked to the other heavy chain by one or more covalent disulfide bonds. Also, each heavy chain and each light chain may have one or more intra-chain disulfide bonds. As is known to those skilled in the art, each heavy chain typically comprises a variable domain (V ), followed by several constant domains. Each light chain typically comprises a variable domain (V ) at one end and a constant domain. As is known to those skilled in the art, antibodies typically have a selective affinity for their target molecules, i.e., antigens. H ) and several constant domains following it. Each light chain typically comprises a variable domain (V L ) and a constant domain at one end. As is known to those skilled in the art, antibodies typically have a selective affinity for their target molecules, i.e., antigens.

[0172]

[0139] The antibodies provided herein may have any antibody form known to those skilled in the art. The antibodies provided herein may be full-length or fragments. Representative full-length antibodies include IgA, IgA1, IgA2, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM, etc. Representative fragments include Fv, Fab, Fc, scFv, scFv-Fc, etc.

[0173]

[0140] In certain embodiments, the conjugated antibody comprises one, two, three, four, five, or six CDR sequences described herein. In certain embodiments, the conjugated antibody comprises the heavy chain variable domain (V ) described herein. In certain embodiments, the conjugated antibody comprises the light chain variable domain (V H ) described herein. In certain embodiments, the conjugated antibody comprises the heavy chain variable domain (V L ) described herein and the light chain variable domain (V H ) described herein. In certain embodiments, the conjugated antibody comprises the heavy chain variable domain (V L) is included. In certain embodiments, the antibody of the conjugate is the pair of heavy and light chain variable domains (V H -V L pair) described herein.

[0174]

[0141] In certain embodiments, the antibody conjugate may be formed from an antibody comprising one or more reactive groups . In certain embodiments, the antibody conjugate may be formed from an antibody consisting entirely of naturally encoded amino acids. One of ordinary skill in the art will recognize that some naturally encoded amino acids contain reactive groups capable of conjugating to a payload or linker. Such reactive groups include cysteine side chains, lysine side chains, and amino-terminal groups. In such embodiments, the antibody conjugate may include a payload or linker linked to the residue of the antibody reactive group. In such embodiments, the payload precursor or linker precursor includes a reactive group capable of forming a bond with the antibody reactive group. Exemplary reactive groups include maleimide groups, activated carbonates (including but not limited to p-nitrophenyl esters), and activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters, and aldehydes). Particularly useful reactive groups include maleimide and succinimide, such as N-hydroxysuccinimide, for forming bonds with cysteine and lysine side chains. Additional reactive groups are described in the sections and examples below.

[0175]

[0142] In further embodiments, the antibody has one reactive group as described herein It contains one or more modified amino acids. Typically, the modified amino acids are not natural encoded amino acids. Such modified amino acids may contain reactive groups useful for forming covalent bonds with the linker precursor or payload precursor. One skilled in the art can use the reactive groups to link the polypeptide to any molecular entity capable of forming a covalent bond with the modified amino acid. Accordingly, provided herein are conjugates comprising antibodies containing modified amino acid residues linked directly or indirectly via a linker to a payload. Representative modified amino acids are described in the following section. Generally, modified amino acids have reactive groups capable of forming bonds with linkers or payloads having complementary reactive groups.

[0176]

[0143] In certain embodiments, the unnatural amino acid is placed at a selected position in the polypeptide chain of the antibody. Such positions have been identified as providing optimal sites for substitution with unnatural amino acids. Each site is capable of retaining the unnatural amino acid by optimal structure, function, and / or method for producing the antibody.

[0144] In certain embodiments, the site-specific positions for substitution provide a stable antibody. Stability can be measured by any technique obvious to one skilled in the art.

[0177]

[0145] In certain embodiments, the site-specific positions for substitution provide an antibody with optimal functional properties. For example, the antibody may show little or no loss of binding affinity to its target antigen compared to an antibody that does not have site-specific unnatural amino acids. In certain embodiments, the antibody can show enhanced binding compared to an antibody that does not have site-specific unnatural amino acids.

[0146] In certain embodiments, the site-specific positions for substitution are advantageously made.

[0178]

[0147] In certain embodiments, the site-specific positions for substitution are advantageously made.

[0148] In certain embodiments, the site-specific positions for substitution are advantageously made.

[0179]

[0149] In certain embodiments, the site-specific positions for substitution are advantageously made. To provide an antibody that can be... For example, in certain embodiments, the antibody exhibits advantageous properties in its synthesis method. In certain embodiments, the antibody may show little or no loss of yield during production compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody can show enhanced yield during production compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody may show little or no loss of tRNA suppression compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody can show enhanced tRNA suppression during production compared to an antibody that does not have site-specific non-natural amino acids.

[0180]

[0147] In certain embodiments, the site-specific position for substitution provides an antibody with advantageous solubility. In certain embodiments, the antibody may show little or no loss of solubility compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody can show enhanced solubility compared to an antibody that does not have site-specific non-natural amino acids. To provide an antibody that has... In certain embodiments, the antibody may show little or no loss of solubility compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody can show enhanced solubility compared to an antibody that does not have site-specific non-natural amino acids.

[0181]

[0148] In certain embodiments, the site-specific position for substitution provides an antibody with advantageous expression. In certain embodiments, the antibody may show little or no loss of expression compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody can show enhanced expression compared to an antibody that does not have site-specific non-natural amino acids. To provide an antibody that has... In certain embodiments, the antibody may show little or no loss of expression compared to an antibody that does not have site-specific non-natural amino acids. In certain embodiments, the antibody can show enhanced expression compared to an antibody that does not have site-specific non-natural amino acids.

[0182]

[0149] In certain embodiments, the site-specific position for substitution provides an antibody with advantageous folding. Provided are antibodies having wings. In certain embodiments, the antibody may show little or no loss of correct folding compared to an antibody having no site-specific unnatural amino acids. In certain embodiments, the antibody may show enhanced folding compared to an antibody having no site-specific unnatural amino acids.

[0183]

[0150] In certain embodiments, the site-specific positions for substitution provide antibodies that allow for advantageous conjugation. As described below, some unnatural amino acids have side chains or functional groups that facilitate conjugation of the antibody with a second agent, either directly or via a linker. In certain embodiments, the antibody may show enhanced conjugation efficiency compared to an antibody having no same or other unnatural amino acids at other positions. In certain embodiments, the antibody may show enhanced conjugation yield compared to an antibody having no same or other unnatural amino acids at other positions. In certain embodiments, the antibody may show enhanced conjugation specificity compared to an antibody having no same or other unnatural amino acids at other positions.

[0151] In some embodiments, one or more unnatural amino acids are located at site-specific positions selected in at least one polypeptide chain of the antibody. The polypeptide chain may be any polypeptide chain of the antibody, including but not limited to any light chain or any heavy chain. The site-specific position may be present in any domain of the antibody, including any variable domain and any constant domain.

[0184]

[0152] In certain embodiments, the antibodies provided herein contain one or more unnatural amino acids at the site-specific position. In certain embodiments, the antibodies provided herein contain two unnatural amino acids at the site-specific position. In certain embodiments,

[0185]

[0152] In certain embodiments, the antibodies provided herein contain two unnatural amino acids at the site-specific position. or more unnatural amino acids at the site-specific position. In certain embodiments, the antibodies provided herein contain two unnatural amino acids at the site-specific position. In certain embodiments, The antibodies provided herein contain three non-natural amino acids at site-specific positions. In certain embodiments, the antibodies provided herein contain more than three non-natural amino acids at site-specific positions.

[0186]

[0153] In certain embodiments, the antibodies provided herein are Kabat or C hothia or contain one or more non-natural amino acids or their post-translational modification variants at positions independently selected from the group consisting of heavy chain residues or light chain residues HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, LC-T22, LC-S7, LC-N152, LC-K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, or HC-S70 according to the EU numbering scheme. In certain embodiments, the antibodies provided herein contain one or more non-natural amino acids or their post-translational modification variants at positions independently selected from the group consisting of HC-180, HC-222, LC-7, or LC-42 according to the Kabat or Chothia or EU numbering scheme. In these designations, HC represents a heavy chain residue and LC represents a light chain residue. In certain embodiments, the non-natural amino acid is at HC-F404. In certain embodiments, the non-natural amino acid is at HC-Y180. In certain embodiments, the non-natural amino acids are at HC-F404 and HC-Y180. In certain embodiments, the non-natural amino acid is at HC-K222. In certain embodiments, the non-natural amino acid is at LC-S7. In certain embodiments, the non-natural amino acid is at LC-K42. In certain embodiments, the non-natural amino acids are at HC-Y180, HC-K222, LC-S7, and / or LC-K42. In certain embodiments, the non-natural amino acids are at HC-F241, HC-K121, and / or HC-S190. In certain embodiments, the non-natural amino acids are the same. In certain embodiments, the non-natural amino acids are different. In certain embodiments, the non-natural amino acid is the residue of formula (30) herein.

[0187]

[0154] In some embodiments, the antibody sequence may include a Q-tag sequence that is compatible with transglutaminase conjugation. In some embodiments, one or more glutamine residues are in a Q-tag independently selected from the group consisting of LLQGA, YAHQAHY, YRYRQ, PNPQLPF, PKPQQFM, GQQQLG, WALQRPH, WELQRPY, YPMQGWF, LSLSQG, GGGLLQGG, GLLQG, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR, LLQGPA, LLQGPP or GGLLQGPP.

[0188]

[0155] In some embodiments, the acyl donor glutamine-containing tag includes at least one Gln. In some embodiments, the acyl donor glutamine-containing tag includes the amino acid sequence XXQX, where X is any amino acid (e.g., the normal amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or non-conventional amino acids). In some embodiments, the acyl donor glutamine-containing tag (Q-tag) includes an amino acid sequence selected from the group consisting of LLQGG, LLQG, LSLSQG, GGGLQGG, GLLQG, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR. In some embodiments, the acyl donor glutamine-containing tag (Q-tag) includes an amino acid sequence selected from the group consisting of LLQGPA, LLQGPP or GGLLQGPP. In some embodiments, the acyl donor glutamine-containing tag (Q-tag) includes an amino acid sequence selected from the group consisting of LLQGPA, LLQGPP or GGLLQGPP. In some embodiments, the acyl donor glutamine-containing tag (Q-tag) ) comprises an amino acid sequence selected from the group consisting of LLQGG and LLQGA. In such embodiments, the linker-payload that retains an amino group can be conjugated to the side chain of one or more glutamine (Q) residues of the antibody in the presence of transglutaminase.

[0189]

[0156] In certain embodiments, provided herein is a conjugate of formula (C1) or (C2):

[0190]

Chemical formula

[0191]

[0157] In some embodiments, n is an integer selected from 1 to 8. In some embodiments In this state, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8.

[0192] 3.1 Attachment group

[0158] The attachment group facilitates the incorporation of the eliminator group, release-inducing group, hydrophobic group, spacer group, and / or conjugate group into the compound. Useful attachment groups are known and apparent to those skilled in the art. Examples of useful attachment groups are provided herein. In certain embodiments, the attachment group is W 1 、W 2 、W 3 、W 4 、or W 5 so-called. In certain embodiments, the attachment group may include a divalent ketone, divalent ester, divalent ether, divalent amide, divalent amine, alkylene, arylene, sulfide, disulfide, carbonylene, or a combination thereof. In certain embodiments, the attachment group is -C(O )-, -O-, -C(O)NH-, -C(O)NH-alkyl-, -OC(O)NH-, -SC(O)NH-, -NH-, -NH-alkyl-, -C(O)N(CH3)-, -C(O)N(CH3)-alkyl-, -N(CH3)-, -N(CH3)-alkyl-, -N(CH3)CH2CH2N(CH3)-, -C(O)CH2CH2CH2C(O)-, -S-, -S-S-, -OCH2CH2O-, or the reverse thereof (e.g., -NHC(O)-), or a combination thereof.

[0193] 3.2 Eliminator (leaving) group

[0159] The eliminator group is the biology of the compounds or conjugates described herein The active moiety is facilitated to be separated from the remaining portion of the compound or conjugate in vivo and / or in vitro. Also, the eliminator group, in conjunction with the release-inducing group, can facilitate the separation of the biologically active moiety of the compounds or conjugates described herein. For example, the eliminator group and the release-inducing group can react in a release reaction to release the biologically active moiety of the compounds or conjugates described herein from the compound or conjugate in vivo and / or in vitro. When the release reaction is initiated by a release inducer, the eliminator group cleaves the biologically active moiety or the prodrug form of the biologically active moiety to form a stable and non-toxic entity that does not further affect the activity of the biologically active moiety.

[0194]

[0160] In certain embodiments, the eliminator group is referred to herein as EG . Useful eliminator groups include those described herein. In certain embodiments, the eliminator group is

[0195]

Chemical formula

[0196] [Chemical formula] as shown. In certain embodiments, the eliminator group is

[0197] [Chemical formula] as shown. In certain embodiments, the eliminator group is

[0198] [Chemical formula] as shown. In certain embodiments, the eliminator group is

[0199] [Chemical formula] as shown.

[0200]

[0161] In some embodiments, the eliminator group is

[0201] [Chemical formula] as shown, wherein Z may be CH or N, and each R EGis independently selected from the group consisting of hydrogen, alkyl, biphenyl, -CF3, -NO2, -CN, fluoro, bromo, chloro, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylaminoC(O)-. In each structure, the phenyl ring may have one, two, three, or in some cases, four R EG groups attached thereto. In the first and second structures, one of ordinary skill in the art will recognize that, as shown in the above description of formula (C1), EG is attached to RT which is not within the backbone of formula (C1). In some embodiments, each R EG is independently selected from the group consisting of hydrogen, alkyl, biphe nyl, -CF3, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylaminoC(O)-. In further embodiments, R EG is selected from the group consisting of hydrogen, -NO2, -CN, fluoro, bromo, and chloro. In some embodiments, each R of EG EG is hydrogen. In certain embodiments, the eliminator group is

[0202]

Chemical formula

[0203]

Chemical formula

[0204]

Chemical formula

[0205] 3.3 Release-inducing groups

[0162] The release-inducing group is the biological activity of the compounds or conjugates described herein The moiety facilitates separation of the remainder of the compound or conjugate in vivo and / or in vitro. Further, the release-inducing group, in conjunction with the eliminator group, can facilitate separation of the biologically active moiety of the compounds or conjugates described herein. For example, the eliminator group and the release-inducing group react in the release reaction to release the biologically active moiety of the compounds or conjugates described herein in vivo and / or in vitro. In certain embodiments, the release inducer can act by a biologically-driven reaction having high tumor:non-tumor specificity, such as proteolysis of an enzyme overexpressed in the tumor environment.

[0206]

[0163] In certain embodiments, the release-inducing group is referred to herein as RT. In certain embodiments, RT is divalent and is attached within the backbone of formula (C1). In other embodiments, RT is monovalent and is attached to the EG as shown above. Useful release-inducing groups include those described herein. In certain embodiments, the release-inducing group includes a natural or unnatural amino acid residue or a residue of a sugar ring. In certain embodiments, the release-inducing group is

[0207]

Chemical formula

[0208]

[0164] One of ordinary skill in the art will recognize that the first structure can be divalent and attached within the backbone of formula (C1) or as shown in formula (C2), and the second structure can be monovalent and attached to the EG as shown in the above formula (C1 ). In certain embodiments, the release-inducing group is

[0209]

Chemical formula

[0210]

Chemical formula

[0211]

[0165] In some embodiments, the release-inducing group is

[0212]

Chemical formula

[0213]

Chemical formula

[0214]

Chemical formula

[0215]

Chemical formula

[0216]

Chemical formula

[0217] [Chemical formula] is monovalent and may be bonded to EG as shown in the above formula (C1).

[0218] 3.4 Hydrophilic group

[0166] The hydrophilic group promotes an increase in the hydrophilicity of the compounds described in this specification. The increase in hydrophilicity is thought to enable greater solubility in aqueous solutions such as those found in biological systems. Also, the hydrophilic group can function as a spacer group described in more detail herein.

[0219]

[0167] In certain embodiments, the hydrophilic group is referred to herein as HP. Useful hydrophilic groups include those described herein. In certain embodiments, the hydrophilic group is divalent poly(ethylene glycol). In certain embodiments, the hydrophilic group has the formula:

[0220] [Chemical formula] is divalent poly(ethylene glycol) according to, where m is an integer selected from 1 to 13, optionally 1 to 4, optionally 2 to 4, or optionally 4 to 8.

[0221]

[0168] In some embodiments, the hydrophilic group has the following formula:

[0222] [Chemical formula] is divalent poly(ethylene glycol) according to.

[0223]

[0169] In some other embodiments, the hydrophilic group has the following formula:

[0224] [Chemical formula] is the divalent poly(ethylene glycol) according to

[0225]

[0170] In other embodiments, the hydrophilic group is a divalent poly(ethylene glycol) according to the following formula: :

[0226]

Chemical formula

[0227]

[0171] In other embodiments, the hydrophilic group is the following formula:

[0228]

Chemical formula

[0229]

[0172] In some embodiments, the hydrophilic group is the formula:

[0230]

Chemical formula

[0231] 3.5 Spacer group

[0173] The spacer group facilitates the separation between the conjugate group and other groups of the compounds described herein. This separation can lead to more efficient conjugation of the compounds described herein with a second compound, as well as more efficient cleavage of the active metabolite. Also, the spacer group can stabilize the conjugate group and lead to an improvement in the overall antibody-drug conjugate properties.

[0232]

[0174] In certain embodiments, the spacer group is referred to herein as SG. There Examples of useful spacer groups include those described herein. In certain embodiments, the spacer group is

[0233]

Chemical formula

[0234]

Chemical formula

[0235]

[0175] In some embodiments, SG is

[0236]

Chemical formula

[0237]

[0176] In some embodiments, the divalent poly(ethylene glycol) has the following formula:

[0238]

Chemical formula

[0239]

[0177] In some other embodiments, the divalent poly(ethylene glycol) has the following formula:

[0240]

Chemical formula

[0241]

[0178] In other embodiments, the divalent poly(ethylene glycol) has the following formula:

[0242] [ka] has.

[0243] In other embodiments, the divalent poly(ethylene glycol) has the following formula:

[0244] [ka] has.

[0245]

[0180] In some embodiments, the hydrophilic group has the formula:

[0246] [ka] This allows the sulfonic acid presented on the chain to be retained.

[0247] 3.6 Conjugate groups and their residues The conjugate group may be a combination of a payload as described herein and an antibody as described herein. In certain embodiments, the conjugate group is referred to herein as R. The conjugate group can react by any suitable reaction mechanism known to those skilled in the art. In certain embodiments, the conjugate group reacts by [3+2] alkyne-azide cycloaddition reaction, inverse electron demand Diels-Alder ligation reaction, thiol-electrophilic reaction, or carbonyl-oxyamine reaction, as described in detail herein. In certain embodiments, the conjugate group comprises an alkyne, a strained alkyne, a tetrazine, a thiol, a para-acetyl-phenylalanine residue, an oxyamine, a maleimide, or an azide. In certain embodiments, the conjugate group comprises an alkyne, a strained alkyne, a tetrazine, a thiol, a para-acetyl-phenylalanine residue, an oxyamine, a maleimide, or an azide. , the conjugate group is

[0248] [ka] , -N3, or -SH, wherein R 201 is lower alkyl. In one embodiment, R 201 is methyl, ethyl, or propyl. In one embodiment, R 201 is methyl. Additional conjugate groups are described, for example, in U.S. Patent Application Publication No. 2014 / 0356385, U.S. Patent Application Publication No. 2013 / 0189287, U.S. Patent Application Publication No. 2013 / 0251783, U.S. Patent No. 8,703,936, U.S. Patent No. 9,145,361, U.S. Patent No. 9,222,940, and U.S. Patent No. 8,431,558.

