5H-Pyrrolo[3,2-d]pyrimidine-2,4-diamino compounds and their antibody conjugates

Abstract

The present disclosure relates to 5H-pyrrolo[3,2-d]pyrimidine-2,4-diamino compounds, and / or antibody conjugates thereof, pharmaceutical compositions thereof, methods of making the conjugates, and methods of using the conjugates and compositions for therapy.

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 hereby incorporated by reference in its entirety.

[0002]

[0001] This specification provides 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 of treating and preventing cell proliferation and cancer, methods of detecting cell proliferation and cancer, and methods of diagnosing cell proliferation and cancer. The compounds, conjugates, and compositions are also useful in methods of 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. Activation of TLRs induces innate immune (rapid, non-specific) and / or adaptive immune (slower, more specific) responses such as cytokine induction and / or co-stimulation of phagocytes and / or activation of T cell responses. Among the TLRs, TLR3, 7, 8, and 9 are expressed in intracellular endosomes, while the other TLRs (TLR1, 2, 4, 5, 6, 10, and 11) are localized to the plasma membrane. Each TLR elicits a cell response specific to the pathogen 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 inflammatory responses. 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 instances disease progression is promoted while in others 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. The injection of TLR7 agonists such as imiquimod, loxoribine, CL264 (a 9-benzyl-8-hydroxyadenine derivative containing glycine in the benzyl group), ssRNA40, R848, and SM-276001, either alone or as a vaccine adjuvant, induces a strong immunity that leads to antitumor therapeutic effects 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 may 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 producing 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 comprising 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, there is provided 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. In some or any embodiments, the conjugate is of formula (V)

[0013]

Chemical formula

[0014]

[0010] In another aspect, there is provided a method of using a compound of formula (I-P), formula (I), (II), or (III), or an embodiment thereof, or an antibody-drug conjugate described herein. 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 inflammatory diseases or conditions.

[0016] [

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

[0017] [[Chemical]] of linker payloads are provided, wherein R, SG, W 6 , HP, X, W 1 and PA are as defined in the detailed description section herein. [[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] ] A 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] ] A graph showing in vitro data demonstrating the ability of Compound 10 to produce cytokine release - IL-6 (Figure 4A), MCP-1 (Figure 4B), and IL1Ra (Figure 4C) from human PBMC.

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), TNFα (Figure 6C) and IP-10 (Figure 6D) from mouse splenocytes.

Figure 7

[0019] Graph showing in vivo data regarding the anti-tumor 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 dosing). The anti-tumor effect of treatment with 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 antibody conjugates thereof 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 having 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 difference from what is generally 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, for example, using conventional methodologies such as 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 in accordance with 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 above and below the stated value and includes 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. 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 immunoglobulin", all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins is well-characterized. See, for example, 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 H or VH) and a heavy chain constant region (C H or CH). The heavy chain constant region typically contains three domains abbreviated as C H1 (or CH1), C H2 (or CH2), and C H3 (or CH3). Each light chain typically contains a light chain variable region (V L 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 in more conserved regions. The more conserved regions are called framework regions (FR). Each V H and V LGenerally 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 reference is incorporated herein 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 designated 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 CDRs can be determined by one of several known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme); MacCallum 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), which are known to those skilled in the art. Each of these references is incorporated herein by reference in its entirety.

[0029]

[0030] CDRs can be assigned, for example, using antibody numbering software such as Abnum, available at www.bioinf.org.uk / abs / abnum / , and described in 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 antibody heavy chain constant region (e.g., as reported by Kabat et al., supra). Unless otherwise stated, 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 an antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fv fragments, Fab fragments, F(ab')2 fragments, Fab’ fragments, single-chain Fv (scFv) fragments, and scFv-Fc fragments.

[0032]

[0033] The "Fv" fragment contains a dimer linked by non-covalent bonds of one heavy chain variable domain and one light chain variable domain.

[0033]

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

[0034]

[0035] The "F(ab')2" fragment contains two Fab' fragments joined by disulfide bonds near the hinge region. The F(ab')2 fragment can be produced, for example, by recombinant methods or by pepsin digestion of the 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 V H domains and V L domains 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 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 has the directionality of the variable domain of the scFv (i.e., V H -V L or V L -V H) in response to, V H or V L may subsequently be present. 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.

[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 includes 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 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" is one that 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, the isolated antibody is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, for example, by using a spinning cup sequenator. 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. An isolated antibody includes an in situ antibody in a recombinant cell because at least one component of the natural environment of the antibody is absent. In some aspects, 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 the 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 is the dissociation constant (K D) can be represented by. Affinity can be measured by common 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" to a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean a measurable binding that is different 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 changes. In one embodiment, the affinity matured antibody has a nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies can be produced using various methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783. This document is incorporated by reference in its entirety) describe H and V LAffinity maturation by domain shuffling is described. 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 by reference in its entirety.

[0046]

[0047] The term "amino acid" refers to the 20 commonly occurring natural amino acids. Natural 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 post-translational variants of 22 naturally occurring amino acids such as prenylated amino acids, isoprenylated amino acids, myristoylated 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" also includes non-natural (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, phenylalanyl, tryptophanyl, methioninyl, glycyl, serynyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaryl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolynyl, β-phenylalanyl, β-tryptophanyl, β-methioninyl, β-glycyl, β-serynyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaryl, β-lysiny, β-argininyl, or β-histidinyl. The unnatural amino acid is not a nascent protein amino acid or a post-translational modified variant thereof. 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 antibody described herein. The payload may be any payload 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 a 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, a linker can form more than one covalent bond to an antibody. In certain embodiments, a linker may form more than one covalent bond to a payload or may form covalent bonds to more than one payload. The remaining structure of the linker after it has formed a bond to the antibody or payload or both, i.e., the residue of the linker after one or more covalent bonds have been 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 to an antibody or payload or both. In some embodiments, the linker is a cleavable linker. For example, a cleavable linker may be a linker released by a biodegradation function that may or may not have been genetically engineered. In some embodiments, the linker is a non-cleavable linker. For example, a 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 terms in this specification, this section shall be given precedence unless otherwise specified.

[0052]

[0053] As used herein, the term "alkyl" refers to saturated straight-chain or branched hydrocarbons 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 10 alkyl. In certain embodiments, the alkyl group includes saturated straight-chain or branched hydrocarbons having 1 to 6 carbon atoms, i.e., 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 by which an 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 include 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] In certain embodiments, "alkenyl" refers to an olefinically unsaturated hydrocarbon group having up to about 11 carbon atoms or 2 to 6 carbon atoms that can be linear or branched and having at least one or one to two alkenyl unsaturation sites.

[0055]

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

[0056]

[0057] In certain embodiments, "alkynyl" refers to an acetylenically unsaturated hydrocarbon group having up to about 11 carbon atoms or 2 to 6 carbon atoms that can be linear or branched and having at least one or one to two 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 the 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, a cycloalkyl group can be saturated, and / or bridged, and / or unbridged, and / or a fused bicyclic group. In certain embodiments, a 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 ), 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 by heteroatoms independently selected from N, O, and S(O) 0~2 selected independently therefrom.

[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, wherein 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, may include a fused ring system or a bridged ring system, a nitrogen atom or a sulfur atom may optionally be oxidized, a nitrogen atom 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 heterocyclic ring that is a monocyclic or polycyclic non-aromatic ring system. In some or any embodiments, the heterocycloalkyl is a monocyclic, polycyclic, fully saturated ring system.Such complex cyclic and / or heterocycloalkyl radicals include, but are not limited to, 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, 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, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl. In certain embodiments, the complex cyclic may be optionally substituted as described herein. In some or any embodiments, the complex cyclic 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 one or two heteroatoms, a 5- to 6-membered heterocycloalkyl may generally contain one, two, or three heteroatoms, and a 7- to 10-membered heterocycloalkyl may generally contain one, two, three, or four 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 a 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, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazolothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thienopyridyl. Tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In certain embodiments, heteroaryl may optionally be substituted as described herein."Replacement heteroaryl" is heteroaryl substituted as defined for aryl.

[0076]

[0077] The term "heteroarylene" refers to a divalent heteroaryl group as defined herein. "Replacement 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.A partially saturated heteroaryl radical consists of, or includes 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 be optionally substituted as described herein.