[0249]

[0182] After conjugation, a divalent residue of the conjugate group is formed and binds to the residue of the second compound. The structure of the divalent residue is determined by the type of conjugation reaction used to form the conjugate. The structure of the divalent residue is determined by the type of conjugation reaction used to form the conjugate.

[0250]

[0183] In certain embodiments, when the conjugate is formed by a [3+2] alkyne-azide cycloaddition reaction, the divalent residue of the conjugate group comprises a triazole ring or a fused cyclic group containing a triazole ring. In certain embodiments, when the conjugate is formed by a strain-promoted [3+2] alkyne-azide cycloaddition (SPAAC) reaction , the divalent residue of the conjugate group is is.

[0251]

Chemical formula

[0252]

[0184] In certain embodiments, when the conjugate is formed by a tetrazine inverse electron demand Diels-Alder ligation reaction, the divalent residue of the conjugate group comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In certain embodiments In an embodiment, when the conjugate is formed by a tetrazine inverse electron demand Diels-Alder ligation reaction, the divalent residue of the conjugate group is

[0253]

Chemical formula

[0254]

[0185] In certain embodiments, when the conjugate is formed by a thiol-maleimide reaction, the divalent residue of the conjugate group includes succinimidylene and a sulfur linkage. In certain embodiments, when the conjugate is formed by a thiol-male imide reaction, the divalent residue of the conjugate group is

[0255]

Chemical formula

[0256]

[0186] In certain embodiments, the conjugate is formed by a thiol-N-hydroxysuccinimide reaction using the following group:

[0257]

Chemical formula

[0258]

Chemical formula

[0259]

Chemical formula

[0260] ​

[0187] In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group comprises the divalent residue of an unnatural amino acid. In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group is as follows. In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group is

[0261]

Chemical formula

[0262]

[0188] In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group comprises an oxime linkage. In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group is as follows.

[0263]

Chemical formula

[0264]

[0189] In some embodiments, provided herein are conjugates of formula (C1) or (C2), or pharmaceutically acceptable salts, solvates, stereoisomers, or tautomers thereof, wherein EG comprises phenylene, carboxylene, amine, or combinations thereof. In one embodiment, provided herein are conjugates of formula (C1) or (C2), or pharmaceutically acceptable salts, solvates, stereoisomers, or tautomers thereof, wherein EG is as follows. In one embodiment, provided herein are conjugates of formula (C1) or (C2), or pharmaceutically acceptable salts, solvates, stereoisomers, or tautomers thereof, wherein EG is

[0265]

Chemical formula

[0266]

[0190] In some embodiments, provided herein is a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein EG comprises phenylene, carboxylene, amine, or combinations thereof. In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein EG is .wherein Z may be CH or N, and each R

[0267]

Chemical formula

[0268]

[0191] In some embodiments, provided herein is a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein RT comprises a residue of a natural or unnatural amino acid or a residue of a sugar. In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein RT is

[0269]

Chemical formula

[0270] In some embodiments, the present specification provides a conjugate according to formula (C1) or (C2): Provided herein is a conjugate according to formula (C1) or (C2), or a pharma- ceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein RT comprises a residue of a natural or unnatural amino acid or a residue of a sugar. In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharma-ceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein RT is

[0271] [ka] (wherein R1 is a bond to the remainder of the compound, or

[0272] [ka] and R2 is -CH3, -CH2CH2CO2H, or -(CH2)3NHCONH2;

[0273] [ka] (wherein Z is OH or NH2). or a legumain-cleavable Ala-Ala-Asn (AAN) or Ala-Ala-Asp (AAD) peptide according to the structure:

[0274] [ka] A person skilled in the art would recognize that the β-glucuronidase cleavable β-glucuronide has the structure:

[0275] [ka] is a divalent structure and is attached within the backbone of formula (C1) or as shown in formula (C2). will recognize that it may be. Structure

[0276]

Chemical formula

[0277]

[0193] In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein HP contains poly(ethylene glycol). In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein HP is and in the formula, m is an integer selected from 1 to 13.

[0278]

Chemical formula

[0279]

[0194] In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein SG contains C1-C alkylene, C4-C6 alkylene, carbonylene, or a combination thereof. In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein SG is 10 and in the formula, m is an integer selected from 1 to 13.

[0280]

Chemical formula

[0281]

[0195] In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein W 1 and W 2 and W 3 and W 4 and W 5 are each independently a single bond, absent, or contain a divalent ketone, divalent ester, divalent ether, divalent amide, divalent amine, alkylene, arylene, sulfide, disulfide, carbonylene, or a combination thereof. In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein W 1 and W 2 and W 3 and W 4 and W 5 are each independently a single bond, absent, or contain -C(O)-, -O-, -C(O)NH-, -C(O)NH-alkyl-, -OC(O)NH-, -SC(O)NH-, -NH-, -NH-alkyl-, -C(O)N(CH3)-, -C(O)N(CH3)-alkyl-, -N(CH3)-, -N(CH3)-alkyl-, -N(CH3)CH2CH2N(CH3)-, -C(O)CH2CH2CH2C(O)-, -S-, -S-S-, -OCH2CH2O-, or the reverse thereof (e.g., -NHC(O)-), or a combination thereof.

[0282]

[0196] In one embodiment, the present specification provides a conj A gate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided, wherein R’ contains a triazolyl ring. In one embodiment, herein is provided a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R’ is a triazolyl ring or a fused cyclic group containing a triazolyl ring. In one embodiment, herein is provided a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R’ is

[0283]

Chemical formula

[0284]

[0197] In one embodiment, herein is provided a conjugate according to formula (C1) or (C2), A gate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided, wherein R’ contains a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In one embodiment, herein is provided a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R’ is

[0285]

Chemical formula

[0286]

[0198] In one embodiment, herein is provided a conjugate according to formula (C1) or (C2), A gate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided, wherein R’ contains a sulfur linkage. In one embodiment, herein is provided a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R’ is

[0287] [ka] It is.

[0288] In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2): or a pharma- ceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R' comprises a divalent residue of a non-natural amino acid. In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharma- ceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R' is

[0289] [ka] It is.

[0290]

[0200] In one embodiment, the present specification provides a compound of formula (C1) or (C2 In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharma- ceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R' is

[0291] [ka] It is.

[0292]

[0201] In one embodiment, the present specification provides a compound of formula (C1) or (C2) which includes an oxime linkage. A conjugate according to [reference], or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided. In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R’ is

[0293]

Chemical formula

[0294]

[0202] In one embodiment, provided herein is a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R’ is

[0295]

Chemical formula

[0296]

[0203] In one embodiment, provided herein is a compound according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of any compound known to be useful for conjugation to a payload described herein, and an optional linker as described herein. In one embodiment, provided herein is a compound according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of an antibody chain, or an antigen-binding fragment thereof.

[0297]

[0204] In one aspect, provided herein is a payload described herein, and conjugated to an antibody An antibody conjugate comprising an optionally selected linker as described in the present specification, wherein Ab is a residue of an antibody. In one embodiment, the present specification provides an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of an antibody and R' comprises a triazole ring or a fused cyclic group containing a triazole ring. In one embodiment, the present specification provides an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of an antibody , R' is

[0298]

Chemical formula

[0299]

[0205] In one embodiment, the present specification provides an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of an antibody or an antigen-binding fragment thereof and R' comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In one embodiment, the present specification provides an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of an antibody or an antigen-binding fragment thereof and R' is

[0300]

Chemical formula

[0301]

[0206] In one embodiment, the present specification provides an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof A gate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided, wherein Ab is a residue of a polypeptide and R' comprises a divalent residue of a sulfur linkage. In one embodiment, provided herein is an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of a polypeptide and R' is

[0302]

Chemical formula

[0303]

[0207] In one embodiment, provided herein is an antibody conjugate according to formula (C1) or (C2) A gate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided, wherein Ab is a residue of a polypeptide and R comprises a divalent residue of a non-natural amino acid. In one embodiment, provided herein is an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of a polypeptide and R' is

[0304]

Chemical formula

[0305]

[0208] In one embodiment, provided herein is an antibody conjugate according to formula (C1) or (C2) A gate, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, is provided, wherein Ab is a residue of a polypeptide and R' comprises an oxime linkage. In one embodiment, provided herein is an antibody conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of a polypeptide and R' is

[0306] [ka] It is.

[0307] In one embodiment, the antibody conjugate according to formula (C1) or (C2) is In one embodiment, provided herein is an antibody conjugate according to formula (C1) or (C2), or a pharma- ceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Ab is a residue of a polypeptide and R' comprises an oxime linkage.

[0308] [ka] It is.

[0309] In one embodiment, the present specification provides a conjugate according to any of the following formulas: and positional isomers thereof are provided, where Ab represents a residue of an antibody or antigen-binding fragment thereof, and PA represents a payload moiety. One skilled in the art will recognize that Ab can be attached at more than one position. Each positional isomer and mixtures thereof are provided herein.

[0310] [ka]

[0311] [ka] It is.

[0312] In one embodiment, the present specification provides a conjugate according to any of the following formulas: where Ab represents the antibody residue and PA represents the payload moiety.

[0313] [Chemical formula]

[0314]

[0212] In one embodiment, the present specification provides a conjugate according to any of the following formulas: wherein Ab represents a residue of an antibody or an antigen-binding fragment thereof, and PA represents a payload moiety.

[0315] [Chemical formula]

[0316] [Chemical formula]

[0317]

[0213] In one embodiment, the present specification provides a conjugate according to any of Formulas 101a to 105b, wherein Ab represents a residue of an antibody or an antigen-binding fragment thereof, and PA represents a payload moiety. wherein Ab represents a residue of an antibody or an antigen-binding fragment thereof, and PA represents a payload moiety.

[0318] [Chemical formula]

[0319] [Chemical formula]

[0320]

[0214] In any of the above embodiments, the conjugate comprises n PA moieties. m and n are integers selected from 1 to 8. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. Those skilled in the art will recognize that formula (101a) and formula (101b) are positional isomers based on the nitrogen atom in the triazole to which the antibody binds. Similarly, formula (102a) and formula (102b), formula (103a) and formula (103b), formula (104a) and formula (104b), formula (105a) and formula (105b) are pairs of positional isomers.

[0321]

[0215] In certain embodiments, this specification provides by any one of formulas 101a to 105b There is provided an antibody conjugate, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30). In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30) at position 404 of the heavy chain according to the EU numbering system. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30) at position 180 of the heavy chain according to the EU numbering system. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30) at position 241 of the heavy chain according to the EU numbering system. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30) at position 222 of the heavy chain according to the EU numbering system. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30) at position 7 of the light chain according to the Kabat or Chothia numbering system. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab contains a residue of a non-natural amino acid according to the following formula (30) at position 42 of the light chain according to the Kabat or Chothia numbering system. In certain embodiments, PA is a residue of a compound of formula (I) described herein is a residue of the compound.

[0322] [Chemical formula]

[0323]

[0216] Those skilled in the art will recognize that amino acids such as formula (30) are incorporated as residues into polypeptides and antibodies will be recognized. For example, the residue of formula (30) is the following formula (30'):

[0324] [ka] For example, further modifications at -N3 are also encompassed by the term residue herein.

[0325]

[0217] In certain embodiments, the present specification provides a compound according to any one of Formulas 101a to 105b. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to formula (56) below at heavy chain position 404 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to formula (56) below at heavy chain position 180 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to formula (56) below at heavy chain position 241 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to formula (56) below at position 222 of the heavy chain according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to formula (56) below at position 7 of the light chain according to the Kabat or Chothia numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to formula (56) below at position 42 of the light chain according to the Kabat or Chothia numbering system. In certain embodiments, the PA comprises a PA of formula (IP) as described herein. A non-natural amino acid according to formula (56) is a residue of a compound of formula (I), (II), and / or (III):

[0326] [ka]

[0327]

[0218] In certain embodiments, the present specification provides a compound according to any one of Formulas 101a to 105b. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises the unnatural amino acid residue paraazidomethyl-L-phenylalanine at heavy chain position 404 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises the unnatural amino acid residue paraazidomethyl-L-phenylalanine at heavy chain position 180 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises the unnatural amino acid residue paraazidomethyl-L-phenylalanine at heavy chain position 241 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises the non-natural amino acid residue paraazidomethyl-L-phenylalanine at heavy chain position 222 according to the EU numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises the non-natural amino acid residue paraazidomethyl-L-phenylalanine at light chain position 7 according to the Kabat or Chothia numbering system. In certain embodiments, provided herein is an antibody conjugate according to any of Formulas 101a-105b, wherein Ab comprises the non-natural amino acid residue paraazidomethyl-L-phenylalanine at light chain position 42 according to the Kabat or Chothia numbering system. In certain embodiments, PA is a residue of a compound of Formula (I) as described herein.

[0328]

[0219] In certain embodiments, the present specification provides antibody-drug conjugates of the compounds of formula (I-P), formula (I ), formula (II), and / or formula (III). In one aspect, the present specification provides a compound of formula (V):

[0329]

Chemical Structure

[0330]

[0331]

[0220] In some examples of formula (V),

Chemical Structure

[0332] is

Chemical Structure

[0333] W 1 , W 2 , W 3 , W 4 , SG, RT, HP, EG, and R’ are as defined herein for formulas (C1) and (C2) in some or any embodiments. In some other examples of formula (V), ​​​ is,

[0334] [Chemical formula] wherein W 1 , W 6 , SG, X, HP, and R’ are as defined herein for formula (VI) in some or any embodiments.

[0335]

[0221] In another aspect, provided herein is an antibody conjugate having the structure of formula (VI)

[0336] [Chemical formula] or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or positional isomer mixture thereof, wherein W 1 is, in each occurrence, independently a single bond, absent, or a divalent linking group, X is, in each occurrence, independently absent,

[0337] [Chemical formula] where subscript b is an integer selected from 1 to 10, R A is, when present, in each occurrence, independently selected from C 1~3 alkyl, RT is, when present, in each occurrence, independently a release-inducing group, HP is, when present, in each occurrence, independently a hydrophilic group, W 6 is, in each occurrence, independently a residue of a peptide or absent, SG, in each occurrence, independently, is absent or is a bivalent spacer group, and R’, in each occurrence, independently, is a bivalent residue of a conjugated group, The subscript n is an integer selected from 1 to 30, Ab is an antibody or an antigen-binding fragment thereof,

[0222] PA, in each occurrence, independently, is a residue of a compound of formula (I-P), (I), (II), or (III), wherein PA is -NR 3a -, -C(R 3c )2NH- of -NH-, the nitrogen of the heterocycloalkyl of R 3 , the nitrogen of the partially saturated heteroaryl of R 3 , the -NH- of -O-CH2-(phenyl)-CH2-NH-, or is attached to the remainder of the molecule via the nitrogen of ring B. In another embodiment, provided herein is a conjugate of formula (VI-P):

[0338]

Chemical formula

[0339]

Chemical formula

[0340]

Chemical formula

[0341]

Chemical formula

[0342]

[0223] In some embodiments, the compound according to formula (VI) is according to formula (VIa), (VIb), (VIc), (VId), or (VIe):

[0343]

Chem.

[0344]

Chem.

[0345]

[0224] In some examples of formulae (VI), (VIa), (VIb), (VIc), (VId), and (VIe), SG is absent, or

[0346]

Chem.

[0347]

Chem.

[0348] [Chemical formula] and each

[0349] [Chemical formula] indicates the attachment point to the rest of the formula.

[0350]

[0225] In some examples of formula (VI), (VIa), (VIb), (VIc), (VId), and (VIe), W 1 when present,

[0351] [Chemical formula] where the subscript e is an integer selected from 1 to 10, and each

[0352] [Chemical formula] indicates the attachment point to the rest of the formula. In some examples, W 1 when present,

[0353] [Chemical formula] where each

[0354] [Chemical formula] indicates the attachment point to the rest of the formula.

[0355]

[0226] In some examples of formula (VI), (VIa), (VIb), (VIc), (VId), and (VIe), when W 6 is a residue of a peptide, the residue of the peptide may contain natural and / or non-natural amino acid residues. In some examples of formula (VI), when W 6 is present, it is a tripeptide residue. In some of such examples, W 6 is

[0356]

Chem.

[0357]

Chem.

[0358]

Chem.

[0359]

Chem.

[0360]

[0227] In some examples of formula (VI), (VIa), (VIb), (VIc), (VId), and (VIe), RT is

[0361]

Chem.

[0362]

Chem.

[0363]

[0228] In some examples of formula (VI), (VIa), (VIb), (VIc), (VId), and (VIe), HP, when present, is a PEG group. In some examples of formula (VI), HP, when present,

[0364]

Chem.

[0365]

Chemical formula

[0366]

[0229] In some examples of formula (VI), (VIa), (VIb), (VIc), (VId), and (VIe), R’ is

[0367]

Chemical formula

[0368]

Chemical formula

[0369]

Chemical formula

[0370]

Chemical formula

[0371]

[0230] In certain embodiments, the antibody conjugates described herein are

[0372]

Chemical formula

[0373]

Chemical formula

[0374]

Chem.

[0375]

Chem.

[0376]

[0231] In some embodiments, the antibody-drug conjugate of formula (VI) described herein is

[0377]

Chem.

[0378]

Chem.

[0379]

[0232] As used herein, when an antibody is conjugated to a linker precursor, for convenience, the conjugate is, in some or any embodiment, as follows:

[0380]

Chem.

[0381]

Chem.

[0382]

Chem.

Chem.

[0383]

[0233] In some examples, the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-BCMA, anti-Muc16, trastuzumab, sofitizumab, anti-GFP, and anti-FolRa, or an antigen-binding fragment thereof.

[0384]

[0234] In some examples, the antibody or antigen-binding fragment thereof comprises the Y180(pAMF) mutation, the F404 pAMF mutation, or both.

[0385]

[0002] In any of the preceding embodiments of formula (V) or (VI), the subscript n is 1 to 3 0, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2. In some examples, the subscript n is 1. In some examples, the subscript n is 2. In some examples, the subscript n is 3. In some examples, the subscript n is 4. In some examples, the subscript n is 5. In some examples, the subscript n is 6. In some examples, the subscript n is 7. In some examples, the subscript n is 8. In some examples, the subscript n is a number greater than 8.