[0078]

[0079] "Spiro heterocyclic" or "spiro heterocycle" or "spiroheterocycloalkyl" refers to a heterocycle as defined herein that includes two rings bonded to each other through a common atom. Non-limiting examples of spiro heterocycles include an azetidinyl ring, a morpholinyl ring, and / or a piperidinyl ring bonded 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] "Pharmaceutically acceptable salts" refers 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.Examples of 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; or (2) salts formed when the acidic proton present in the parent compound is replaced by (a) a metal ion such as an alkali metal ion, alkaline earth metal ion or aluminum ion, or an alkali metal or alkaline earth metal hydroxide such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxides, or ammonia, or (b) coordinated with an organic base such as an aliphatic, alicyclic, or aromatic organic amine such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, etc.

[0082]

[0082] Pharmaceutically acceptable salts further include, by way of example only and not limitation, 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, such as 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, and the like.

[0083]

[0083] The terms "substantially free of" or "substantially absent from" with respect to a composition refer to a composition that contains at least 85% by weight or 90% by weight, in certain embodiments 95% by weight, 98% by weight, 99% by weight or 100% by weight 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]

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

[0085]

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

[0086]

[0086] "Isotope composition" refers to the amount of each isotope present for a given atom, and "natural isotope composition" refers to the composition or abundance of the isotopes that are naturally present for a given atom. Atoms containing these natural isotope compositions may also be referred to herein as "non-enriched" atoms. Unless otherwise specified, the 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 isotope 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 about 0.0156%, the deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is about 0.0156%. The isotope enrichment of the compounds provided herein can be determined using conventional analytical methods known to those skilled in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.

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

[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 may 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 isotopic composition.

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

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

[0092] As used herein, IC 50refers 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 responses.

[0093]

[0093] As used herein, the terms "subject" and "patient" are used interchangeably herein. The term "subject" (singular and plural) refers to mammals, e.g., humans, 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., horse, cow, pig, etc.) or a pet (e.g., dog or cat). In certain embodiments, the subject is 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 being 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 in treating 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 in preventing or ameliorating the disease or progression of the disease, or results in an improvement in symptoms. A "therapeutically effective amount" varies, 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., referring to cells such as tumor cells) is intended to include measurably reducing 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 reduction in cell growth can occur by various 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 that is known to be useful, or has been used, or is currently being used to prevent or inhibit the onset, development, progression, and / or severity of a disorder.

[0099] As used herein, the phrase "preventive effective amount" refers to an amount of a treatment (e.g., a preventive agent) sufficient to effect 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] In some of the chemical structures shown herein, certain substituents, chemical groups, 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, for example, recombinantly or synthetically, at that sequence position. Similarly, a moiety may be "site - specifically" linked to a residue at a particular 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 that can modulate 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] The 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, the present specification provides 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] [ka] and -CH2-aryl-CH2NH2; 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 a cycloalkyl, R 4 is C 1~6 is alkyl, R 5 is C 3~6 Cycloalkyl or C 1~6 alkyl, which are each independently halo, hydroxy, alkoxy, amino, C 1~6 Alkylamino, C 1~6 Dialkylamino, C 3~6 one, two, or three R independently selected from cycloalkyl, aryl, and heteroaryl; 5a group, where heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, where R 5a C 3~6 Any of the cycloalkyl, aryl, and heteroaryl groups are optionally substituted with one or two (in some embodiments, one) groups independently selected from halo, hydroxy, alkyl, and haloalkyl.

[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), in ring A, at least one -OR 4 is in the ortho position relative to the group

[0118]

Chemical formula

[0119]

Chemical formula

[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]

Chemical formula

[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 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 4 is 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 4is isopropyl. In some embodiments of the compounds of formula (I-P), (I), formula (II) and / or formula (III), 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 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 one, two, or three R 1~6 groups, wherein heteroaryl contains one, two, three, or four heteroatoms independently selected from N, S, and O, and wherein 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.

[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 groups independently selected from halo, hydroxy, alkoxy, amino, C 1~6 alkylamino, and C 5a dialkylamino, optionally substituted with one, two, or three R 1~6 groups, and is alkyl. 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 one or two hydroxy. In some such examples, R 5 is branched C 1~6 alkyl optionally substituted with one or two hydroxy.

[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]

Chem.

[0130]

Chem.

[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), R 5 is C 1~6 alkyl optionally substituted with aryl or heteroaryl, where heteroaryl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and where aryl and heteroaryl are optionally further substituted with halo, alkyl, or haloalkyl.

[0133] 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] In some embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), R 5 is selected independently from 1, 2, or 3 R 1~6 groups of halo, hydroxy, alkoxy, amino, C 1~6 alkylamino, C 3~6 dialkylamino, C 5a cycloalkyl, aryl, and heteroaryl optionally substituted C 3~6 alkyl, 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 substituted with halo, hydroxy, alkyl, or haloalkyl.

[0137] 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] In some or any embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B has 1 to 2 R3 is a 4-membered N-linked heterocycloalkyl substituted with, wherein 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 Rs attached to the same carbon 3 together with the carbon atom to which they are attached form a spiroheterocycloalkyl, 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 1-2 C 1~3 alkyls.

[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 1-3 Rs, wherein R 3 is, in each occurrence, independently, -N(R 3 )2, -OR 3a , -C(R 3b )2NH2, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon 3c together with the carbon atom to which they are attached form a spiroheterocycloalkyl, 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 1-2 C 3 alkyls. 1~3

[0140]

[0123] 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 1-3 Rs​3 7- to 10-membered N-linked heterocycloalkyl or 1 to 3 R's substituted with 3 5- to 10-membered N-linked heteroaryl substituted with, 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's attached to the same carbon 3 form spiroheterocycloalkyl together with the carbon atom to which they are attached, 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 substituted with 1 to 2 C 1~3 alkyl.

[0141]

[0124] 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 fully saturated heterocycloalkyl ring substituted with 1 to 3 R 3 's. In some or any embodiments of the compounds of formula (I), formula (II), and / or formula (III), ring B is a fully saturated heterocycloalkyl ring substituted with two R 3 's that are attached to the same carbon and form spiroheterocycloalkyl together with the attached carbon atom, where the spiroheterocycloalkyl contains 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O, and is optionally further substituted with 1 to 2 C 1~3 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 's, an N-linked azetidinyl ring substituted with 1 to 2 R 3An 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, or 1 to 2 R 3 An N-linked piperazinyl ring substituted with. In some or any preceding embodiments of the compounds of formula (I-P), formula (I), formula (II) and / or formula (III), ring B is 2 R 3 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, or 2 R 3 An N-linked piperazinyl ring substituted with, wherein 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]pyradinyl, -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 1 to 2 R 3It is an N-linked azetidinyl ring substituted with. 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 R 3 It is a piperidine ring or a morpholinyl ring substituted with. 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 piperidinyl ring substituted with 1 to 3 R 3 It is a piperazinyl ring substituted with. 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 R 3 It is a 5- to 6-membered heteroaryl substituted with.

[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]

Chem.

[0151]

Chem.

[0152]

Chem.

[0153]

Chem.

[0154]

[0130] In some or any 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, together with the atoms to which they are attached, forms a spiroheterocycloalkyl with two R 3It is an N-linked azetidinyl ring substituted with. In some 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 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, each independently, -OH, -NH2, -CH3,

[0155]

Chemical formula

[0156]

Chemical formula

[0157] In some or any 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 N-linked morpholinyl or piperidinyl substituted with two R 3 which together with the attached atoms forms a spiroheterocycloalkyl. In some of such embodiments, the spiroheterocycloalkyl is an azetidinyl ring or a piperidinyl ring. In some or any 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 N-linked piperidinyl ring substituted with a partially saturated heteroaryl. In some or any 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 a piperazinyl ring substituted with a heteroaryl ring optionally substituted with C 1~3 alkyl. In some or any 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 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 preceding embodiments of the compounds of formula (I-P), formula (I), formula (II), and / or formula (III), R 3is methyl. 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 N-linked ring substituted with any combination of R 3 described herein and / or in this paragraph.

[0158]

[0132] 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 A is a phenyl ring substituted at the ortho position with one methoxy group with respect to the group

[0159]

Chem.

[0160]

Chem.

[0161]

Chem.

[0162]

Chem.

[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 used as payloads in the antibody-drug conjugates described herein. 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 skilled 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-(ethoxymethyl)-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-trimethylpent-4-en-1-yl)-1H-imidazo[4,5-c]quinolin-4-amine (S-34240), Roxolibin, CL264, ssRNA40, R848, and SM-276 001.