[0386]

[0003] Also contemplated within the scope of the embodiments presented herein are antibody drug conjugates in which the antibody is selected from a variety of therapeutic antibodies that have been approved for use, are in clinical trials, or are in development for clinical use. Such therapeutic antibodies include, but are not limited to, rituximab (Rituxan®, IDEC / Genentech / Roche). (see, e.g., U.S. Pat. No. 5,736,137), chimeric anti-CD20 antibodies approved to treat non-Hodgkin's lymphoma, HuMax-CD20, an anti-CD20 currently being developed by Genmab, the anti-CD20 antibodies described in U.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769 (PCT Application No. PCT / US2003 / 040426), trastuzumab (Herceptin® , Genentech) (see, e.g., U.S. Pat. No. 5,677,171) to treat breast cancer. pertuzumab (rhuMab-2C4, Omnitarg®), currently in development by Genentech; anti-Her2 antibodies (U.S. Patent No. 4,753,894); cetuximab (Erbitux®, Imclone), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers (U.S. Patent No. 4,943,533; PCT Publication No. WO 96 / 40210); ABX-EGF (U.S. Patent No. 6,235,883), currently in development by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Patent No. 7,247,301), currently in development by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy, et al. al. (1987) Arch. Biochem. Biophys. 25 2(2): 549-60; Rodeck, et al. (1987) J. Cell. Biochem. 35(4): 315-20; Kettleborough, et al. (1991) Protein Eng. 4(7): 773-83); ICR62 (Institute of Cancer Research) (PCT Publication No. WO95 / 20045; Modjtahedi, et al. (1993) J. Cell. Biophys. 22(I-3): 129-46; Modjtahedi, et al. (1993) Br. J. Cancer 67(2): 247-53; Modjtahedi, et al. (1996) Br. J. Cancer 73(2): 228-35; Modjtahedi, et al. (2003) Int. J. Cancer 105(2): 273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Patent No. 5,8 91,996; U.S. Patent No. 6,506,883; Mateo, et al. (1997) Immunotechnol. 3(1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci. USA. 100 (2): 639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO01 / 62931A2); and SC100 (Scancell) (PCT Publication No. WO01 / 88138); Alemtuzumab (Campath®, Millenium), a humanized mAb currently approved for the treatment of B-cell chronic lymphocytic leukemia; Muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson, ID ​ Ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed by EC / Schering AG, developed by Celltech / Wyeth Gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Alefacept (Amevive®), an anti-LFA-3 Fc fusion developed by Biogen, developed by Centocor / Lilly Abciximab (ReoPro®), developed by Novartis, Basiliximab (Simulect®), developed by Medimmune, Palivizumab (Synagis®), an anti-TNF alpha antibody developed by Centocor Infliximab (Remicade®), an anti-TNF alpha antibody developed by Abbott, Adalimumab (Humira®), an anti-TNF alpha antibody developed by Celltech, Humicade®, an anti-TNF alpha antibody developed by Centocor, Golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, Etanercept (Enbrel®), a p75 TNF receptor Fc fusion developed by Immunex / Amgen, Ienercept, a p55 TNF receptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD147 antibody developed by Abgenix, ABX-IL8, an anti-IL8 antibody developed by Abgenix, ABX-MA1, an anti-MUC18 antibody developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 under development by Antisoma, Therex (R1550), an anti-MUC1 antibody developed by Antisoma, AngioMab (AS1405), developed by Antisoma, HuBC-1, developed by Antisoma, by Antisoma Thioplatin (AS1407) being developed, A, an anti-alpha-4-beta-1 (VLA-4) and anti-alpha-4-beta-7 antibody being developed by Biogen ntegren® (natalizumab), VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-β antibody being developed by Cambridge Antibody Technology, ABT874 (J695), an anti-IL-12p40 antibody being developed by Abbott, CAT-192, an anti-TGFβ1 antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-eotaxin 1 antibody being developed by Cambridge Antibody Technology, LymphoStat-B®, an anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1 mAb, an anti-TRAIL-R1 antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., Avastin® bevacizumab (rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, anti-HER receptor family antibodies being developed by Genentech,, anti-tissue factor (ATF), an anti-tissue factor antibody being developed by Genentech, Xolair® (omalizumab), an anti-IgE antibody developed by Genentech; Raptiva® (efalizumab), an anti-CD11a antibody developed by Genentech and Xoma; MLN-02 antibody (formerly LDP-02) developed by Genentech and Millennium Pharmaceuticals; HuMaX CD4, an anti-CD4 antibody developed by Genmab; HuMax-IL15, an anti-IL15 antibody developed by Genmab and Amgen; HuMax-Inflam developed by Genmab and Medarex; HuMax-Cancer, an anti-heparanase I antibody developed by Genmab, Medarex and Oxford GlycoSciences; HuMax-Lymphoma developed by Genmab and Amgen; HuMax-TAC developed by Genmab; IDEC-131, an anti-CD40L antibody developed by IDEC Pharmaceuticals; IDEC-151 (clenoliximab), an anti-CD4 antibody developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody developed by IDEC Pharmaceuticals; IDEC-152, an anti-CD23 developed by IDEC Pharmaceuticals; an anti-macrophage migration inhibitory factor (MIF) antibody developed by IDEC Pharmaceuticals; an anti-idiotype antibody developed by Imclone such as BEC2; IMC-1C11, an anti-KDR antibody developed by Imclone; DC101, an anti-flk-1 antibody developed by Imclone; an anti-VE cadherin antibody developed by Imclone; CEA-Cide® [Iabetuzumab], an anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics (Iabetuzumab) developed by Immunomedics LymphoCide® (epratuzumab), an anti-CD22 antibody; AFP-Cide, developed by Immunomedics; MyelomaCide, developed by Immunomedics; LkoCide, developed by Immunomedics; ProstaCide, developed by Immunomedics; MDX-010, an anti-CTLA4 antibody developed by Medarex; MDX-060, an anti-CD30 antibody developed by Medarex; MDX-070, developed by Medarex; MDX-018, developed by Medarex; Osidem® (IDM-1), an anti-Her2 antibody developed by MedareX and Immuno-Designed Molecules; HuMax®-CD4, an anti-CD4 antibody developed by MedareX and Genmab; HuMax-IL15, an anti-IL15 antibody developed by MedareX and Genmab; CNTO148, an anti-TNFα antibody developed by MedareX and Centocor / J&J; CNTO1275, an anti-cytokine antibody developed by Centocor / J&J; MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies developed by MorphoSys; MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody developed by MorphoSys; Nuvion® (visilizumab), an anti-CD3 antibody developed by Protein Design Labs; HuZAF®, an anti-gamma interferon antibody developed by Protein Design Labs; anti-α5β1 integrin, developed by Protein Design Labs Design Labs; HuMax®-CD4, an anti-CD4 antibody developed by MedareX and Genmab; HuMax-IL15, an anti-IL15 antibody developed by MedareX and Genmab; CNTO148, an anti-TNFα antibody developed by MedareX and Centocor / J&J; CNTO1275, an anti-cytokine antibody developed by Centocor / J&J; MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies developed by MorphoSys; MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody developed by MorphoSys; Nuvion® (visilizumab), an anti-CD3 antibody developed by Protein Design Labs; HuZAF®, an anti-gamma interferon antibody developed by Protein Design Labs; Protein Design Labs; anti-α5β1 integrin, developed by Prote Anti-IL-12 developed by Design Labs, ING-1, an anti-Ep-CAM antibody developed by Xoma, Xolair® (omalizumab), a humanized anti-IgE antibody developed by Genentech and Novartis, and MLN01, an anti-beta2 integrin antibody developed by Xoma. In another embodiment, the therapeutic agents include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO95 (alphaV integrin, Centocor); MEDI-522 (alphaVbeta3 integrin , Medimmune); brolucizumab (alphaVbeta1 integrin, Biogen / PDL); human mAb216 (B cell glycosolated epitope, NCl); BiTE MT103 (bispecific CD19×CD3, Medimmune); 4G7×H22 (bispecific B cell×Fc gammaR1, Medarex / Merck KGa) ; rM28 (bispecific CD28×MAPG, European Patent No. 1444268); MDX447 (EMD82633) (bispecific CD64×EGFR, Medarex); catumaxomab (removab) (bispecific EpCAM×anti-CD3, Trion / Fres); ertumaxomab (bispecific HER2 / CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® )(WX G250) (carbonic anhydrase IX, Wilex); CNTO888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Bristol Myers Squibb); MDX-1342 (CD19, Medarex); sipuleucel-T (MEDI-507) (CD2, Medimmune); ofatumumab (Humax-CD20) (CD20, Genmab); rituximab Rituxan (CD20, Genentech); Belzutifan (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); Lumiliximab (IDEC152) (CD23, Biogen); Muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi -1, CD30, NCl); MDX-060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genentics); Campath1h (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093 / D93) (Cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); Ipilimumab (MDX-010) (CTLA4, Bristol Myers Squibb); Tremelimumab (Tici limumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4 TRAIL-R1 agonist, Human Genome Science / Glaxo Smith Kline); AMG-655 (DR5 , Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo Co., Ltd.); HGS-ETR2 (Lexatumumab) (DR5 TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8 (EGFR, Imclone); Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); Adecatumumab (MT201) (Epcam, Merck); Edrecolomab (Panorex, 17-1A) (Epcam, Glaxo / Centocor); MORAb-003 (Folate receptor a, Morphotech); KW-2871 ( Ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex) ; Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); Apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); Anti-IGF-1R R1507 (IGF1-R, Roche); CP751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122) , Hoffman LaRoche); CNTO328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTβR, Biogen); HuHMFG1 (MUC1, Antisoma / NCl); RAV12 (N-linked sugar chain epitope, Raven); CAL (parathyroid Parathyroid-related protein (PTH-rP), University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1, Medarex / Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); Bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa, Centocor); A27.15 (Transferrin receptor, Salk Institute, INSERN WO2005 / 111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT publication number WO / 2000 / 034337, University of Texas); IMC-18 F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone) and the like.

[0387]

[0004] Examples of useful bispecific parental antibodies include, but are not limited to, those having one antibody against a tumor cell antigen and the other antibody against a cytotoxicity-inducing molecule, for example, anti-FcγRI / anti-CD15, anti-p185 HER2 / FcγRIII (CD16), anti-CD3 / anti-malignant B cell (1D10), anti-CD3 / anti-p185 HER2, anti-CD3 / anti-p97, anti-CD3 / anti-renal cell carcinoma, anti-CD3 / anti-OVCAR-3, anti-CD3 / L-D1 (anti-colorectal cancer), anti-CD3 / anti-melanocyte-stimulating hormone analog, anti-EGF receptor / anti-CD3, anti-CD3 / anti-CAMA1, anti-CD3 / anti-CD19, anti-CD3 / MoV18, anti-neural cell adhesion molecule (NCAM) / anti-CD3, anti-folate binding protein (FBP) / anti-CD3, anti-pan-cancer associated antigen (AMOC-31) / anti-CD3; bispecific antibodies having one antibody that specifically binds to a tumor antigen and another antibody that binds to a toxin, for example, anti-saporin / anti-Id-1, anti-CD22 / anti-saporin, anti-CD7 / anti-saporin, anti-CD38 / anti-saporin, anti-CEA / anti-ricin A chain, anti-interferon α (IFN-α) / anti-hybridoma idiotype, anti-CEA / anti-vinca alkaloid; bispecific antibodies for converting enzyme-activated prodrugs, for example, anti-CD30 / anti-alkaline phosphatase (catalyzing the conversion of mitomycin phosphate prodrug to mitomycin alcohol); bispecific antibodies that can be used as fibrinolytic agents, for example, anti-fibrin / anti-tissue plasminogen activator (tPA), anti-fibrin / anti-urokinase-type plas minogen activator (uPA); bispecific antibodies for targeting immune complexes to cell surface receptors, for example, anti-low density lipoprotein (LDL) / anti-Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII); bispecific antibodies for use in the treatment of infectious diseases, for example, anti-CD3 / anti-simple herpes virus (HSV), anti-T cell receptor:CD3 complex / anti-in fluenza, anti-FcγR / anti-HIV; bispecific antibodies for tumor detection in vitro or in vivo, for example anti-CEA / anti-EOTUBE, anti-CEA / anti-DPTA, anti-p185 / anti-hapten; bispecific antibodies as vaccine adjuvants (see Fanger, M W et al., Crit Rev Immunol. 1992; 12(34):101-24, incorporated herein by reference); and bispecific antibodies as diagnostic tools, for example, anti-rabbit Ig HER2 ​G / Anti-ferritin, anti-horseradish peroxidase (HRP) / Anti-hormone, anti-somatostatin Examples of trispecific antibodies include anti-antibody / anti-substance P, anti-HRP / anti-FITC, and anti-CEA / anti-β-galactosidase (see Nolan, O et R. O'Kennedy, Biochim Biophys Acta. 1990 Aug. 1; 1040(1):1-11, which is incorporated herein by reference). These include CD3 / anti-CD4 / anti-CD37, anti-CD3 / anti-CD5 / anti-CD37, and anti-CD3 / anti-CD8 / anti-CD37.

[0388] In the above-mentioned embodiment, the antibody conjugate has a structure according to formula (V) and (VI). In any embodiment, the bracketed structure may be covalently attached to one or more unnatural amino acids of the antibody, the one or more unnatural amino acids being located at a site independently selected from the group consisting of HC-F241, HC-F404, HC-Y180, and LC-K42, and combinations thereof, according to the Kabat numbering scheme or the EU numbering scheme of Kabat. In some embodiments, the bracketed structure is covalently linked to one or more unnatural amino acids at site HC-F404 of the antibody. In some embodiments, the bracketed structure is covalently linked to one or more unnatural amino acids at site HC-Y180 of the antibody. In some embodiments, the bracketed structure is covalently linked to one or more unnatural amino acids at site HC-F241 of the antibody. In some embodiments, the bracketed structure is covalently linked to one or more unnatural amino acids at site LC-K42 of the antibody. In some embodiments, the bracketed structure is covalently linked to one or more unnatural amino acids at sites HC-F404 and HC-Y180 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently attached to one or more non-natural amino acids at positions HC-F241, HC-F404, and HC-Y180 of the antibody. In some embodiments, at least one structure enclosed in square brackets is covalently attached to a non-natural amino acid at position HC-F404 of the antibody, and at least one structure enclosed in square brackets is covalently attached to a non-natural amino acid at position HC-Y180 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently attached to one or more non-natural amino acids at positions HC-Y180 and LC-K42 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently attached to one or more non-natural amino acids at positions HC-F404 and LC-K42 of the antibody. In certain embodiments, each non-natural amino acid is a residue according to formula (30).

[0389]

[0236] In additional embodiments, the antibody conjugate is selected from the group consisting of It can have additional payloads: labels, dyes, polymers, water-soluble polymers, polyethylene glycol, derivatives of polyethylene glycol, photo-crosslinking agents, cytotoxic compounds, radionuclides, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, a second protein or polypeptide or polypeptide analog, antibodies or antibody fragments, metal chelating agents, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, peptides, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that interact covalently or non-covalently with other molecules, photocaged moieties, photo-isomerizable moieties, biotin, derivatives of biotin, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox-active agents, amino acids, toxic moieties, isotope-labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, high electron density groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, biologically active agents, detectable labels, small molecules, or any combination thereof. In one embodiment, the payload is a label, dye, polymer, cytotoxic compound, radionuclide, drug, affinity label, resin, protein, polypeptide, polypeptide analog, antibody, antibody fragment, metal chelating agent, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, peptide, fluorophore, or carbon-linked sugar. In another embodiment, the payload is a label, dye, polymer, drug, antibody, antibody fragment, DNA, RNA, or peptide.

[0390]

[0237] In certain embodiments, the conjugate comprises one or more water-soluble polymers -including. A wide variety of macromolecular polymers and other molecules can be linked to the polypeptides described herein to modulate the biological properties of the polypeptides and / or provide new biological properties to the polypeptides. Such macromolecular polymers can be linked to the polypeptide via natural encoded amino acids, via non-natural encoded amino acids, or via any functional substituent of natural or modified amino acids, or via any substituent or functional group added to natural or modified amino acids. The molecular weight of the polymer may be in a wide range, including but not limited to, about 100 Da to about 100,000 Da or greater.

[0391]

[0238] The selected polymer may be water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment such as a physiological environment The polymer may be branched or unbranched. Preferably, for therapeutic use of the final product preparation, the polymer will be pharmaceutically acceptable.

[0392]

[0239] In certain embodiments, the ratio of polyethylene glycol molecules to polypeptide molecules will vary. Similarly, their concentrations in the reaction mixture will vary. In general, the optimal ratio (from the perspective of reaction efficiency where there is a minimum of excess unreacted protein or polymer) can be determined by the molecular weight of the selected polyethylene glycol and the number of available reactive groups. With respect to molecular weight, typically, the higher the molecular weight of the polymer, the fewer the number of polymer molecules that can attach to the protein. Similarly, the branching of the polymer should be taken into account when optimizing such parameters. In general, the higher the molecular weight (or the more branched), the higher the polymer:protein ratio.

[0393]

[0240] Water-soluble polymers include, but are not limited to, linear, dendritic, or branched ​It may have any structural form. Typically, the water-soluble polymer is a poly(alkylene glycol) such as poly(ethylene glycol) (PEG), but other water-soluble polymers can also be used. As an example, PEG is used to illustrate certain embodiments.

[0394]

[0241] PEG is a well-known water-soluble polymer that can be commercially available or prepared by ring-opening polymerization of ethylene glycol according to methods well-known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to include any polyethylene glycol molecule regardless of the size or end modification of PEG, and can be represented as being linked to the polypeptide by the formula: X’O-(CH2CH2O) -CH2CH2-Y, where n is from 2 to 10,000, X is an end modification including but not limited to H or C n alkyl, and Y is the attachment point to the polypeptide. 1~4

[0242] In some cases, PEG is terminated with hydroxy or methoxy at one end

[0395] , that is, X is H or CH3 ("methoxy PEG"). Alternatively, PEG ​may be terminated with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups include those commonly used to react with functional groups found in the 20 common amino acids (maleimide groups, activated carbonates including but not limited to p-nitrophenyl esters, activated esters including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters, and aldehydes), and functional groups that are inert to the 20 common amino acids but specifically react with complementary functional groups present in non-natural encoded amino acids (including but not limited to azide groups, alkyne groups). Note that the other end of the PEG represented by Y in the above formula will be attached to the polypeptide either directly or indirectly via a naturally occurring amino acid or a non-natural encoded amino acid. For example, Y may be an amide linkage with an amine group of the polypeptide (including but not limited to the epsilon amine of lysine or the N-terminus), a car bamate linkage, or a urea linkage. Alternatively, Y may be a maleimide bond with a thiol group (including but not limited to the thiol group of cysteine). Alternatively, Y may be a linkage with a residue that is not generally accessible via the 20 common amino acids. For example, an azide group in the PEG may be reacted with an alkyne group of the polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group in the PEG may be reacted with an azide group present in a non-natural encoded amino acid such as a modified amino acid described herein to form a similar product. In some embodiments, a strong A nucleophile (including, but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in an unnatural encoded amino acid to form a hydrazone, oxime, or semicarbazone, and, if desired, they can be further reduced in some cases by treatment with a suitable reducing agent. Alternatively, a strong nucleophile can be incorporated into a polypeptide via an unnatural encoded amino acid and used to preferentially react with a ketone or aldehyde group present in a water-soluble polymer.

[0396]

[0243] Although not limited thereto, any molecular weight of PEG, including from about 100 Daltons (Da) to 100,000 Da, or, if desired, greater molecular weights (optionally including 0.1 - 50 kDa or 10 - 40 kDa, but not limited thereto), can be used as actually desired. Branched-chain PEGs can also be used that include, but are not limited to, PEG molecules having an MW in the range of 1 - 100 kDa (including 1 - 50 kDa or 5 - 20 kDa, but not limited thereto). Although not limited thereto, a wide range of PEG molecules are described in the catalogs of Shearwater Polymers, Inc. and Nektar Therapeutics. These catalogs are incorporated herein by reference.

[0244] Generally, at least one end of the PEG molecule is available for reaction with an antibody

[0397]

[0244] Generally, at least one end of the PEG molecule is available for reaction with an antibody For example, PEG can be attached to the non-naturally encoded amino acid as described herein using PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains. If the non-naturally encoded amino acid contains an azide, the PEG will typically contain either an alkyne moiety to effect formation of a [3+2] cycloaddition product, or an activated PEG species (i.e., ester, carbonate) that contains a phosphine group to effect formation of an amide linkage. Alternatively, if the non-naturally encoded amino acid contains an alkyne, the PEG will typically contain an azide moiety to effect formation of a [3+2] Huisgen cycloaddition product. If the non-naturally encoded amino acid contains a carbonyl group, the PEG will typically contain a strong nucleophile (including but not limited to hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) to effect formation of a hydrazone linkage, an oxime linkage, and a semicarbazone linkage, respectively. In another alternative, the reverse orientation of the reactive groups described herein can be used. That is, the azide moiety of the non-naturally encoded amino acid can be reacted with an alkyne-containing PEG derivative.