[0168] 3. Conjugate

[0135] This specification provides 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 can 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 can 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 comprising multiple polypeptide chains. 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 a 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 of ordinary skill in the art, each heavy chain typically comprises a variable domain (V H ) followed by several constant domains. Each light chain typically comprises a variable domain (V L) and include a steady domain. 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 H ) described herein. In certain embodiments, the conjugated antibody comprises the light chain variable domain (V L ) described herein. In certain embodiments, the conjugated antibody comprises the heavy chain variable domain (V H ) and the light chain variable domain (V L ) described herein. In certain embodiments, the conjugated antibody comprises a pair of heavy chain variable domain and light chain variable domain (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 a 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 side chains and lysine side chains. Additional reactive groups are described in the sections and examples below.

[0175]

[0142] In a further embodiment, the antibody comprises one or more modified amino acids having a reactive group, as described herein. Typically, the modified amino acids are not naturally encoded amino acids. Such modified amino acids may contain reactive groups useful for forming covalent bonds with a linker precursor or a 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 an antibody comprising a modified amino acid residue linked directly or indirectly via a linker to a payload. Representative modified amino acids are described in the section below. Generally, the modified amino acids have reactive groups capable of forming bonds with a linker or payload 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 the unnatural amino acid. Each site is capable of retaining the unnatural amino acid by virtue of the optimal structure, function, and / or method for producing the antibody.

[0177]

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

[0178]

[0145] In certain embodiments, the site-specific positions for substitution provide an antibody having optimal functional properties. For example, the antibody may show little or no loss of binding affinity for 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.

[0179]

[0146] In certain embodiments, the site-specific positions for substitution provide antibodies that can be advantageously produced. For example, in certain embodiments, the antibodies exhibit advantageous properties in their synthesis method. In certain embodiments, the antibodies may show little or no loss of yield upon production compared to antibodies that do not have site-specific unnatural amino acids. In certain embodiments, the antibodies can show enhanced yield upon production compared to antibodies that do not have site-specific unnatural amino acids. In certain embodiments, the antibodies may show little or no loss of tRNA suppression upon production compared to antibodies that do not have site-specific unnatural amino acids. In certain embodiments, the antibodies can show enhanced tRNA suppression upon production compared to antibodies that do not have site-specific unnatural amino acids.

[0180]

[0147] In certain embodiments, the site-specific positions for substitution provide antibodies with advantageous solubility. In certain embodiments, the antibodies may show little or no loss of solubility compared to antibodies that do not have site-specific unnatural amino acids. In certain embodiments, the antibodies can show enhanced solubility compared to antibodies that do not have site-specific unnatural amino acids.

[0181]

[0148] In certain embodiments, the site-specific positions for substitution provide antibodies with advantageous expression. In certain embodiments, the antibodies may show little or no loss of expression compared to antibodies that do not have site-specific unnatural amino acids. In certain embodiments, the antibodies can show enhanced expression compared to antibodies that do not have site-specific unnatural amino acids.

[0182]

[0149] In certain embodiments, the site-specific positions for substitution provide antibodies with favorable folding. In certain embodiments, the antibody may show little or no loss of correct folding as compared to an antibody that does not have site-specific unnatural amino acids. In certain embodiments, the antibody can show enhanced folding as compared to an antibody that does not have site-specific unnatural amino acids.

[0183]

[0150] In certain embodiments, the site-specific positions for substitution provide antibodies capable of favorable conjugation. As described below, some unnatural amino acids have side chains or functional groups that facilitate the conjugation of the antibody with a second agent, either directly or via a linker. In certain embodiments, the antibody can show enhanced conjugation efficiency as compared to an antibody that does not have the same or other unnatural amino acids at other positions. In certain embodiments, the antibody can show enhanced conjugation yield as compared to an antibody that does not have the same or other unnatural amino acids at other positions. In certain embodiments, the antibody can show enhanced conjugation specificity as compared to an antibody that does not have the same or other unnatural amino acids at other positions.

[0184]

[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.

[0185] In certain embodiments, the antibodies provided herein contain one or more non-natural amino acids at site-specific positions. In certain embodiments, the antibodies provided herein contain two non-natural amino acids at site-specific positions. 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 include one or more non-natural amino acids or post-translational modification variants thereof at each position 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 Kabat or Chothia or EU numbering scheme. In certain embodiments, the antibodies provided herein include one or more non-natural amino acids or post-translational modification variants thereof at each position 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 indicates a heavy chain residue and LC indicates 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 a 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 contains at least one Gln. In some embodiments, the acyl donor glutamine-containing tag contains 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 an unnatural amino acid). In some embodiments, the acyl donor glutamine-containing tag (Q tag) contains an amino acid sequence selected from the group consisting of LLQGG, LLQG, LSLSQG, GGGLLQGG, 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) contains 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) contains an amino acid sequence selected from the group consisting of LLQGG and LLQGA. In such embodiments, the linker-payload retaining 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, 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 incorporation of the compound into an eliminator group, release-inducing group, hydrophobic group, spacer group, and / or conjugate group. Useful attachment groups are known and apparent to those of ordinary skill 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 5It is referred to as. 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 combinations thereof. In certain embodiments, the attachment group may include -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 combinations thereof.

[0193] 3.2 Eliminator (leaving) group

[0159] The eliminator group facilitates the separation of the biologically active moiety of the compounds or conjugates described herein from the remainder 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 a 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] and in the formula, each R EG is 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 dialkylamino C(O)-. In each structure, the phenyl ring may have one, two, three, or in some cases, four R EG groups attached. In the second and third structures, those skilled in the art will recognize that, as shown in the above description of formula (C1), EG is attached to RT that is not within the skeleton of formula (C1). In some embodiments, each R EG is independently selected from the group consisting of hydrogen, alkyl, biphenyl, -CF3, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O-, alkylamino-C(O)-, and dialkylamino C(O)-. In further embodiments, each R EG is independently selected from the group consisting of hydrogen, -NO2, -CN, fluoro, bromo, and chloro. In certain embodiments, the eliminator group is

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

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

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

[0199] [Chemical formula] is as follows.

[0200]

[0161] In some embodiments, the eliminator group is

[0201]

Chemical Formula

[0202]

Chemical Formula

[0203]

Chemical Formula

[0204] [Chem.] is.

[0205] 3.3 Release-inducing group

[0162] The release-inducing group facilitates the separation of the biologically active moiety of the compounds or conjugates described herein from the remainder of the compound or conjugate in vivo and / or in vitro. Further, the release-inducing group, in conjunction with an eliminator 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. In certain embodiments, the release inducer can act by a biologically driven reaction having high tumor:non-tumor specificity, such as the 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 comprises a natural or unnatural amino acid residue or a residue of a sugar ring. In certain embodiments, the release-inducing group is

[0207] [Chem.] is.

[0208] One skilled in the art will recognize that the first structure is divalent and may be bonded within the backbone of formula (C1) or as shown in formula (C2), and the second structure is monovalent and may be bonded to 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] has a divalent structure and will recognize that it may be bound within the backbone of formula (C1) or as shown in formula (C2). Structure:

[0217] [Chemical formula] is monovalent and may be bound 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 herein. The increase in hydrophilicity is thought to allow for greater solubility in aqueous solutions such as those found in biological systems. Also, the hydrophilic group can function as a spacer group as 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) of, wherein 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] It is a divalent poly(ethylene glycol) according to .

[0223]

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

[0224]

Chemical formula

[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 has the following formula:

[0228]

Chemical formula

[0229]

[0172] In some embodiments, the hydrophilic group has 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 result in more efficient conjugation between the compounds described herein and a second compound, as well as more efficient cleavage of the active metabolite. In addition, 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. Useful spacer groups include those described herein. In certain embodiments, the spacer group

[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]

Chemical formula

[0243]

[0179] In other embodiments, the divalent poly(ethylene glycol) has the following formula:

[0244]

Chemical formula

[0245]

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

[0246]

Chemical formula

[0247] 3.6 Conjugate groups and their residues

[0181] The conjugate group facilitates conjugation of the payloads described herein with a second compound such as an antibody 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 a [3+2] alkyne-azide cycloaddition reaction, an inverse electron demand Diels-Alder ligation reaction, a thiol-electrophile reaction, or a 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 is

[0248]

Chemical Structure

[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.

[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

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

[0255]

Chemical formula

[0256]

[0186] In certain embodiments, the conjugate is the following group:

[0257]

Chem.

[0258]

Chem.

[0259]

Chem.

[0260]

[0187] In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group includes the divalent residue of a non-natural amino acid. In certain embodiments, when the conjugate is formed by a carbonyl-oxyamine reaction, the divalent residue of the conjugate group is

[0261]

Chem.