[0398] In some embodiments, the polypeptide variants having PEG derivatives are The side chain of a cyclic amino acid includes a chemical functionality that is reactive with the chemical functionality present on the side chain of the cyclic amino acid.

[0399] In certain embodiments, the payload is from about 800 Da to about 100,000 Da. It is an azide-containing or acetylene-containing polymer comprising a water-soluble polymer backbone having an average molecular weight of a. The polymer backbone of the water-soluble polymer may be poly(ethylene glycol). However, various water-soluble polymers including, but not limited to, poly(ethylene) glycol and other related polymers including poly(dextran) and poly(propylene glycol) are also suitable for use, and the use of the term PEG or poly(ethylene glycol) is understood to be intended to encompass and include all such molecules. The term PEG includes, but is not limited to, all forms of poly(ethylene glycol) including bifunctional PEG, multi-arm PEG, derivatized PEG, star PEG, branched PEG, pendant PEG (i.e., PEG or related polymer having one or more functional groups pendant from the polymer backbone), or PEG having a cleavable linkage therein.

[0400]

[0247] The polymer backbone may be linear or branched. Branched poly mer backbones are generally known in the art. Typically, a branched polymer has a central branched core portion and a plurality of linear polymer chains linked to the central branched core. PEG is generally used in a branched form that can be prepared by adding ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol, and sorbitol. Also, the central branched portion may be derived from some amino acids such as lysine. Branched poly(ethylene glycol) has a basic form of R(-PEG-OH) mIt can be represented as, in the formula, R is derived from a core part such as glycerol, glycerol oligomer, or pentaerythritol, and m represents the number of arms. Also, multi-arm PEG molecules such as those described in U.S. Patent Nos. 5,932,462, 5,643,575; 5,229,490; 4,289,872; U.S. Patent Application Publication No. 2003 / 0143596; WO96 / 21469; and WO93 / 21259 can be used as the polymer backbone. Each of these documents is hereby incorporated by reference in its entirety.

[0401]

[0248] Also, branched PEG can be in the form of star-shaped PEG represented by PEG(-YCHZ2) n wherein Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of a specified length.

[0402]

[0249] Another branched form, pendant PEG, has a reactive group such as carboxyl along the PEG backbone rather than at the end of the PEG chain.

[0403]

[0250] In addition to these forms of PEG, the polymer can also be prepared to have a weak or degradable linkage in the backbone. For example, PEG having an ester linkage in a polymer backbone susceptible to hydrolysis can be prepared. As shown herein, this hydrolysis results in the polymer being cleaved into lower molecular weight fragments: -PEG-CO2-PEG- + H2O → PEG-CO2H + HO-PEG-. Those skilled in the art will understand that the term poly(ethylene glycol) or PEG represents or includes all forms known in the art including but not limited to those disclosed herein.

[0404]

[0251] Many other polymers are also suitable for use. In some embodiments, 2 to about 300 ​​A polymer backbone of a water-soluble polymer having ends is particularly suitable. Examples of suitable polymers include, but are not limited to, poly(propylene glycol) (“PPG”), etc. Other poly(alkylene glycols), their copolymers (including, but not limited to, copolymers of ethylene glycol and propylene glycol), their terpolymers, and their mixtures, etc. The molecular weight of each chain of the polymer backbone may vary, but typically ranges from about 800 Da to about 100,000 Da, and in many cases from about 6,000 Da to about 80,000 Da.

[0405]

[0252] Those skilled in the art will recognize that the above list of backbones that are substantially water-soluble is by no means exhaustive and is merely illustrative, and that all polymer materials having the qualities described herein are intended to be suitable for use.

[0406]

[0253] In some embodiments, the polymer derivative is “multifunctional,” which means that the polymer backbone has at least two ends, perhaps even up to about 300 ends, that are functionalized or activated with functional groups. Examples of multifunctional polymer derivatives include, but are not limited to, linear polymers having two ends each attached to a functional group that may be the same or different.

[0407] 4. Linker

[0254] In certain embodiments, the antibody may be linked to the payload by one or more linkers capable of reacting with the antibody amino acids and the payload group. The one or more linkers may be any linker apparent to those skilled in the art. The one or more linkers may be any linker apparent to those skilled in the art.

[0408]

[0255] The term “linker” is used herein to generally refer to a group or bond that is formed as a result of a chemical reaction and is typically a covalent linkage. The term “linker” is used herein to generally refer to a group or bond that is formed as a result of a chemical reaction and is typically a covalent linkage.

[0409]

[0256] Useful linkers include those described in this specification. Certain In particular embodiments, the linker is any divalent or polyvalent linker known to those skilled in the art. Useful divalent linkers include alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene, and substituted heteroarylene. In certain embodiments, the linker is C 1~10 alkylene or C 1~10 heteroalkylene. In some embodiments, the C 1~10 heteroalkylene is PEG.

[0410]

[0257] In certain embodiments, the linker is hydrolytically stable. A hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water at useful pH values under conditions including, but not limited to, physiological conditions over a long period of time, perhaps even indefinitely. In certain embodiments, the linker is hydrolytically unstable. A hydrolytically unstable or cleavable linkage means that the linkage is cleavable in water or in an aqueous solution such as blood. An enzymatically unstable or cleavable linkage means that the linkage can be cleaved by one or more enzymes as such.

[0411]

[0258] As understood in the art, PEG and related polymers may contain cleavable linkages in the polymer backbone or in a linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. For example, an ester linkage formed by the reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group on a biologically active agent generally hydrolyzes under physiological conditions to release the agent.

[0412]

[0259] Other hydrolyzable linkages include, but are not limited to, the following ones: carbonate linkages; imine linkages resulting from the reaction of amines and aldehydes; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages that are reaction products of hydrazides and aldehydes; acetal linkages that are reaction products of aldehydes and alcohols; orthoester linkages that are reaction products of formates and alcohols; peptide linkages formed by an amine group, including but not limited to those at the ends of polymers such as PEG, and the carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to those at the ends of polymers, and the 5'-hydroxyl group of an oligonucleotide.

[0413]

[0260] Many different cleavable linkers are known to those skilled in the art. See U.S. Pat. Nos. 4,61 8,492, 4,542,225, and 4,625,014. Mechanisms for releasing an agent from such linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis. For example, U.S. Pat. No. 4,671,958 includes a description of an immunoconjugate that includes a linker that is cleaved at a target site in vivo by a proteolytic enzyme of a patient's complement system. The length of the linker can be predetermined or selected according to the desired spatial relationship between the polypeptide and the molecule linked thereto. One of ordinary skill in the art would be expected to be able to determine a suitable method for attaching a given agent to a polypeptide in light of the numerous reported methods for attaching various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to polypeptides. A suitable method for attaching a given agent to a polypeptide is expected to be determined.

[0414]

[0261] The linker may have a wide range of molecular weights or molecular lengths. Larger or Linkers with smaller molecular weights can be used to provide the desired spatial relationship or conformation between the polypeptide and the linking entity. Linkers with longer or shorter molecular lengths can also be used to provide the desired space or flexibility between the polypeptide and the linking entity. Similarly, linkers with specific shapes or conformations can be used to impart specific shapes or conformations to the polypeptide or the linking entity, either before or after the polypeptide reaches its target. The functional groups present at each end of the linker can be selected to modulate the release of the polypeptide or payload under desired conditions. Such optimization of the spatial relationship between the polypeptide and the linking entity can provide the molecule with new, modulated, or desired properties.

[0415] In some embodiments, the present disclosure provides a polymer comprising: a) at least a first terminal of a polymer backbone; Provided is a water-soluble bifunctional linker having a dumbbell structure comprising: a) an azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety at one end; and b) at least a second functional group at a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group is not reactive with the first functional group. In some embodiments, provided is a water-soluble compound comprising at least one arm of a branched molecular structure. For example, the branched molecular structure may be a dendritic structure.

[0416] In some embodiments, the linker is selected from the group consisting of: - Derived from precursors: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) , N-Succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-Succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (Sulfo-SPDB) , N-Succinimidyl iodoacetate (SIA), N-Succinimidyl (4-iodo acetyl) aminobenzoate (SIAB), maleimide PEG NHS, N-Succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-Sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (Sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5 -dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazapentadecan-1-oate (CX1-1). In certain embodiments , the linker is derived from the linker precursor N-Succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC).

[0417]

[0264] In some embodiments, the linker is derived from a linker precursor selected from the group consisting of dipeptides, tripeptides, tetrapeptides , and pentapeptides. In such embodiments, the linker can be cleaved by proteases. Representative dipeptides include, but are not limited to, valine-citrulline (vc or val-cit) , alanine-phenylalanine (AF or ala-phe), phenylalanine-lysine (FK or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Representative tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit), glycine-glycine -glycine (gly-gly-gly), and glycine-methoxyethoxyethyl) serine-valine -alanine (gly-val-citalanine OMESerValAla).

[0418]

[0265] In some embodiments, the linker comprises a self-cleaving spacer. In certain embodiments, the self-cleaving spacer comprises p-aminobenzyl. In some embodiments, p-aminobenzyl alcohol is attached to an amino acid unit via an amide bond to create a carbamate, methylcarbamate, or carbonate between the benzyl alcohol and the payload (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103 ). In some embodiments, the linker comprises p-aminobenzyloxycarbonyl (PAB). Other examples of self-cleaving spacers include, but are not limited to, 2-aminoimidazole-5-methanol derivatives (U.S. Patent No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237), and aromatic compounds electronically similar to the PAB group such as ortho- or para-aminobenzyl acetate . In some embodiments, substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2 ring systems (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amide (Amsberry, et al. (1990) J. Org. Chem. 55:5867), etc., spacers that cyclize upon amide bond hydrolysis can be used. The linkage of a drug to the α-carbon of a glycine residue is another example of a self-cleaving spacer that can be useful in conjugates (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

[0266] In certain embodiments, linker precursors are combined to form larger linkers .

[0266] In certain embodiments, linker precursors are combined to form larger linkers .

[0419]

[0266] In certain embodiments, linker precursors are combined to form larger linkers can be formed. For example, in certain embodiments, the linker comprises the dipeptide valine-citrulline and p-aminobenzyloxycarbonyl, which are also referred to as the citValCit--PAB linker.

[0420]

[0267] In certain embodiments, the payload may be linked to the linker, which may be referred to herein as the linker-payload, and one or more linker groups are capable of reacting with antibody amino acid groups. The one or more linkers may be any linker that is apparent to one of ordinary skill in the art or as shown herein.

[0421]

[0268] The linker precursor can be prepared as described in the Examples section herein and / or by standard techniques, or may be obtained from commercial sources. See, for example, WO2019 / 055931, WO2019 / 055909, WO2017 / 132617, WO2017 / 132615, which are hereby incorporated by reference in their entireties, respectively.

[0422]

[0269] For example, linker precursors (A)-(H) and (J)-(M ) and other additional linkers as described below are disclosed herein.

[0423] 4.1 Linker-Payload

[0270] In one aspect, provided herein is a linker-payload compound of formula (IV):

[0424]

Chemical Formula

[0425] [Chem.] and subscript b is an integer selected from 1 to 10, each R A , when present, in each occurrence, is independently selected from C 1~3 alkyl, and RT, when present, is a release-inducing group, each HP, when present, is a hydrophilic group, W 6 is a residue of a peptide or is absent, SG is absent or is a divalent spacer group, R is hydrogen or a terminal conjugate group,

[0271] PA is a residue of a compound of formula (I-P), (I), (II), or (III), wherein PA is -NR 3a -, -C(R 3c )2NH- of -NH-, nitrogen of a heterocycloalkyl of R 3 , nitrogen of a partially saturated heteroaryl of R 3 , -O-CH2-(phenyl)-CH2-NH- of -NH-, or is attached to the remainder of the molecule via the nitrogen of ring B. In one embodiment, herein, formula (IV-P):

[0426] [Chem.] is provided, a linker payload compound thereof, or a mixture of its pharmaceutically acceptable salts, solvates, stereoisomers, tautomers, or positional isomers, wherein W 1 is a single bond, is absent, or is a divalent attachment group, X is absent or

[0427] [Chem.] and The subscript letter b is an integer from 1 to 10, R A when present, in each occurrence, independently, is selected from C 1~3 alkyl, RT, when present, is a release-inducing group, HP, when present, is a hydrophilic group, W 6 is a peptide or is absent, SG is absent or is a divalent spacer group, R is hydrogen, a terminal conjugate group, or a divalent residue of a terminal conjugate group, PA is of formula (I)

[0428]

Chemical formula

[0429]

Chemical formula

[0430]

[0272] In some embodiments of formula (IV), PA is any residue of a compound of formula (I), (II), or (III) described herein or any group of a compound.

[0431]

[0273] In some embodiments, the compound according to formula (IV) is of formula (IVa), (IVb), (IVc), (IVd), or (IVe):

[0432]

Chem.

[0433]

Chem.

[0434]

[0274] In some examples of formula (IV), SG is absent or

[0435]

Chem.

[0436]

Chem.

[0437]

[0275] In some examples of formula (IV), SG is

[0438]

Chem.

[0439]

Chem.

[0440]

[0276] In some examples of formula (IV), W 1 when present,

[0441]

Chem.

[0442]

Chem.

[0443]

[0277] In some examples of formula (IV), W 1 when present,

[0444]

Chem.

[0445]

Chem.

[0446]

[0278] In some examples of formula (IV), W 6 is a residue of a peptide, natural and / or contains non-natural amino acids. In some examples of formula (IV), W 6 is, when present, a tripeptide residue. In some examples of formula (IV), W 6 is, when present,

[0447]

Chemical formula

[0448]

Chemical formula

[0449]

[0279] In some examples of formula (IV), W 6 is, when present, a dipeptide residue. In formula (IV), in some examples, W 6 is, when present,

[0450]

Chemical formula

[0451]

Chemical formula

[0452]

[0280] In some examples of formula (IV), RT is

[0453]

Chemical formula

[0454]

Chemical formula

[0455]

[0281] In some examples of formula (IV), HP, when present, is a PEG group. Formula (IV) In some examples, HP, when present,

[0456]

Chemical formula

[0457]

Chemical formula

[0458]

[0282] In some examples of formula (IV), R is

[0459]

Chemical formula

[0460]

Chemical formula

[0461]

[0283] In some embodiments, the linker-payload compound of formula (VI) is

[0462]

Chemical formula

[0463]

Chemical formula

[0464] 5. Antibody Specificity

[0284] The conjugate includes an antibody that selectively binds to a human antigen. In some embodiments the antibody binds to a homolog of the human antigen. In some aspects, the antibody binds to a homolog of the human antigen that is derived from a species selected from monkey, mouse, dog, cat, rat, cow, horse, goat, and sheep. In some aspects, the homolog is a cynomolgus monkey homolog. In some aspects, the homolog is a mouse or mouse homolog.

[0465]

[0285] In some embodiments, the antibody includes a light chain. In some aspects, the light chain is a kappa light chain In some aspects, the light chain is a lambda light chain.

[0466]

[0286] In some embodiments, the antibody includes a heavy chain. In some aspects, the heavy chain is IgA In some aspects, the heavy chain is IgD. In some aspects, the heavy chain is IgE. In some aspects, the heavy chain is IgG. In some aspects, the heavy chain is IgM. In some aspects, the heavy chain is IgG1. In some aspects, the heavy chain is IgG2. In some aspects, the heavy chain is IgG3. In some aspects, the heavy chain is IgG4. In some aspects, the heavy chain is IgA1. In some aspects, the heavy chain is IgA2.

[0467]

[0287] In some embodiments, the antibody is an antibody fragment. In some aspects, the antibody fragment is an Fv fragment. In some aspects, the antibody fragment is a Fab fragment. In some aspects, the antibody fragment is an F(ab')2 fragment. In some aspects, the antibody fragment is a Fab’ fragment. In some aspects, the antibody fragment is a scFv (sFv) fragment. In some aspects, the antibody fragment is an s cFv-Fc fragment.

[0468]

[0288] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0469]

[0289] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an affinity matured antibody.

[0470]

[0290] The antibody conjugates provided herein may be useful for the treatment of various diseases and conditions including cancer (e.g., any cancer described herein). In some embodiments, the antibody conjugates provided herein may be useful for the treatment of solid tumor cancers.

[0471] 6. Glycosylation Variants

[0291] In certain embodiments, the antibody may be modified to increase, decrease, or eliminate the degree of glycosylation. Glycosylation of polypeptides is typically either “N-linked” or “O-linked”.

[0472]

[0292] “N-linked” glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of carbohydrate moieties to asparagine side chains. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.

[0473]

[0293] “O-linked” glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0474] ​​​​

[0294] Addition or deletion of N-linked glycosylation sites to the antibody can be achieved by altering the amino acid sequence such that one or more of the tripeptide sequences are created or removed. Addition or deletion of O-linked glycosylation sites can be achieved by adding, deleting, or substituting one or more serine or threonine residues within or relative to the antibody sequence (where applicable).

[0475] 7. Modified Amino Acids

[0295] If the antibody conjugate contains a modified amino acid, the modified amino acid can be any modified amino acid that the person skilled in the art deems suitable. In certain embodiments, the modified amino acid contains a reactive group useful for forming a covalent bond with a linker precursor or payload precursor. In certain embodiments, the modified amino acid is a non-natural amino acid. In certain embodiments, the reactive group is selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazide, semicarbazide, sulfanyl, azide, and alkynyl. Modified amino acids are also described, for example, in WO2013 / 185115 and WO2015 / 006555. Each of these documents is hereby incorporated by reference in its entirety into this specification.

[0476]

[0296] The terms "residue of an amino acid" and "amino acid residue" refer to the product of amide coupling or peptide coupling of an amino acid to a suitable coupling partner. For example, a water molecule is expelled after amide or peptide coupling of an amino acid, resulting in a product in which the amino acid residue is incorporated. In some embodiments, the amino acid residue is

[0477]

Chem.

[0478]

Chem.

[0479]

[0297] The terms "residue of a peptide" and "peptide residue" refer to the product of amide coupling or peptide coupling to a suitable coupling partner of an amino acid. For example, a water molecule is eliminated after amide or peptide coupling of an amino acid, resulting in a product in which a peptide residue is incorporated. In some embodiments, the peptide residue is

[0480]

Chem.

[0481]

Chem.

[0482]

[0298] In certain embodiments, the amino acid residue has the following formula:

[0483]

Chem.

[0484]

[0299] In the above formula, the wavy line indicates a bond connecting to the rest of the polypeptide chain of the antibody. Such non - natural amino acids can be incorporated into the polypeptide chain in exactly the same manner as natural amino acids when incorporated into the same polypeptide chain. In certain embodiments, the non - natural amino acid is incorporated into the polypeptide chain via an amide bond as shown in the formula.

[0485]

[0300] In the above formula, R represents any functional group, without limitation, provided that the amino acid residue is not the same as a natural amino acid residue. In certain embodiments, R may be a hydrophobic group, a hydrophilic group, a polar group, an acidic group, a basic group, a chelating group, a reactive group, a therapeutic moiety, or a labeling moiety. In certain embodiments, R is selected from the group consisting of R , NR 1 NR 2z R 3z , R 1z C(=O)R 2z , R 1z C(=O)OR 2z , R 1z N3, R 1z C(≡CH). In such embodiments, R 1z is selected from the group consisting of a bond, alkylene, heteroalkylene, arylene, heteroarylene. R 2z and R 3z are each independently selected from the group consisting of hydrogen, alkyl, and heteroalkyl.

[0486]

[0301] In some embodiments, the non - natural encoded amino acid is not found among the 20 common amino acids The non-naturally encoded amino acid comprises a side chain functional group that reacts efficiently and selectively with non-naturally encoded functional groups (including, but not limited to, azide groups, ketone groups, aldehyde groups, and aminooxy groups) to form a stable conjugate. For example, an antigen-binding polypeptide comprising a non-naturally encoded amino acid containing an azide functional group can be conjugated to a polymer (including, but not limited to, poly(ethylene glycol)). The azide functional group can be reacted with a second polypeptide that does not contain an azide moiety (or a second polypeptide that instead contains an alkyne moiety) to form a stable conjugate, resulting in selective reaction of the azide functional group and the alkyne functional group to form a Huisgen [3+2] cycloaddition product.

[0487] Representative examples of compounds that may be suitable for use in the present invention and are useful for reacting with water-soluble polymers include: Suitable non-naturally encoded amino acids include, but are not limited to, those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In some embodiments, the non-naturally encoded amino acid comprises a saccharide moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-linkage between the amino acid and the saccharide is replaced with a covalent linkage not normally found in nature, including, but not limited to, an alkene, an oxime, a thioether, and an amide. Examples of such amino acids also include saccharides not normally found in naturally occurring proteins, such as 2-deoxy-glucose and 2-deoxygalactose.

[0488]

[0303] Many of the non-naturally encoded amino acids provided herein are available from, e.g., Sigma-Aldrich Available from Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington , MA, USA). Those not commercially available are optionally synthesized as provided herein or using standard methods known to those skilled in the art. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic C hemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Ca rey and Sundberg's Advanced Organic Chemistry (Third Edition, Parts A and B, 1990, Plenum Press, New York). See also U.S. Patent Application Publication Nos. 2003 / 0082575 and 2003 / 0108885. These documents are incorporated by reference. In addition to non-natural amino acids containing non-natural side chains, non-natural amino acids that may be suitable for use in the present invention optionally include, but are not limited to, the structures of Formulas II and III:

[0489]

Chemical Formula

[0490]

[0304] Many unnatural amino acids are based on tyrosine, glutamine, and phenylalanine and are suitable for use in the present invention. Examples of tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, and the substituted tyrosine includes, but is not limited to, a keto group (including but not limited to an acetyl group), a benzoyl group, an amino group, hydrazine, hydroxylamine, a thiol group, a carboxyl group, an isopropyl group, a methyl group, C6~C 20 linear or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl group, polyether group, nitro group, or alkynyl group, etc. In addition, multiply substituted aryl rings are also contemplated. Examples of glutamine analogs that may be suitable for use in the present invention include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives Examples of phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, and the substituents include, but are not limited to, hydroxy group, methoxy group, methyl group, allyl group, aldehyde, azide, iodine, bromo, keto group (including but not limited to acetyl group), benzoyl, or alkynyl group, etc. Specific examples of unnatural amino acids that may be suitable for use in the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-azido-methyl-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine, etc. Examples of the structures of various unnatural amino acids that may be suitable for use in the present invention are provided, for example, in WO2002 / 085923 entitled "In vivo incorporation of unnatural amino acids". For additional methionine analogs, see also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24.

[0491]

[0305] Many of the unnatural amino acids suitable for use in the present invention are, for example, Sigma (USA or commercially available from Aldrich (Milwaukee, Wisconsin, USA). Those not commercially available are optionally synthesized as provided herein, or as provided in various publications, or using standard methods known to those skilled in the art. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of unnatural amino acids include the following: for example, WO2002 / 085923 entitled "In vivo incorporation of Unnatural Amino Acids"; Matsoukas et al., as provided, or using standard methods known to those skilled in the art, are optionally synthesized. For organic synthesis techniques, see, for example, Organic Chemistry by Fessendon and Fessendon (1982, Second Edition, Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of unnatural amino acids include the following: for example, WO2002 / 085923 entitled "In vivo incorporation of Unnatural Amino Acids"; Matsoukas et al. See Advanced Organic Chemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York). Additional publications describing the synthesis of unnatural amino acids include the following: for example, WO2002 / 085923 entitled "In vivo incorporation of Unnatural Amino Acids"; Matsoukas et al.,(1995)J.Med.Chem.,38,4660-4669;King,F.E.&Kidd,D.A.A.(1949)A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates.J.Chem.Soc.,3315-3319;Friedman,O.M.&Chatterrji,R.(1959)Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents.J.Am.Chem.Soc.81,3750-3752;Craig,J.C.et al.(1988)Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine).J.Org.Chem.53,1167-1170;Azoulay,M.,Vilmont,M.&Frappier,F.(1991)Glutamine analogues as Potential Antimalarials,Eur.J.Med.Chem.26,201-5;Koskinen,A.M.P.&Rapoport,H.(1989)Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues.J.Org.Chem.54,1859-1866;Christie,B.D.&Rapoport,H.(1985)Synthesis of Optically Pure Pipecolates from L-Asparagine. Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization.J.Org.Chem.1989:1859-1866;Barton et al.,(1987)Synthesis of Novel a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of. L- and D-a-Amino-Adipic Acids,L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives.Tetrahedron Lett.43:4297-4308;and Subasinghe et al.,(1992)Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site.J.Med.Chem.35:4602-7.See also U.S. Patent Application No. 10 / 744,899, entitled "Protein Arrays," filed December 22, 2003, and No. 60 / 435,821, filed December 22, 2002.

[0492]

[0306] Amino acids having a carbonyl-reactive group enable various reactions for linking molecules (including but not limited to PEG or other water-soluble molecules) by nucleophilic addition or aldol condensation reactions.

[0307] Representative carbonyl-containing amino acids are

[0493]

[0307] Representative carbonyl-containing amino acids are

[0494]

Chemical formula

[0495]

[0308] In some examples, a non-natural coded amino acid that retains adjacent hydroxyl and amino groups can be incorporated into a polypeptide as a "masked" aldehyde functionality. For example, 5-hydroxylysine retains a hydroxyl group adjacent to the epsilon amine. The reaction conditions for generating the aldehyde typically include adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction includes adding about a 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, for example, U.S. Patent No. 6,423,685. This document is incorporated herein by reference.

[0496]

[0309] The carbonyl functionality can be converted to a hydrazine-containing reagent under mild conditions in an aqueous solution, It can react selectively with hydrazide-, hydroxylamine-, or semicarbazide-containing reagents to form the corresponding hydrazone, oxime, or semicarbazone linkages, respectively, that are stable under physiological conditions. See, e.g., Jencks, WP, J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117: 3893-3899 (1995). Furthermore, the unique reactivity of the carbonyl group allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, VW, et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, et al., Science 276:1125-1128 (1997).

[0497]

[0310] Non-naturally occurring cobalt compounds containing nucleophilic groups such as hydrazine, hydrazide, or semicarbazide. The amino acids react with a variety of electrophilic groups to allow for the formation of conjugates (PEG or other water soluble polymers).

[0498]

[0311] Representative hydrazine-containing amino acids, hydrazide-containing amino acids, or semical Basidio-containing amino acids are

[0499] [ka] where n is 0-10; R1 is alkyl, aryl, substituted alkyl, substituted aryl, or absent; X is O, N, S, or absent; R2 is H, an amino acid, a polypeptide, or an amino terminus modification group; and R3 is H, an amino acid, a polypeptide, or a carboxy terminus modification group.

[0500]

[0312] In some embodiments, n is 4, R1 is absent, and X is N. In some embodiments, n is 2, R1 is absent, and X is absent. In some embodiments, n is 1, R1 is phenyl, X is O, and the oxygen atom is para to the aliphatic group on the aryl ring.

[0501]

[0313] Hydrazide-containing amino acids, hydrazine-containing amino acids, and semicarbazide-containing amino acids are available from commercial suppliers. For example, L-glutamate-γ-hydrazide is available from Sigma Chemical (St. Louis, Missouri). Other amino acids that are not commercially available can be prepared by those skilled in the art. See, for example, U.S. Patent No. 6,281,211. This document is incorporated herein by reference.

[0502]

[0314] Polypeptides containing unnatural coded amino acids that retain hydrazide functionality, hydrazine functionality, or semicarbazide functionality can react efficiently and selectively with various molecules containing aldehydes or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995). Hydrazide functional groups, hydrazine functional groups, and semicarbazide functional groups are significantly more reactive towards aldehydes, ketones, and other electrophilic groups compared to the nucleophilic groups (including, but not limited to, the hydroxyl groups of serine or threonine, or the amino groups of lysine and the N-terminus) present in the 20 common amino acids due to their unique reactivity.

[0503]

[0315] Unnatural coded amino acids containing an aminooxy (also called hydroxylamine) group react with various electrophilic groups to conjugate (PEG or other water-soluble polymers to It is possible to form (including but not limited to these). Similar to hydrazine, hydrazide, and semicarbazide, the enhanced nucleophilicity of the aminooxy group enables efficient and selective reactions with various molecules containing aldehydes or other functional groups with similar chemical reactivity. See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893 - 3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727 - 736 (2001). However, the result of the reaction with a hydrazine group is the corresponding hydrazone, while the reaction of an aminooxy group with a carbonyl - containing group such as a ketone generally yields an oxime.

[0504]

[0316] Representative amino acids containing an aminooxy group are

[0505]

Chemical formula

[0506]

[0317] Aminooxy - containing amino acids can be prepared from readily available amino acid precursors (homoserine , and can be prepared from serine and threonine). See, for example, M. Carrasco and R. Brown, J. Org. Chem. 68:8853 - 8858 (2003). Certain aminooxy - containing amino acids, such as L - 2 - amino - 4-(aminooxy)butyric acid, have been isolated from natural sources (Rosenthal, G. et al., Life Sci. 60: 1635 - 1641 (1997)). One of ordinary skill in the art can prepare other aminooxy - containing amino acids can do.

[0507]

[0318] Azide and alkyne functional groups are extremely useful for the selective modification of polypeptides peptides and other biological molecules due to their unique reactivity. Organic azides, especially aliphatic azides, and alkynes are generally stable to common reaction chemical conditions. In particular, both azide and alkyne functional groups are inert to the side chains (i.e., R - groups) of the 20 common amino acids found in naturally occurring polypeptides. However, when in close proximity, the "spring - loaded" nature of the azide and alkyne groups is exerted, and they react selectively and efficiently by the Huisgen [3 + 2] cycloaddition reaction to produce the corresponding triazole. See, for example, Chin J., et al., Science 301:964 - 7 (2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192 - 3193 (2003); Chin, J.W., et al., J. Am. Chem. Soc. 124:9026 - 9027 (2002).

[0508]

[0319] The Huisgen cycloaddition reaction involves a selective cycloaddition reaction rather than nucleophilic substitution, so (See, for example, Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176), incorporation of unnatural code amino acids that hold azide-containing side chains and alkyne-containing side chains enables the resulting polypeptide to be selectively modified at the positions of the unnatural code amino acids. The cycloaddition reaction involving an azide-containing antibody or an alkyne-containing antibody can be carried out at room temperature under aqueous conditions by adding a catalytic amount of Cu(II) (including but not limited to the form of catalytic amount of CuSO4) in the presence of a reducing agent for reducing Cu(II) to Cu(I) in situ. See, for example, Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al., Angew. Chem. Int. Ed. 41:2596-2599 (2002). Representative reducing agents include, but are not limited to, ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe 2+ , Co 2+ , and applied potential.

[0509]

[0320] In some cases, when the Huisgen [3+2] cycloaddition reaction between azide and alkyne is desired, the antigen-binding polypeptide contains an unnatural code amino acid containing an alkyne moiety, and the water-soluble polymer to be attached to the amino acid contains an azide moiety. Alternatively, the reverse reaction (i.e., between the azide moiety in the amino acid and the alkyne moiety present in the water-soluble polymer) can also be carried out.

[0510] ​​

[0321] Also, the azide functional group includes an aryl ester and can selectively react with a water-soluble polymer appropriately functionalized at the arylphosphine moiety to form an amide linkage. The arylphosphine group reduces the azide in situ, and the resulting amine then efficiently reacts with a neighboring ester linkage to form the corresponding amide. See, for example, E. Saxon and C. Bertozzi, Science 287, 2007-2010 (2000). The azide-containing amino acid can be either an alkyl azide (including but not limited to 2-amino-6-azido-1-hexanoic acid) or an aryl azide (p-azido-phenylalanine).

[0511]

[0322] Representative water-soluble polymers containing an aryl ester and a phosphine moiety are

[0512]

Chemical formula

[0513]

[0323] Further, the azide functional group contains a thioester and can selectively react with a water-soluble polymer functionalized appropriately with an arylphosphine moiety to form an amide linkage. The arylphosphine group reduces the azide in situ, and the resulting amine then efficiently reacts with the thioester linkage to produce the corresponding amide. The thioester And representative water-soluble polymers containing phosphine moieties are,

[0514] [Chemical formula] which can be represented as, where n is from 1 to 10, X is O, N, S, or may be absent, Ph is phenyl, and W is a water-soluble polymer.

[0515]

[0324] Representative alkyne-containing amino acids are,

[0516] [Chemical formula] which can be represented as, where n is from 0 to 10, R1 is alkyl, aryl, substituted alkyl, or substituted aryl, or is absent, X is O, N, S, or is absent, m is from 0 to 10, R2 is H, an amino acid, a polypeptide, or an amino-terminal modifying group, and R3 is H, an amino acid, a polypeptide, or a carboxy-terminal modifying group. In some embodiments, n is 1, R1 is phenyl, X is absent, m is 0, and the acetylene moiety is para to the alkyl side chain. In some embodiments, n is 1, R1 is phenyl, X is O, m is 1, and the propargyloxy group is para to the alkyl side chain (i.e., O-propargyl-tyrosine). In some embodiments, n is 1, R1 and X are absent, and m is 0 (i.e., propargylglycine).

[0517]

[0325] Alkyne-containing amino acids are commercially available. For example, propargylglycine is, It is commercially available from Peptech (Burlington, Massachusetts). Instead, alkynyl-containing amino acids can be prepared according to standard methods. For example, for instance, p-propynyloxyphenylalanine can be synthesized as described in Deiters, A., et al., J. Am. Chem. Soc. 125:11782-11783 (2003), and 4-alkynyl-L-phenylalanine can be synthesized as described in Kayser, B., et al., Tetrahedron 53(7):2475-2484 (1997). Those skilled in the art can prepare other alkynyl-containing amino acids.

[0518]

[0326] Representative azide-containing amino acids are

[0519]

Chemical formula

[0520]

[0327] Azide-containing amino acids are available from commercial suppliers. For example, 4-azi Diphenylalanine can be obtained from Chem-Impex International, Inc. (Wood Dale, Illinois). In the case of azide-containing amino acids that are not commercially available, the azide group can be relatively easily prepared using standard methods known to those skilled in the art, including but not limited to replacing suitable leaving groups (including but not limited to halides, mesylates, tosylates) or ring-opening a suitably protected lactone. See, for example, Advanced Organic Chemistry by March (Third Edition, 1985, Wiley and Sons, New York).

[0521]

[0328] Due to its unique reactivity, the beta-substituted aminothiol functional group is extremely useful for the selective modification of polypeptides and other biological molecules containing an aldehyde group by the formation of thiazolidine. See, for example, J. Shao and J. Tam, J. Am. Chem. Soc. 1995, 117(14) 3893 - 3899. In some embodiments, a beta-substituted aminothiol amino acid can be incorporated into an antibody and then reacted with a water-soluble polymer containing an aldehyde functionality. In some embodiments, a water-soluble polymer, drug conjugate, or other payload can be coupled to an antibody polypeptide containing a beta-substituted aminothiol amino acid by the formation of thiazolidine.

[0522]

[0329] Specific examples of useful unnatural amino acids include, but are not limited to, the following Examples include: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc b-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-methyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propynyloxy-phenylalanine. Further useful examples include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine.

[0523]

[0330] In certain embodiments, the non-natural amino acid is selected from p-acetyl-phenylalanine, p-ethynyl-phenylalanine, p-propynyloxyphenylalanine, p-azido-methyl-phenylalanine, and p-azido-phenylalanine. One particularly useful non-natural amino acid is p-azidophenylalanine. It is known to those skilled in the art that this amino acid residue facilitates, for example, the Huisgen [3+2] cycloaddition reaction (so-called "click" chemical reaction) with a compound bearing an alkynyl group. By this reaction, those skilled in the art can easily and rapidly conjugate the antibody at the site-specific position of the non-natural amino acid.

[0524]

[0331] In certain embodiments, the first reactive group is an alkynyl moiety (but not limited to However, it contains the unnatural amino acid p-propynyloxyphenylalanine, where the propynyl group may also be referred to as an acetylene moiety), and the second reactive group is an azide moiety, and [3+2] cycloaddition chemistry can be used. In certain embodiments, the first reactive group is an azide moiety (including but not limited to containing the unnatural amino acid p-azido-L-phenylalanine), and the second reactive group is an alkynyl moiety.

[0525]

[0332] In the above formula, each L represents a divalent linker. The divalent linker can be any divalent linker known to those skilled in the art. Generally, the divalent linker can form a covalent bond with the functional moiety R and the analogous reactive group (e.g., the alpha carbon) of the unnatural amino acid. Useful divalent linkers include bonds, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene, and substituted heteroarylene. In certain embodiments, L is C alkylene or C 1~10 alkylene or C 1~10 heteroalkylene.

[0526]

[0333] The unnatural amino acids used in the methods and compositions described herein are the following four Having at least one of the following characteristics: (1) at least one functional group in the side chain of the unnatural amino acid has at least one feature and / or activity and / or reactivity that is orthogonal to the chemical reactivity of the 20 commonly genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or at least orthogonal to the chemical reactivity of the naturally occurring amino acids present in the polypeptide containing the unnatural amino acid; (2) the introduced unnatural amino acid is chemically substantially inert with respect to the 20 commonly genetically encoded amino acids; (3) the unnatural amino acid can preferably be stably incorporated into the polypeptide with a stability equivalent to that of the naturally occurring amino acids or under typical physiological conditions, and more preferably, such incorporation can be caused by an in vivo system; (4) the unnatural amino acid contains a functional group that can be converted to an oxime group by reacting with a reagent under conditions that do not disrupt the biological properties of the polypeptide containing the unnatural amino acid (of course, unless such disruption of biological properties is for the purpose of modification / transformation), or the conversion can be caused under aqueous conditions at a pH between about 4 and about 8, or the reactive site in the unnatural amino acid is an electrophilic site. Any number of unnatural amino acids can be introduced into the polypeptide. Also, the unnatural amino acid may contain a protecting or shielding oxime, or a protecting or shielding group that can be converted to an oxime group after deprotection of the protecting group or deprotection of the shielding group. Also, the unnatural amino acid may contain a protecting or shielding carbonyl group or dicarbonyl group, which can be converted to a carbonyl group or dicarbonyl group after deprotection of the protecting group or deprotection of the shielding group, thereby being available for reaction with hydroxylamine or oxime to form an oxime group.

[0527]

[0334] In a further embodiment, non-natural amino acids that can be used in the methods and compositions described herein include, but are not limited to, the following: amino acids containing photoactivatable crosslinkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids having novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photo-caged and / or photo-isomerizable amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other carbohydrate-modified amino acids, keto-containing amino acids, aldehyde-containing amino acids, amino acids containing polyethylene glycol or other polyethers, heavy atom-substituted amino acids, chemically and / or photocleavable amino acids, amino acids having extended side chains compared to natural amino acids, including, but not limited to, polyethers, or long-chain hydrocarbons containing more than about 5 or more than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothio acids containing amino acids, and amino acids containing one or more toxic moieties. Examples of non-natural amino acids that can be used in the methods and compositions described herein include, but are not limited to, the following: amino acids containing photoactivatable crosslinkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids having novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photo-caged and / or photo-isomerizable amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other carbohydrate-modified amino acids, keto-containing amino acids, aldehyde-containing amino acids, amino acids containing polyethylene glycol or other polyethers, heavy atom-substituted amino acids, chemically and / or photocleavable amino acids, amino acids having extended side chains compared to natural amino acids, including, but not limited to, polyethers, or long-chain hydrocarbons containing more than about 5 or more than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothio acids containing amino acids, and amino acids containing one or more toxic moieties.