[0262]

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

[0263]

Chem.

[0264]

[0189] 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 includes 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

[0265]

Chemical formula

[0266]

[0190] 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

[0267]

Chemical formula

[0268]

[0191] In some embodiments, the present specification provides 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, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein RT is

[0269]

Chemical Structure

[0270]

[0192] In some embodiments, the present specification provides 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, the present specification provides a conjugate according to formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein RT is

[0271]

Chemical Structure

[0272]

Chemical Structure

[0273]

Chemical Structure

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

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

[0276] [ka] is monovalent and may be attached to EG as shown in formula (C1) above.

[0277] 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 HP comprises poly(ethylene glycol). 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 HP comprises

[0278] [ka] In the formula, m is an integer selected from 1 to 13.

[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 is C1-C 10 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

[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 , W 2 , W 3 , W 4 , and W 5 are each independently a single bond, absent, or include 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 , W 2 , W 3 , W 4 , and W 5Each is independently a single bond, absent, or contains -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 combinations thereof.

[0282]

[0196] In one embodiment, provided herein is a conjugate of formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R' contains a triazolyl ring. In one embodiment, provided herein is a conjugate of 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, provided herein is a conjugate of 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, provided herein is a conjugate of formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R' contains a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In one embodiment, provided herein is a conjugate of formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein R' is

[0285] [Chemical formula] is as follows.

[0286]

[0198] 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 R' includes a sulfur linkage. 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 R' is

[0287] [Chemical formula] is as follows.

[0288]

[0199] 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 R' includes a divalent residue of a non-natural amino acid. 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 R' is

[0289] [Chemical formula] is as follows.

[0290]

[0200] In one embodiment, the present specification provides a conjugate according to formula (C1) or (C2) that includes an oxime linkage, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer 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 R' is

[0291] [Chemical formula] is.

[0292]

[0201] In one embodiment, provided herein are conjugates of formula (C1) or (C2) that include an oxime linkage, or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof. In one embodiment, provided herein are conjugates of 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 are conjugates of 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 are compounds of 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 are compounds of 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 an antibody conjugate comprising a payload described herein and an optional linker described herein conjugated to an antibody, wherein Ab is a residue of an antibody. 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 an antibody and R' comprises a triazole ring or a fused cyclic group containing a triazole ring. 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 an antibody and R' is

[0298]

Chemical Structure

[0299]

[0205] 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 an antibody or an antigen-binding fragment thereof and R' comprises a fused bicyclic ring, the fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. 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 an antibody or an antigen-binding fragment thereof and R' is

[0300]

Chemical Structure

[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, wherein Ab is a residue of a polypeptide and R' includes a divalent residue of a sulfur linkage. 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 a polypeptide and R' is

[0302]

Chemical formula

[0303]

[0207] 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 a polypeptide and R includes a divalent residue of a non-natural amino acid. 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 a polypeptide and R' is

[0304]

Chemical formula

[0305]

[0208] 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 a polypeptide and R' includes an oxime linkage. 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 a polypeptide and R' is

[0306]

Chem.

[0307]

[0209] 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’ 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

[0308]

Chem.

[0309]

[0210] In one embodiment, provided herein are conjugates according to any of the following formulas and their regioisomers, wherein Ab represents a residue of an antibody or an antigen-binding fragment thereof, and PA represents a payload moiety. One of ordinary skill in the art will recognize that Ab can bind at two or more positions. Each regioisomer and mixtures thereof are provided herein.

[0310]

Chem.

[0311]

Chem.

[0312]

[0211] In one embodiment, provided herein are conjugates according to any of the following formulas, wherein Ab represents a residue of an antibody and PA represents a payload moiety.

[0313] [Chemistry]

[0314]

[0212] In one embodiment, provided herein is a conjugate according to any of the following formulas, where Ab represents the residue of an antibody or an antigen-binding fragment thereof, and PA represents a payload moiety.

[0315] [Chemistry]

[0316] [Chemistry]

[0317]

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

[0318] [Chemistry]

[0319] [Chemistry]

[0320] In any of the above embodiments, the conjugate includes n PA moieties, where n is an integer 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. One of ordinary skill in the art will recognize that Formulas (101a) and (101b) are positional isomers based on the nitrogen atom in the triazole to which the antibody binds. Similarly, Formulas (102a) and (102b), Formulas (103a) and (103b), Formulas (104a) and (104b), and Formulas (105a) and (105b) are pairs of positional isomers.