[0528]

[0335] In some embodiments, the non-natural amino acid comprises a saccharide moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the naturally occurring N-linked or O-linked between the amino acid and the saccharide is replaced by a covalent linkage not normally found in nature, including, but not limited to, alkenes, oximes, thioethers, and amides. Examples of such amino acids also include those containing saccharides not normally found in naturally occurring proteins, such as 2-deoxy-glucose and 2-deoxygalactose.

[0529]

[0336] Incorporation of chemical moieties incorporated into antibodies by non-natural amino acids provides various advantages and manipulations of polypeptides. For example, carbonyl or dicarbonyl functional groups (including keto or aldehyde functional groups), due to their unique reactivity, enable selective modification of antibodies having any of several hydrazine-containing or hydroxylamine-containing reagents both in vivo and in vitro. Heavy atom non-natural amino acids can be useful, for example, for phasing of X-ray structural data. Site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing the position of the heavy atoms. Photo-reactive non-natural amino acids (including but not limited to amino acids having benzophenone and aryl azide (including but not limited to phenyl azide) side chains) enable, for example, efficient in vivo and in vitro photocrosslinking of polypeptides. Examples of photo-reactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Antibodies having photo-reactive non-natural amino acids can then be freely crosslinked by excitation of the photo-reactive groups to provide temporal control. In non-limiting examples, the methyl group of non-natural amino can be replaced with, but is not limited to, an isotope-labeled methyl group and used as a probe for local structure and dynamics using, but not limited to, nuclear magnetic resonance and vibrational spectroscopy.

[0530]

[0337] Amino acids having electrophilic reactive groups enable various reactions for linking molecules by various chemical reactions including but not limited to nucleophilic addition reactions. Such electrophilic reactive groups include carbonyl or dicarbonyl groups (including keto or aldehyde groups), carbonyl-like or dicarbonyl-like groups (having reactivity similar to carbonyl or dicarbonyl groups and structurally similar to carbonyl or dicarbonyl groups), masked carbonyl or masked dicarbonyl groups (which can be easily converted to carbonyl or dicarbonyl groups), or protected carbonyl or protected dicarbonyl groups (which has the same reactivity as a carbonyl group or a dicarbonyl group upon deprotection) can be mentioned. Such amino acids include the structure of formula (AA):

[0531] [Chemical formula] including the amino acids represented by, wherein A is optional and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, lower alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -, wherein k is 1, 2, or 3; -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’’)-, -NR’’-(alkylene or substituted alkylene)-, -C(O)N(R’’)-, -CON(R’’)-(alkylene or substituted alkylene)-, -CSN(R’’)-, -CSN(R’’)-(alkylene or substituted alkylene)-, -N(R’’)CO-(alkylene or substituted alkylene)-, -N(R’’)C(O)O-, -S(O) k N(R’’)-, -N(R’’)C(O)N(R’’)-, -N(R’’)C(S)N(R’’)-, -N(R’’)S(O) ka linker selected from the group consisting of -N(R'')-, -N(R'')-N=, -C(R'')=N-, -C(R'')=N-N(R''), -C(R'')=N-N=, -C(R'')2-N=N-, and -C(R'')2-N(R'')-N(R''), wherein each R'' in B is independently H, alkyl, or substituted alkyl, and J is

[0532]

Chemical formula

[0533]

[0338] In certain embodiments, the compound of formula (AA) is at least 1 under weakly acidic conditions It is stable in an aqueous solution over several months. In certain embodiments, the compound of formula (AA) is stable for at least two weeks under weakly acidic conditions. In certain embodiments, the compound of formula (AA) is stable for at least five days under weakly acidic conditions. In certain embodiments, such acidic conditions are pH 2-8.

[0534]

[0339] In certain embodiments of the compound of formula (AA), B is lower alkylene, substituted lower alkylene, -O-(alkylene or substituted alkylene)-, -C(R'')=N-N(R'')-, -N(R'')CO-, -C(O)-, -C(R'')=N-, -C(O)-(alkylene or substituted alkylene)-, -CON(R'')-(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In certain embodiments of the compound of formula (AA), B is -O(CH2)-, -CH=N-, -CH=N-NH-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CH2)-, -SCH2-, -S(=O)CH2-, or -S(O)2CH2-. In certain embodiments of the compound of formula (AA), R is C1-6 alkyl or cycloalkyl. In certain embodiments of the compound of formula (AA), R is -CH3, -CH(CH3)2, or cyclopropyl. In certain embodiments of the compound of formula (AA), R1 is H, tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetraflu It is Oroacetyl (TFA) or Benzyloxycarbonyl (Cbz). In certain embodiments of the compound of formula (AA), R1 is a resin, an amino acid, a polypeptide, or a polynucleotide. In certain embodiments of the compound of formula (AA), R2 is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of the compound of formula (AA), R2 is a resin, an amino acid, a polypeptide, or a polynucleotide. In certain embodiments of the compound of formula (AA), R2 is a polynucleotide. In certain embodiments of the compound of formula (AA), R2 is ribonucleic acid (RNA). In certain embodiments of the compound of formula (AA), R2 is tRNA. In certain embodiments of the compound of formula (AA), the tRNA specifically recognizes a selector codon. In certain embodiments of the compound of formula (AA), the selector codon is selected from the group consisting of an amber codon, an ochre codon, an opal codon, a unique codon, a rare codon, a non-natural codon, a five-base codon, and a four-base codon. In certain embodiments of the compound of formula (AA), R2 is an inhibitory tRNA.

[0535]

[0340] In certain embodiments of the compound of formula (AA),

[0536]

Chemical formula

[0537] [Chemistry] and each R' in J is, independently, H, alkyl, or substituted alkyl, R1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, R2 is optional and when present is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide, each R3 and R4 is, independently, H, halogen, lower alkyl, or substituted lower alkyl, and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0538]

[0341] In certain embodiments, the unnatural amino acid is of formula BB:

[0539] [Chemistry] or a salt thereof, wherein D is -Ar-W3- or -W1-Y1-C(O)-Y2-W2-, Ar is

[0540] [Chemistry] and each of W1, W2, and W3 is, independently, a single bond or lower alkylene, each X1 is, independently, -NH-, -O-, or -S-, each Y1 is, independently, a single bond, -NH-, or -O-, each Y2 is, independently, a single bond, -NH-, -O-, or N-linked or C-linked pyrrolidinylene, one of Z1, Z2, and Z3 is -N-, and the others of Z1, Z2, and Z3 are, independently, -CH-. In certain embodiments, the unnatural amino acid is of formula BBa:

[0541] [Chemistry] which is as defined in the context of formula BB, wherein D is as defined in the context of formula BB. In certain embodiments, the unnatural amino acid is of formula BBb:

[0542] [Chemical formula] or a salt thereof, wherein W4 is C1-C 10 alkylene. In further embodiments, W4 is C1-C5 alkylene. In one embodiment, W4 is C1-C3 alkylene. In one embodiment, W4 is C1 alkylene. In certain embodiments, the unnatural amino acid is

[0543] [Chemical formula]

[0544] [Chemical formula] or a salt thereof. Such unnatural amino acids may be in salt form, or incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may optionally be post-translationally modified.

[0545]

[0342] In certain embodiments, the modified amino acid is of formula CC:

[0546] [Chemical formula] or a salt thereof, wherein Ar is

[0547] [Chemical formula] and V is a single bond, lower alkylene, or -W1-W2-, where one of W1 and W2 is absent or is lower alkylene and the other is -NH-, -O-, or -S-, each X1 is independently -NH-, -O-, or -S-, one of Z1, Z2, and Z3 is -CH- or -N-, the others of Z1, Z2, and Z3 are each independently -CH-, and R is lower alkyl. In certain embodiments, Ar is

[0548] [Chemical formula] and V is -NH-, in which case one of Z1, Z2, and Z3 is -N-. In certain embodiments, V is a single bond, -NH-, or -CH2NH-.

[0549]

[0343] In certain embodiments, Ar is

[0550] [Chemical formula] and Z1, Z2, Z3, and X1 are as defined in the context of Formula CC. In certain embodiments according to this paragraph, V is -W1-W2-, where one of W1 and W2 is absent or is -CH2- and the other is -NH-, -O-, or -S-. In certain embodiments according to this paragraph, V is a single bond, -NH-, or -CH2NH-. In certain embodiments according to this paragraph, Z1 is N. In certain embodiments according to this paragraph, Z2 is N. In certain embodiments according to this paragraph, Z3 is N. In certain embodiments according to this paragraph, Z1 is CH, Z3 is CH, and X1 is S.

[0551]

[0344] In certain embodiments, Ar is

[0552] [Chemical formula] and Z1, Z2, and Z3 are as defined in the context of formula CC. In certain embodiments according to this paragraph, V is -W1-W2-, where either W1 or W2 is absent or is -CH2-, and the other is -NH-, -O-, or -S-. In certain embodiments according to this paragraph, V is a single bond, -NH-, or -CH2NH-. In certain embodiments according to this paragraph, Z1 is N. In certain embodiments according to this paragraph, Z2 is N. In certain embodiments according to this paragraph, Z3 is N.

[0553]

[0345] In certain embodiments, Ar is

[0554]

Chem.

[0555]

[0346] In certain embodiments, the modified amino acid is of formula CCa:

[0556]

Chem.

[0557]

[0347] In one embodiment, a compound of either formula CC or CCa is provided, wherein V is a single bond. In another embodiment, a compound of either formula CC or CCa is provided, wherein V is -NH-. In another embodiment, a compound of either formula CC or CCa is provided, wherein V is -CH2NH-.

[0558]

[0348] In certain embodiments, the modified amino acid is of formula DD:

[0559]

Chem.

[0560]

[0349] In certain embodiments, the modified amino acid is of formula EE:

[0561]

Chem.

[0562]

[0350] In certain embodiments, the modified amino acid is of formula FF:

[0563]

Chem.

[0564]

[0351] In certain embodiments, the modified amino acid is of formula GG:

[0565]

Chemical formula

[0566]

[0352] In certain embodiments, the modified amino acid is of formula HH:

[0567]

Chemical formula

[0568]

[0353] In certain embodiments, the modified amino acid is of formula JJ:

[0569]

Chemical formula

[0570]

[0354] In certain embodiments, the modified amino acid is of formula KK:

[0571]

Chemical formula

[0572]

[0355] In certain embodiments, the modified amino acid is of formula LL:

[0573]

Chemical formula

[0574]

[0356] In certain embodiments, the modified amino acid is of Formulae 51 - 62:

[0575]

Chemical formula

[0576]

[0357] In certain embodiments, the non-natural amino acid is selected from the group consisting of the above compounds 30, 53, 56 , 59, 60, 61, and 62. In certain embodiments, the non-natural amino acid is compound 30. In certain embodiments, the non-natural amino acid is compound 56. In some embodiments, the non-natural amino acid is compound 61. In some embodiments, the non-natural amino acid is compound 62.

[0577] 8. Forms and formulations of the compounds

[0358] In some embodiments, this specification includes (a) Compounds of formula I and / or II and / or III and / or V and / or VI as described herein, and pharmaceutically acceptable salts and compositions thereof; (b) Use for the treatment and / or prevention of cancer (e.g., pancreatic cancer, multiple myeloma) of compounds of formula I and / or II and / or III and / or V and / or VI as described herein, and pharmaceutically acceptable salts and compositions thereof; (c) Use for the manufacture of a medicament for the treatment and / or prevention of cancer (e.g., pancreatic cancer, multiple myeloma) of compounds of formula I and / or II and / or III and / or V and / or VI as described herein, and pharmaceutically acceptable salts and compositions thereof; (d) A medicament comprising a compound of formula I and / or II and / or III and / or V and / or VI as described herein, and pharmaceutically acceptable salts and compositions thereof, for use in the treatment and / or prevention of cancer (e.g., pancreatic cancer, multiple myeloma); (c) More detailed descriptions can be found elsewhere in this specification and / or in the Examples section of the processes for the preparation of the compounds described herein, for example, of formula I and / or II and / or III and / or V and / or VI; (d) The compounds described herein, for example, of formula I and / or II and / or III and / or V and / or VI, or pharmaceutically acceptable salts thereof, pharmaceutical formulations comprising the same together with a pharmaceutically acceptable carrier or diluent; (e) The compounds described herein, for example, of formula I and / or II and / or III and / or V and / or VI, or pharmaceutically acceptable salts thereof, pharmaceutical formulations comprising the same together with one or more other active anti-cancer agents, optionally in a pharmaceutically acceptable carrier or diluent; (f) Use of the compounds of formula I and / or II and / or III and / or V and / or VI, or pharmaceutical compositions comprising formula I and / or II and / or III and / or V and / or VI for the treatment of cancer and / or inflammatory conditions. The use includes administration of an effective amount of the compounds described herein, for example, of formula I and / or II and / or III and / or V and / or VI, pharmaceutically acceptable salts thereof or compositions; or (g) A method for treating cancer and / or inflammatory conditions comprising administering the compounds described herein, for example, of formula I and / or II and / or III and / or V and / or VI, pharmaceutically acceptable salts or compositions thereof, in combination with and / or alternating with one or more active anti-cancer agents is provided.

[0578] Optically active compounds

[0359] The compounds provided herein have several chiral centers and are optically active and It is understood that they may exist in racemic form or may be isolated. Some compounds may exhibit polymorphism. It should be understood that any racemate, optically active form, diastereomer, polymorph, or stereoisomeric form of the compounds provided herein, or mixtures thereof, having the useful properties described herein are within the scope of the present invention. Methods for preparing optically active forms (e.g., resolution of racemic forms by recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using chiral stationary phases) are well known in the art.

[0579]

[0360] Similarly, most amino acids are chiral (designated as L or D, and the L enantiomer is the naturally occurring configuration) and can exist as distinct enantiomers.

[0361] Examples of methods for obtaining optically active materials are known in the art and include, at least

[0580] the following.

[0361] Examples of methods for obtaining optically active materials are known in the art and include, at least i) Physical separation of crystals - a technique for manually separating macroscopic crystals of individual enantiomers. This technique can be used when crystals of distinct enantiomers are present, i.e., when the substance is an aggregate and the crystals are visually distinguishable; ii) Simultaneous crystallization - a technique for separately crystallizing individual enantiomers from a solution of a racemate, which is only possible when the racemate is an aggregate in the solid state; iii) Enzymatic resolution - a technique for partially or completely separating a racemate by the enantiomers reacting with an enzyme at different rates; iv) Enzymatic asymmetric synthesis - a synthetic technique that uses an enzyme reaction in at least one step of the synthesis to obtain a synthetic precursor of the desired enantiomer that is enantiomerically pure or enriched; v) Chemical asymmetric synthesis - a synthetic technique for synthesizing the desired enantiomer from an achiral precursor under conditions that generate asymmetry (i.e., chirality) in the product, which can be achieved using a chiral catalyst or a chiral auxiliary; vi) Diastereomer separation - A technique of reacting a racemic compound with an enantiomerically pure reagent (chiral auxiliary) that converts individual enantiomers into diastereomers. Subsequently, the obtained diastereomers are separated by chromatography or crystallization based on the more distinct structural differences, and later the chiral auxiliary is removed to obtain the desired enantiomer. vii) Primary and secondary asymmetric transformation - A technique in which diastereomers from a racemate equilibrate to predominate in a solution of diastereomers from the desired enantiomer, or the preferential crystallization of diastereomers from the desired enantiomer disrupts the equilibrium and ultimately, in principle, all materials are converted into crystalline diastereomers from the desired enantiomer. The desired enantiomer is then released from the diastereomer. viii) Kinetic resolution - This technique refers to achieving partial or complete resolution of a racemate (or further resolution of a partially resolved compound) by the different reaction rates of enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) Enantioselective synthesis from non-racemic precursors - A synthetic technique for the desired enantiomer from achiral starting materials, where stereochemical integrity is not or minimally compromised during the synthesis process; x) Chiral liquid chromatography - A technique for separating enantiomers of a racemate in a liquid mobile phase by different interactions with a stationary phase. The stationary phase can be made from a chiral material, or the mobile phase can contain an additional chiral material to induce different interactions. xi) Chiral gas chromatography - A technique for volatilizing a racemate and separating enantiomers by different interactions with a column containing a fixed non-racemic chiral adsorption phase in a gaseous mobile phase; xii) Extraction with chiral solvents - A technique for separating enantiomers by preferentially dissolving one enantiomer in a specific chiral solvent; xiii) Transport across chiral membranes - a technique in which a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids containing the racemate, and a driving force such as concentration or pressure difference causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemate to pass.

[0581]

[0362] In some embodiments, the present specification provides a method for producing a specified enantiomer of a compound. Qualitatively free of the formula (IP) and / or formula I and / or II and / or II Compositions of compounds of formula I and / or V and / or VI are provided. In certain embodiments, in the methods and compounds of the present invention, the compounds are substantially free of enantiomers. In some embodiments, compositions comprising the compounds contain at least 85% by weight of the compound. %, 90%, 95%, 98% and 99% to 100% by weight, with the remainder being other species or enantiomers.

[0582] isotopically enriched compounds

[0583]

[0363] Also provided herein are isotopically enriched compounds, for example, but not limited to, isotopically enriched compounds of Formula (IP) and / or Formula I and / or II and / or III and / or V and / or VI.

[0584]

[0364] Pharmaceuticals can be isotopically enriched (e.g., deuterized) to assess pharmacokinetics ("PK"), pharmacodynamics, and Improving the function ( "PD") and toxicity profile has been demonstrated in the past with several classes of drugs. See, for example, Lijinsky et.al., Food Cosmet.Toxicol., 20:393 (1982); Lijinsky et. al., J.Nat.Cancer Inst., 69:1127 (1982); Mangold et.al., Mutation Res. 308:33 (1994); Gordon et.al., Drug Metab.Dispos., 15:589 (1987); Zello et.al., Metabolism, 43:487 (1994); Gately et. al., J.Nucl.Med., 27:388 (1986); Wade D, Chem.Biol.Interact. 117:191 (1999).

[0585]

[0365] Using isotope enrichment of a drug, for example, (1) reducing or eliminating unwanted metabolites or (2) extending the half-life of the parent drug, or (3) reducing the number of administrations required to produce the desired effect, or (4) reducing the amount required to produce the desired effect, or (5) increasing the formation of any active metabolites if formed, and / or (6) reducing the production of harmful metabolites in a particular tissue, and / or for combination therapy, whether or not the combination therapy is intended, drugs with improved efficacy and / or drugs with improved safety can be produced.

[0586]

[0366] Replacing an atom with one of its isotopes often changes the reaction rate of a chemical reaction This phenomenon is known as the kinetic isotope effect ( "KIE"). For example, C- When an H bond is broken during the rate-determining step (i.e., the step with the highest transition state energy) in a chemical reaction, replacing that hydrogen with deuterium slows down the reaction rate and the reaction process. This phenomenon is known as the deuterium kinetic isotope effect ("DKIE") (see, for example, Foster et al., Adv. Drug Res., vol.14, pp.1-36(1985); Kushner et al., Can. J. Physiol. Pharmacol., vol.77, pp.79-88(1999)).

[0587]

[0367] The degree of DKIE can be expressed as the ratio of the rate of a given reaction in which a C-H bond is broken to the rate of the same reaction when the hydrogen is replaced with deuterium. DKIE can range from about 1 (no isotope effect) to a very large number, e.g., 50 or more, meaning that replacing hydrogen with deuterium can slow the reaction down by 50-fold or more. High DKIE values are in part thought to be due to a phenomenon known as tunneling, which is a result of the uncertainty principle. Tunneling occurs because of the small mass of the hydrogen atom, and can occur when a transition state involving a proton is formed without the required activation energy. Since the mass of deuterium is heavier than that of hydrogen, statistically, the probability of this phenomenon occurring is much lower than that of hydrogen.

[0588]

[0368] Tritium ("T") is a radioactive isotope of hydrogen and is used in research, fusion reactors, neutron generators, and radiopharmaceuticals. Tritium is a hydrogen atom with two neutrons in its nucleus and has an atomic weight close to 3. Tritium does not occur naturally in the environment ​​It exists at the lowest concentration within and is most commonly seen as T2O. Tritium decomposes slowly (half-life = 12.3 years) and emits low-energy beta particles that cannot pass through the outer layer of human skin. The main danger associated with this isotope is internal exposure, but large amounts must be ingested to cause significant health risks. Compared to deuterium, tritium requires less consumption to reach dangerous levels. When hydrogen is replaced by tritium ("T"), a stronger bond is obtained than with deuterium, and an isotope effect numerically larger than that of deuterium is obtained. Similarly, substitution of other elements with their isotopes, for example, but not limited to, carbon's 13 C or 14 substitution with C, sulfur's 33 S, 34 S, 36 substitution with S, nitrogen's 15 substitution with N, and oxygen's 17 O or 18 substitution with O results in a similar kinetic isotope effect.

[0589]

[0369] For example, DKIE has been used to reduce the hepatotoxicity of halothane by perhaps limiting the formation of reactive species. However, this method may not be applicable to all drug classes. For example, incorporating deuterium can lead to metabolic switching. The concept of metabolic switching postulates that when a foreign gene is sequestered by a Phase I enzyme, it transiently binds prior to a chemical reaction (e.g., oxidation) and can recombine in various steric configurations. This hypothesis is supported by the relatively large size of the binding pockets in many Phase I enzymes and the chaotic nature of many metabolic reactions. Metabolic switching can lead to different ratios of not only known metabolites but also entirely new metabolites. This new metabolic profile can be more or less toxic.

[0590]

[0370] In an animal's body, for the purpose of eliminating foreign substances such as therapeutic agents from its circulatory system, various A variety of enzymes are expressed. Examples of such enzymes include cytochrome P450 enzymes ("CYP"), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases that react with these foreign substances to convert them into intermediates or metabolites with improved polarity for renal excretion. Some of the more common pharmaceutical compound metabolic reactions involve the oxidation of a carbon - hydrogen (C - H) bond to either a carbon - oxygen (C - O) or a carbon - carbon (C - C) π bond. The resulting metabolites can be either stable or unstable under physiological conditions and can have pharmacokinetic, pharmacodynamic, and acute and chronic toxicity profiles that are substantially different from those of the parent compound. For many drugs, such oxidation is rapid. Thus, for these drugs, it is often necessary to administer multiple doses or increase the daily dose.

[0591]

[0371] Thus, when isotopically enriching at specific positions of the compounds provided herein, a detectable kinetic isotope effect (KIE) can be obtained that affects the pharmacokinetic, pharmacological, and / or toxicological profiles of the compounds provided herein compared to similar compounds having a natural isotope composition.

[0592] 9. Preparation of Compounds of Formula (I) and Sub - formulas

[0593]

Chemical Formula

[0594]

[0372] In a group of embodiments, the compounds of formula (I) are prepared as shown in Scheme 1 above. Reaction of Compound 1.1 and Compound 1.2 gives Intermediate 1.3. The reaction can be carried out in the presence of any suitable base (e.g., cesium carbonate, sodium carbonate, potassium carbonate) and any suitable aprotic solvent (e.g., DMF, THF, dioxane). The chloride in Compound 1.3 is reacted with an amine R5 React with -NH2 to obtain intermediate 1.4. This reaction is carried out in the presence of any suitable base (e.g., DIPEA, TEA) and an aprotic solvent (e.g., NMP, DMF). Reduce the ester group of compound 1.3 to an alcohol (compound 1.4) in the presence of any suitable reducing agent (e.g., LAH, DIBAL). . Convert the hydroxy group in compound 1.5 to a leaving group (e.g., chloride, bromide, triflate) in the presence of a suitable reagent (e.g., thionyl chloride, thionyl bromide, trifluoromethanesulfonate) and a solvent (e.g., dichloromethane, dichloroethane) to obtain compound 1.6. React compound 1.6 with an appropriately protected diamine in the presence of a base (e.g., DIPEA, TEA) and a solvent (e.g., dichloromethane, dichloroethane), and then remove the protecting group to obtain the compound of formula (I). Additional methods for the synthesis of the compound of formula (I) and its sub-formulas are described in the Examples section. As used herein, "compounds of formula (I) and its sub-formulas" refers to compounds of formula (I), and / or compounds of formula (II) and / or formula (III).

[0595] 9. Preparation of Antibody Conjugates 9.1. Antigen Preparation

[0373] The protein used for the isolation of the antibody may be an intact antigen or a fragment of the antigen. The intact protein or fragment of the antigen may be in the form of an isolated protein or in the form of a protein expressed by a cell. Other forms of antigens useful for generating antibodies will be apparent to those skilled in the art.

[0596] 9.2. Monoclonal Antibodies

[0374] Monoclonal antibodies are described, for example, in Kohler et al., Nature , first described in 1975, 256:495 - 497 (incorporated in its entirety by reference), using the hybridoma method, and / or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567, which is incorporated in its entirety by reference ). Monoclonal antibodies can also be obtained, for example, using phage - or yeast - based libraries. See, e.g., U.S. Patent Nos. 8,258,082 and 8,691,730. Each of these references is incorporated in its entirety by reference.

[0597]

[0375] In the hybridoma method, a mouse or other suitable host animal is immunized to induce lymphocytes that will produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. See Goding J.W., Monoclonal Antibodies: Principles and Practice 3 ed. (1986) Academic Press, San Diego, CA, which is incorporated in its entirety by reference. rd

[0598]

[0376] The hybridoma cells are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine - guanine phosphoribosyl transferase (HGPRT or HPRT) , the hybridoma medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances prevent the growth of HGPRT - deficient cells .

[0599] ​

[0377] Useful myeloma cells efficiently fuse and support the stable high-level production of antibodies by selected antibody-producing cells and are sensitive to culture conditions such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California), and mouse myeloma lines such as those derived from SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland). Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. See, for example, Kozbor, J. Immunol., 1984, 133:3001. This reference is incorporated herein by reference in its entirety.

[0378] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or biological activity are identified, the selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0379] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). Thus, hybridoma cells can serve as a useful source of DNA encoding antibodies with desired properties. Once isolated, the DNA is placed in an expression vector and then introduced into cells such as bacteria (e.g., E. coli), yeast (e.g.,

[0600]

[0377] Useful myeloma cells efficiently fuse and support the stable high-level production of antibodies by selected antibody-producing cells and are sensitive to culture conditions such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California), and mouse myeloma lines such as those derived from SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland). Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. See, for example, Kozbor, J. Immunol., 1984, 133:3001. This reference is incorporated herein by reference in its entirety.

[0378] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or biological activity are identified, the selected clones may be subcloned by limiting dilution procedures and grown by standard methods. See Goding, supra. Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0601]

[0379] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). Thus, hybridoma cells can serve as a useful source of DNA encoding antibodies with desired properties. Once isolated, the DNA is placed in an expression vector and then introduced into cells such as bacteria (e.g., E. coli), yeast (e.g.,

[0377] Useful myeloma cells efficiently fuse and support the stable high-level production of antibodies by selected antibody-producing cells and are sensitive to culture conditions such as the presence or absence of HAT medium. Among these, preferred myeloma cell lines are MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California), and mouse myeloma lines such as those derived from SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection, Rockville, Maryland). Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. See, for example, Kozbor, J. Immunol., 1984, 133:3001. This reference is incorporated herein by reference in its entirety. Saccharomyces or Pichia sp., COS cells, Chai CHO cells, which do not produce antibodies unless transfected. The vector is then transfected into a host cell, such as a myeloma cell, to produce the monoclonal antibody.

[0602] 9.3. Humanized Antibodies

[0380] A humanized antibody is one in which most or all of the structural portions of a non-human monoclonal antibody are retained. can be generated by replacing the corresponding human antibody sequences, resulting in hybrid molecules in which only the antigen-specific variable portions, i.e., the CDRs, are made up of non-human sequences. Methods for obtaining humanized antibodies include, for example, those described in Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. USA, 1998, 95:8910-8915; Steinberger et al., J. Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86:10029-10033; and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, each of which is incorporated by reference in its entirety.

[0603] 9.4. Human Antibodies

[0381] Human antibodies can be produced by various techniques known in the art, e.g., by transgenic These can be generated by using humanized animals (e.g., humanized mice). See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. U. See S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993, 362:255-258; Bruggermann et al., Year in Immuno, 1993, 7:33; as well as U.S. Patent Nos. 5,591,669, 5,589,369, and 5,545,807. Each of these references is incorporated herein by reference in its entirety. Also, human antibodies can be derived from phage display libraries (e.g., Hoogenboom et al., J. Mol. Biol., 1991, 227: 381-388; Marks et al., J. Mol. Biol., 1991, ...

Claims

1. A compound of formula (I): 【Chemical 1】 or a pharmaceutically acceptable salt, solvate, or N-oxide thereof, wherein R １ａ 、 R １ｂ 、 R ２ａ 、 and R ２ｂ each occur independently and are selected from hydrogen and C １～６ alkyl, ring A is cycloalkyl, heterocycloalkyl, monocyclic aryl, monocyclic heteroaryl, fused bicyclic aryl, or fused bicyclic heteroaryl, wherein each heterocycloalkyl and each heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, Ring B is 1 to 2 R ３ wherein the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O; and R ３ is independently, at each occurrence, -N(R ３ａ ) ２ , -OR ３ｂ , -C(R ３ｃ ) ２ N.H. ２ , C １～６ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two R ３ together with the carbon atom to which they are attached form a spiroheterocycloalkyl, R ３ The heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of the formula (I) contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and optionally 1 to 2 C １～３ Is substituted with alkyl, or, Ring B is a 5- to 6-membered N-linked heterocycloalkyl or 1-3 R ３ substituted 5- to 6-membered N-linked heteroaryl, where the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O, and R ３ is, in each occurrence, independently, -N(R ３ ), -OR ３ａ ), -C(R ２ NH ３ｂ ), heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two R ３ｃ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are attached, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ２ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1-2 C ２ alkyl, or ３ where two R ３ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are attached, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R １～３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1-2 C or, Ring B is a 7- to 10-membered N-linked heterocycloalkyl or a 5- to 10-membered N-linked heteroaryl substituted with 1 to 3 R ３ wherein R is independently, in each occurrence, -N(R ３ ), -OR ３ ), -C(R ３ａ )(R ２ ), -C(R ３ｂ )(R ３ｃ ), -C(R ２ )(R ２ ), -C(R １～６ )(R ３ ), -C(R forms a spiroheterocycloalkyl with the atom, where R ３ the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of １～３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 - 2 C R ３ａ is, in each occurrence, independently selected from hydrogen, C １～６ alkyl, -C(=O)-CH ２ NH ２ , and cycloalkyl; R ３ｂ is, in each occurrence, independently, hydrogen, 【Chemical Formula 2】 (where q1 is 1, 2, or 3), and -CH ２ -aryl-CH ２ NH ２ selected from, R ３ｃ is, in each occurrence, independently selected from hydrogen and C １～６ alkyl, or two R ３ｃ together with the carbon atom to which they are attached form cycloalkyl, and R ４ is C １～６ alkyl, R ５ is C ３～６ cycloalkyl or C １～６ alkyl, each of which is independently selected from halo, hydroxy, alkoxy, amino, C １～６ alkylamino, C １～６ dialkylamino, C ３～６ cycloalkyl, aryl, and heteroaryl, and is optionally substituted with 1, 2, or 3 R ５ａ groups, where heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and any of the C ５ａ cycloalkyl group, aryl group, and heteroaryl group of R ３～６ is optionally further substituted with 1, 2, or 3 groups independently selected from halo, hydroxy, alkyl, and haloalkyl, a compound, or a pharmaceutically acceptable salt, solvate, or N-oxide thereof.

2. ring A is cycloalkyl, heterocycloalkyl, monocyclic aryl, monocyclic heteroaryl, fused bicyclic aryl, or fused bicyclic heteroaryl, wherein each heterocycloalkyl and each heteroaryl contains 1, 2, 3, or 4 heteroatoms selected from N, S, and O, Ring B is a 4-membered N-linked heterocycloalkyl further substituted with 1 to 2 Rs ３ wherein R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ ) ３ｃ NH ２ , C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs １～６ bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are bonded, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl contain 1, 2, 3, or 4 heteroatoms selected from N, S, and O, optionally further substituted with 1 to 2 C ３ alkyls, １～３ or or, Ring B is a 5- to 6-membered N-linked heterocycloalkyl further substituted with 1 to 3 Rs ３ wherein each occurrence of R is independently —N(R ３ ), —OR ３ａ ), —C(R ２ )(R ３ｂ ), —C(R ３ｃ )(R ２ )NH ２ heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon together with the carbon atom to which they are attached form spiroheterocycloalkyl, where heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl contain 1, 2, 3, or 4 heteroatoms selected from N, S, and O and are optionally further substituted with 1 to 2 C ３ alkyls １～３ or or, Ring B is 7- to 10-membered N-linked heterocycloalkyl or 1- to 3 R ３ further substituted with 5- to 10-membered N-linked heteroaryl further substituted with 1- to 3 R ３ wherein R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ ) ３ｃ NH ２ , C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two R １～６ bonded to the same carbon form spiroheterocycloalkyl together with the carbon atom to which they are attached, wherein heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl contain 1, 2, 3, or 4 heteroatoms selected from N, S, and O, and are optionally further substituted with 1 to 2 C ３ alkyl, １～３ ​ R ３ｂ which, in each occurrence, is independently hydrogen, 【Chemical Formula 3】 , and -CH ２ -aryl-CH ２ NH ２ selected from, R ５ halo, hydroxy, alkoxy, amino, C １～６ Alkylamino, C １～６ Dialkylamino, C １～６ C optionally substituted with cycloalkyl, aryl, or heteroaryl １～６ Cycloalkyl or C １～６ 2. The compound of claim 1, wherein the cycloalkyl, aryl, and heteroaryl are optionally further substituted with halo, hydroxy, alkyl, or haloalkyl.

3. A compound of formula (II): 【Chemical Formula 4】 or a pharmaceutically acceptable salt, solvate, or N-oxide thereof, wherein R １ａ 、R １ｂ 、R ２ａ 、and R ２ｂ is, in each occurrence, independently selected from hydrogen and C １～６ alkyl, Ring A is a 6-membered aryl ring or a 6-membered heteroaryl ring, and Y １ , Y ２ , Y ３ , and Y ４ are each independently selected from C and N, Ring B is a 4-membered N-linked heterocycloalkyl substituted with 1 to 2 Rs ３ wherein R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ (R ３ ｃ ) ２ NH ２ , C １～６ is alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs bonded to the same carbon ３ together with the carbon atom to which they are attached form spiroheterocycloalkyl, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and optionally 1 - 2 C １～３ alkyl substituted, or or, Ring B is a 5- to 6-membered N-linked heterocycloalkyl further substituted with 1 to 3 Rs, or a 5- to 6-membered N-linked heteroaryl substituted with 1 to 3 Rs, where each occurrence of R is independently -N(R), -OR, -C(R)NH, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon together with the carbon atom to which they are attached form spiroheterocycloalkyl, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally further substituted with 1 to 2 C alkyls. ３ and is further substituted with 1 to 3 Rs, ３ or a 5- to 6-membered N-linked heteroaryl substituted with 1 to 3 Rs, where R ３ in each occurrence is independently -N(R), ３ａ ), ２ -OR, ３ｂ -C(R), ３ｃ ), ２ NH, ２ heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon ３ together with the carbon atom to which they are attached form spiroheterocycloalkyl, where R ３ 's heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally further substituted with 1 to 2 C １～３ alkyls, or or, Ring B is a 7- to 10-membered N-linked heterocycloalkyl or a 5- to 10-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the 7- to 10-membered N-linked heterocycloalkyl or the 5- to 10-membered N-linked heteroaryl is substituted with 1 to 3 Rs ３ and wherein each R, in each occurrence, is independently —N(R ３ ), —OR ３ａ ), —C(R ２ NH ３ｂ ), C ３ｃ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon together with the carbon atom to which they are attached form a spiroheterocycloalkyl, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ２ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1 to 2 C ２ alkyls １～６ and wherein R ３ is as defined above ３ and wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R １～３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1 to 2 C R ３ａ is, in each occurrence, independently selected from hydrogen, C １～６ alkyl, -C(=O)-CH ２ NH ２ , and cycloalkyl; R ３ｂ is, in each occurrence, independently, hydrogen, 【Chemical Formula 5】 , and -CH ２ -aryl-CH ２ NH ２ selected from, R ３ｃ is, in each occurrence, independently selected from hydrogen and C １～６ alkyl, or two R ３ｃ together with the carbon atom to which they are attached form cycloalkyl, and R ４ is C １～６ alkyl, R ５ is C ３～６ cycloalkyl or C １～６ alkyl, each of which is independently selected from 1, 2, or 3 R １～６ groups optionally substituted with halo, hydroxy, alkoxy, amino, C １～６ alkylamino, C ３～６ dialkylamino, C ５ａ cycloalkyl, aryl, and heteroaryl, where heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and the C ５ａ cycloalkyl group, aryl group, or heteroaryl group of R ３～６ is optionally further substituted with 1 or 2 groups independently selected from halo, hydroxy, alkyl, and haloalkyl, the compound according to claim 1 or 2.

4. The compound according to any one of claims 1 to 3, wherein ring A is a phenyl ring.

5. The compound according to any one of claims 1 to 3, wherein ring A is a heteroaryl ring.

6. The compound according to any one of claims 1 to 3 and 5, wherein ring A is a monocyclic heteroaryl ring.

7. In ring A, at least one -OR ４ is a group 【Chemical Formula 6】 ortho to, and each 【Chemical Formula 7】 represents a point of attachment to the remainder of the formula, the compound according to any one of claims 1 to 6.

8. A compound of formula (III): 【Chemical Formula 8】 wherein wherein R １ａ 、R １ｂ 、R ２ａ 、and R ２ｂ are, in each occurrence, independently, hydrogen and C １ ～６ selected from alkyl, Ring B is an N-linked azetidinyl ring substituted with 1 to 2 Rs ３ wherein R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ )(R ３ｃ )NH ２ , C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs １～６ bonded to the same carbon form spiroheterocycloalkyl together with the carbon atom to which they are bonded, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C ３ alkyl １～３ or or, Ring B is an N-linked piperidinyl, piperazinyl, morpholinyl, or triazolyl ring further substituted with 1 to 3 Rs ３ wherein R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ )(R ３ｃ )NH ２ , heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs ２ bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are bonded, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1 to 2 C ３ alkyls, １～３ or or, ring B is unsubstituted 2,5-diazabicyclo[2.2.2]octanyl or 3,9-diazabicyclo[3.3.2]decanyl, or or, Ring B is a 5- to 10-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ ) ３ｃ NH ２ , C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs １～６ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are attached, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C ３ alkyl, １～３ ​ R ３ａ is, in each occurrence, independently selected from hydrogen, C １～６ alkyl, -C(=O)-CH ２ NH ２ , and cycloalkyl, R ３ｂ is, in each occurrence, independently, hydrogen, 【Chemical Formula 9】 , and -CH ２ -aryl-CH ２ NH ２ selected from, R ３ｃ In each occurrence, independently, is selected from hydrogen and C １～３ alkyl, or two R ３ｃ together with the carbon atom to which they are attached form cyclopropyl, R ５ is C ３～６ cycloalkyl or C １～６ alkyl, each of which is independently selected from 1, 2, or 3 R １～６ groups which are optionally substituted with halo, hydroxy, alkoxy, amino, C １～６ alkylamino, C ３～６ dialkylamino, C ５ａ cycloalkyl, aryl, and heteroaryl, where heteroaryl One contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and R ５ａ Of C ３～６ The compound according to any one of claims 1 to 7, wherein any of the cycloalkyl group, aryl group, and heteroaryl group is optionally substituted with halo, hydroxy, alkyl, or haloalkyl.

9. R １ａ and R １ｂ is hydrogen, respectively, the compound according to any one of claims 1 to 8.

10. R ２ａ and R ２ｂ is hydrogen, respectively, the compound according to any one of claims 1 to 8.

11. R １ａ 、 R １ｂ 、 R ２ａ 、 and R ２ｂ is hydrogen, respectively, the compound according to any one of claims 1 to 10.

12. R ４ The compound according to any one of claims 1 to 11, wherein R is methyl, ethyl, propyl, or isopropyl.

13. R ４ The compound according to any one of claims 1 to 12, wherein R is methyl.

14. R ５ wherein R is selected independently from one or two R groups optionally substituted with halo, hydroxy, alkoxy, amino, C １～６ alkylamino, and C １～６ dialkylamino, and is C ５ａ alkyl, the compound according to any one of claims 1 to 13. １～６ ​

15. R ５ is C １～６ alkyl optionally substituted by hydroxy or alkoxy, a compound according to any one of claims 1 to 13.

16. R ５ is 【Chemical Formula 10】 and each 【Chemical Formula 11】 represents a point of attachment to the remainder of the formula, the compound according to claim 15.

17. R ５ is C alkyl optionally substituted by one aryl or heteroaryl, where the heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and the aryl and heteroaryl are further optionally substituted by halo, alkyl, or haloalkyl, a compound according to any one of claims 1 to 13. １～６ ​

18. R ５ is 【Chemical 12】 and each 【Chemical 13】 represents a point of attachment to the remainder of the formula, the compound according to claim 17.

19. 【Fig. 14】 wherein ring B is a 4-, 5- or 6-membered fully saturated heterocycloalkyl ring substituted with 1 to 3 Rs ３ The compound according to any one of claims 1 to 18, wherein the compound is a 4-, 5- or 6-membered fully saturated heterocycloalkyl ring substituted with 1 to 3 Rs.

20. 【Fig. 15】 In which the ring B is two Rs that are attached to the same carbon and together with the carbon atom to which they are attached form a spiroheterocycloalkyl, a 4-, 5- or 6-membered fully saturated heterocycloalkyl ring substituted with ３ , and the spiroheterocycloalkyl contains 1, 2, 3 or 4 heteroatoms independently selected from N, S and O, and optionally 1 to 2 C １～３ alkyls, the compound according to any one of claims 1 to 18. ３ In which the ring B is two Rs that are attached to the same carbon and together with the carbon atom to which they are attached form a spiroheterocycloalkyl, a 4-, 5- or 6-membered fully saturated heterocycloalkyl ring substituted with ３ , and the spiroheterocycloalkyl contains 1, 2, 3 or 4 heteroatoms independently selected from N, S and O, and optionally 1 to 2 C １～３ alkyls, the compound according to any one of claims 1 to 18. １～３ In which the ring B is two Rs that are attached to the same carbon and together with the carbon atom to which they are attached form a spiroheterocycloalkyl, a 4-, 5- or 6-membered fully saturated heterocycloalkyl ring substituted with ３ , and the spiroheterocycloalkyl contains 1, 2, 3 or 4 heteroatoms independently selected from N, S and O, and optionally 1 to 2 C １～３ alkyls, the compound according to any one of claims 1 to 18.

21. 【Fig. 16】 is 【Chemical 17】 and each 【Chemical Formula 18】 represents a point of attachment to the remainder of the formula, the compound according to any one of claims 1 to 18.

22. 【Fig. 19-1】 [Chemical 19-2] 【Chemical Formula 19-3】 A compound selected from the group consisting of, or a pharmaceutically acceptable salt, solvate, or N-oxide thereof.

23. A pharmaceutical composition comprising the compound according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

24. A method for treating or preventing a target disease or condition in need thereof, the method comprising administering to the subject an effective amount of a compound according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23.

25. The method according to claim 24, wherein the disease or condition is cancer.

26. A compound according to formula (IV): 【Chemical 20】 or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or positional isomer mixture thereof, wherein W １ is a single bond, absent, or a divalent linking group, X is absent or 【Chemical 21】 is subscript b is an integer selected from 1 to 10, R Ａ which, when present, is, in each occurrence, independently selected from C １～３ alkyl, each RT, when present, is a release-inducing group, HP, when present, is a hydrophilic group, W ６ is a residue of the peptide or is absent, SG is absent or is a divalent spacer group, R is hydrogen or a terminal conjugate group, PA is a residue of formula (I): 【Chemical 22】 or a pharmaceutically acceptable salt, solvate, or N-oxide thereof, wherein R １ａ 、R １ｂ 、R ２ａ 、and R ２ｂ is, in each occurrence, independently selected from hydrogen and C １～６ alkyl, ring A is cycloalkyl, heterocycloalkyl, monocyclic aryl, monocyclic heteroaryl, fused bicyclic aryl, or fused bicyclic heteroaryl, where the heterocycloalkyl and each heteroaryl contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, Ring B is a 4-membered N-linked heterocycloalkyl substituted with 1 to 2 Rs ３ wherein the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O, and R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ ) ３ｃ NH ２ , C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs １～６ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are attached, and the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C ３ alkyls, １～３ or or Ring B is a 5- to 6-membered N-linked heterocycloalkyl or 5- to 6-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O, and R ３ is, in each occurrence, independently, -N(R ３ ), -OR ３ａ ), -C(R ２ )(R ３ｂ ), -C(R ３ｃ )(R ２ )NH ２ ), heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs ３ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are bonded, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C １～３ alkyls or Ring B is a 7- to 10-membered N-linked heterocycloalkyl or a 5- to 10-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the 7- to 10-membered N-linked heterocycloalkyl or the 5- to 10-membered N-linked heteroaryl is substituted with 1 to 3 Rs ３ and wherein each R, in each occurrence, is independently —N(R ３ ), —OR ３ａ ), —C(R ２ ), —C(R ３ｂ ), —C(R ３ｃ ), —NH ２ ), C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon together with the carbon atom to which they are attached form a spiroheterocycloalkyl, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R １～６ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C ３ alkyls ３ and wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R １～３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C R ３ａ is, in each occurrence, independently selected from hydrogen, C １～６ alkyl, -C(=O)-CH ２ NH ２ , and cycloalkyl; R ３ｂ is, in each occurrence, independently, hydrogen, 【Chemical 23】 (where q1 is 1, 2, or 3), and -CH ２ -aryl-CH ２ NH ２ selected from, R ３ｃ is, in each occurrence, independently hydrogen and C １～６ alkyl, or two R ３ｃ together with the carbon atom to which they are attached form cycloalkyl, R ４ is C １～６ alkyl and R ５ is C ３～６ cycloalkyl or C １～６ alkyl, each of which is optionally substituted with 1, 2, or 3 R １～６ groups independently selected from halo, hydroxy, alkoxy, amino, C １～６ alkylamino, C ３～６ dialkylamino, C ５ａ cycloalkyl, aryl, and heteroaryl, where heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and any of the C ５ａ cycloalkyl group, aryl group, and heteroaryl group of R ３～６ is further optionally substituted with 1, 2, or 3 groups independently selected from halo, hydroxy, alkyl, and haloalkyl. Here, PA is -NR ３ａ -, -C(R ３ｃ ), ２ NH- of -NH-, R ３ nitrogen of heterocycloalkyl of, R ３ nitrogen of partially saturated heteroaryl of, -O-CH ２ -(phenyl)-CH ２ NH- of -NH-, or a compound bonded to the rest of the molecule via the nitrogen of ring B.

27. A compound according to formula (IVa), (IVb), (IVc), (IVd), or (IVe): 【Chemical 24】 wherein B' is spiroheterocycloalkyl containing 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O); or 【Chemical 25】 (wherein R ３ ' is a heterocycloalkyl or partially saturated heteroaryl, each of which contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, provided that at least one nitrogen is in the R ３ ' ring and is bonded to W １ ; or R ３ ' is -O-CH ２ -(phenyl)-CH ２ -NH-, where NH is bonded to W １ ) The compound according to claim 26, according to

28. SG is absent or 【Chemical Formula 26】 is, subscript d is an integer selected from 1 to 10, and each 【Chemical 27】 represents the point of attachment to the rest of the formula, the compound according to claim 26 or 27.

29. SG is 【Chemical Formula 28】 and each 【Chemical formula 29】 represents the point of attachment to the rest of the formula, the compound according to any one of claims 26 to 28.

30. W １ if present, 【Chemical Formula 30】 is, subscript e is an integer selected from 1 to 10, and each 【Chemical Formula 31】 represents the point of attachment to the rest of the formula, the compound according to any one of claims 26 to 29.

31. W １ if present, 【Chemical Formula 32】 is and each 【Chemical 33】 represents the point of attachment to the rest of the formula, the compound according to any one of claims 26 to 30.

32. W ６ The compound according to any one of claims 26 to 31, wherein, when present, W is a tripeptide residue.

33. W ６ if present, 【Chemical 34】 is and each 【Chemical 35】 represents the point of attachment to the rest of the formula, the compound according to any one of claims 26 to 32.

34. W ６ The compound according to any one of claims 26 to 31, wherein W, when present, is a dipeptide residue.

35. W ６ if present, 【Chemical 36】 and each 【Chemical 37】 is a compound according to any one of claims 26 to 31 and 34, which indicates the attachment point to the rest of the formula. **Claim 36** RT is 【Chemical Formula 38】 and 【Chemical Formula 39】 is a compound according to any one of claims 26 to 35, which indicates the attachment point to the rest of the formula. **Claim 37** When HP is present, 【Chemical 40】 where the subscript b is an integer selected from 1 to 10, 【Chemical Formula 41】 is a compound according to any one of claims 26 to 36, which indicates the attachment point to the rest of the formula. **Claim 38** A compound according to any one of claims 26 to 37, wherein R is a conjugate group. **Claim 39** R is 【Chemical Formula 42】 , -N ３ , or -SH, and R ２０１ is C １～６ alkyl, and each 【Chemical 43】 is a compound according to any one of claims 26 to 38, which indicates the attachment point to the rest of the formula. **Claim 40** PA is 【Chemical Formula 44-1】 【Chemical 44-2】 【Chemical 44-3】 selected from the group consisting of, and each 【Chemical Formula 45】 is a compound according to any one of claims 26 to 39, which indicates the attachment point to the rest of the formula.

41. 【Fig. 46-1】 【Chemical Formula 46-2】 selected from the group consisting of, a compound according to any one of claims 26 to 40, or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or mixture of positional isomers thereof. **Claim 42** An antibody-drug conjugate according to formula (V): 【Chemical 47】 or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or mixture of positional isomers thereof, wherein in the formula, Ab is an antibody or an antigen-binding fragment thereof, L is a linker, PA is a residue of formula (I), and the subscript n is an integer selected from 1 to 30. **Claim 43** An antibody-drug conjugate according to claim 42 according to formula (VI): 【Chemical 48】 or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or mixture of positional isomers thereof, wherein in the formula, Each W １ is independently a single bond, absent, or a divalent attachment group, each X, in each occurrence, is independently either absent or 【Chemical 49】 、 and the subscript b is an integer from 1 to 10, Each R Ａ is, when present, in each occurrence, independently, C １～３ alkyl selected from, and each RT is, when present, in each occurrence, independently, a release-inducing group, each HP, when present, is independently a hydrophilic group, Each W ６ is, independently, a residue of a peptide or is absent, each SG, in each occurrence, is independently either absent or a divalent spacer group, each R', in each occurrence, is independently a divalent residue of a conjugated group, the subscript n is an integer selected from 1 to 30, Ab is an antibody or an antigen-binding fragment thereof, each PA is a residue of formula (I): 【Chemical Formula 50】 or a pharmaceutically acceptable salt, solvate, or N-oxide thereof, in the formula, R １ａ 、 R １ｂ 、 R ２ａ 、 and R ２ｂ is, in each occurrence, independently selected from hydrogen and C １～６ alkyl, Ring A is cycloalkyl, heterocycloalkyl, monocyclic aryl, monocyclic heteroaryl, fused bicyclic aryl, or fused bicyclic heteroaryl, where the heterocycloalkyl and each heteroaryl contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, Ring B is a 4-membered N-linked heterocycloalkyl substituted with 1 to 2 Rs ３ wherein the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O, and R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ ) ３ｃ NH ２ , C ２ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs １～６ bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are bonded, and the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C ３ alkyl, １～３ or or, Ring B is a 5- to 6-membered N-linked heterocycloalkyl or 5- to 6-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O, and R ３ is, in each occurrence, independently, -N(R ３ ), -OR ３ａ ), -C(R ２ NH ３ｂ ), a heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs ３ｃ bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are bonded, where the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ２ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O and are optionally substituted with 1 to 2 C ２ alkyls, or ３ is a 5- to 6-membered N-linked heterocycloalkyl or 5- to 6-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, S, and O, and R １～３ is, in each occurrence, independently, -N(R or, Ring B is a 7- to 10-membered N-linked heterocycloalkyl or 5- to 10-membered N-linked heteroaryl substituted with 1 to 3 Rs ３ wherein the Rs are, in each occurrence, independently, -N(R ３ ), -OR ３ ), -C(R ３ａ )(R ２ ), -NH ３ｂ ), C ３ｃ alkyl, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are attached, wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ２ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1 to 2 C ２ alkyl １～６ and wherein the two Rs attached to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are attached, and wherein the heterocycloalkyl, spiroheterocycloalkyl, heteroaryl, and partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and are optionally substituted with 1 to 2 C ３ alkyl, and １～３ are optionally substituted with 1 to 2 C R ３ａ is, in each occurrence, independently selected from hydrogen, C １～６ alkyl, -C(=O)-CH ２ NH ２ , and cycloalkyl; R ３ｂ is, in each occurrence, independently, hydrogen, 【Chemical 51】 (where q1 is 1, 2, or 3), and -CH ２ -aryl-CH ２ NH ２ selected from, R ３ｃ In each occurrence, independently, is selected from hydrogen and C １～６ alkyl, or two R ３ｃ together with the carbon atom to which they are attached form cycloalkyl, R ４ is C １～６ alkyl, and R ５ is C ３～６ cycloalkyl or C １～６ alkyl, each of which is independently selected from 1, 2, or 3 R １～６ groups selected from halo, hydroxy, alkoxy, amino, C １～６ alkylamino, C ３～６ dialkylamino, C ５ａ cycloalkyl, aryl, or heteroaryl, and heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and the C ５ａ cycloalkyl group, aryl group, or heteroaryl group of R ３～６ is optionally further substituted with 1, 2, or 3 groups independently selected from halo, hydroxy, alkyl, and haloalkyl. Here, PA is -NR ３ａ -, -C(R ３ｃ ) ２ NH- of -NH-, N of the heterocycloalkyl of R ３ , N of the partially saturated heteroaryl of R ３ , -O-CH ２ -(phenyl)-CH ２ NH- of -NH-, or bonded to the rest of the molecule via the nitrogen of ring B an antibody-drug conjugate. **Claim 44** SG is absent, or 【Chemical 52】 where the subscript d is an integer selected from 1 to 10, and each 【Chemical Formula 53】 represents an attachment point to the remainder of the formula, the antibody-drug conjugate according to claim 43. **Claim 45** SG is 【Chemical 54】 where each 【Chemical Formula 55】 represents an attachment point to the remainder of the formula, the antibody-drug conjugate according to any one of claims 43 to 44. **Claim 46** W １ if present, [Chemical Formula 56] where the subscript e is an integer selected from 1 to 10, and each 【Chemical 57】 represents an attachment point to the remainder of the formula, the compound according to any one of claims 43 to 45. **Claim 47** W １ if present, 【Chemical Formula 58】 where each 【Chemical Formula 59】 represents an attachment point to the remainder of the formula, the compound according to any one of claims 43 to 46. **Claim 48** W ６ The antibody-drug conjugate according to any one of claims 43 to 47, wherein, when present, W is a tripeptide residue. **Claim 49** W ６ if present, 【Chemical Formula 60】 where each 【Chemical Formula 61】 represents an attachment point to the remainder of the formula, the antibody-drug conjugate according to any one of claims 43 to 48. **Claim 50** W ６ The antibody-drug conjugate according to any one of claims 43 to 47, wherein W, when present, is a dipeptide residue. **Claim 51** W ６ if present, 【Chemical Formula 62】 where each 【Chemical Formula 63】 represents an attachment point to the remainder of the formula, the antibody-drug conjugate according to any one of claims 43 to 47 and 50. **Claim 52** RT is 【Chemical Formula 64】 where 【Chemical Formula 65】 represents an attachment point to the remainder of the formula, the antibody-drug conjugate according to any one of claims 43 to 51. **Claim 53** When HP is present, 【Chemical Formula 66】 where the subscript b is an integer selected from 1 to 10, 【Chemical 67】 represents an attachment point to the remainder of the formula, the antibody-drug conjugate according to any one of claims 43 to 52. **Claim 54** R' is 【Chemical Formula 68】 and R ２０１ is C １～６ alkyl, and each 【Chemical Formula 69】 represents an attachment point to the remainder of the formula, 【Chemical 70】 represents an attachment point to the antibody or its antigen-binding fragment, 【Chemical Formula 71】 represents an attachment point to the antibody or its antigen-binding fragment via the sulfur atom of a cysteine residue, the antibody-drug conjugate according to any one of claims 43 to 53.

55. 【Fig. 72-1】 【Chemical Formula 72-2】 【Chemical Formula 72-3】 or a mixture thereof selected from the group consisting of its pharmaceutically acceptable salts, solvates, stereoisomers, tautomers, or positional isomers, and each 【Chemical 73】 represents an attachment point to the remainder of the formula; L is a linker, Ab is an antibody or its antigen-binding fragment, the antibody-drug conjugate according to claim 43.

56. 【Fig. 74】 The antibody-drug conjugate according to claim 43, which is selected from the group consisting of or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or positional isomer mixture thereof.

57. The antibody-drug conjugate according to any one of claims 43 to 56, wherein the antibody or an antigen-binding fragment thereof is selected from the group consisting of anti-BCMA, anti-Muc16, trastuzumab, sofituzumab, anti-GFP, and anti-FolRa, or antigen-binding fragments thereof.

58. The antibody-drug conjugate according to any one of claims 43 to 57, wherein the antibody or an antigen-binding fragment thereof comprises the Y180 pAMF mutation, the F404 pAMF mutation, or both.

59. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 43 to 58 and a pharmaceutically acceptable carrier.

60. A method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody-drug conjugate according to any one of claims 43 to 58 or the pharmaceutical composition according to claim 59.

61. A method for diagnosing a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody conjugate according to any one of claims 43 to 58 or the pharmaceutical composition according to claim 59.

62. The method according to claim 60 or 61, wherein the disease or condition is cancer.

63. The method according to claim 60 or 61, wherein the disease or condition is an inflammatory disease or condition.

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