[0321]

[0215] In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to Formula (30) below. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab comprises a residue of a non-natural amino acid according to Formula (30) below 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 comprises a residue of a non-natural amino acid according to Formula (30) below 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 comprises a residue of a non-natural amino acid according to Formula (30) below 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 comprises a residue of a non-natural amino acid according to Formula (30) below 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 comprises a residue of a non-natural amino acid according to Formula (30) below 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 comprises a residue of a non-natural amino acid according to Formula (30) below 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.

[0322]

Chemical formula

[0323]

[0216] One of ordinary skill in the art will recognize that amino acids such as those of Formula (30) can be incorporated as residues into polypeptides and antibodies. For example, the residue of Formula (30) is of the following Formula (30'):

[0324]

Chem.

[0325]

[0217] In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a - 105b, wherein Ab comprises a residue of a non - natural amino acid according to Formula (56) below. In certain embodiments, provided herein are antibody conjugates 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 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 comprises a residue of a non - natural amino acid according to Formula (56) below 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 comprises a residue of a non - natural amino acid according to Formula (56) below 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 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 are antibody conjugates 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 are antibody conjugates 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, PA is a residue of a compound of Formula (I - P), (I), (II), and / or (III) described herein. The non - natural amino acid according to Formula (56) is as follows.

[0326] [Chem.]

[0327]

[0218] In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab comprises an unnatural amino acid residue of para-azidomethyl-L-phenylalanine. In certain embodiments, provided herein are antibody conjugates according to any of Formulas 101a-105b, wherein Ab comprises the unnatural amino acid residue para-azidomethyl-L-phenylalanine 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 comprises the unnatural amino acid residue para-azidomethyl-L-phenylalanine 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 comprises the unnatural amino acid residue para-azidomethyl-L-phenylalanine 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 comprises the unnatural amino acid residue para-azidomethyl-L-phenylalanine 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 comprises the unnatural amino acid residue para-azidomethyl-L-phenylalanine 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 comprises the unnatural amino acid residue para-azidomethyl-L-phenylalanine 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.

[0328]

[0219] In certain embodiments, provided herein are antibody-drug conjugates of the compounds of formula (I-P), formula (I), formula (II), and / or formula (III) described herein. In one aspect, provided herein is formula (V):

[0329]

Chemical formula

[0330]

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

[0331]

Chemical formula

[0332]

Chemical formula

[0333]

Chemical formula

[0334]

Chem.

[0335]

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

[0336]

Chem.

[0337]

Chem.

[0222] PA, in each occurrence, is independently 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 , -O-CH2-(phenyl)-CH2-NH- of -NH-, or is attached to the rest 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] or a payload of its pharmaceutically acceptable salt, solvate, or N-oxide, wherein, R 1a , R 1b , R 2a , and R 2b are, 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, wherein the heterocycloalkyl and each heteroaryl contain 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 or 2 R 3 wherein 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 spiroheterocycloalkyl together with the carbon atom to which they are attached, wherein the 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 or 2 C 1~3 alkyl, or, Ring B is a 5- to 6-membered N-linked heterocycloalkyl further substituted with 1 to 3 R 3 wherein R3 is, in each occurrence, independently, -N(R 3a )2, -OR 3b , -C(R 3c )2NH2, heterocycloalkyl, heteroaryl, or partially saturated heteroaryl, or two Rs attached to the same carbon form, together with the carbon atom to which they are attached, 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 alkyl, 1~3 or, alternatively, ring B is a 7- to 10-membered N-linked heterocycloalkyl further substituted with 1 to 3 Rs 3 or a 5- to 10-membered N-linked heteroaryl further substituted with 1 to 3 Rs 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 Rs attached to the same carbon form, together with the carbon atom to which they are attached, 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 alkyl, 1~3 and R 3a is, in each occurrence, independently, hydrogen, C 1~6 alkyl, -C(=O)-CH2NH2, and cycloalkyl, R 3b is, in each occurrence, independently, hydrogen,

[0341]

Chemical Structure

[0342]

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

[0343]

Chemical formula

[0344]

Chemical formula

[0345]

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

[0346]

Chemical formula

[0347]

Chemical formula

[0348]

Chemical formula

[0349]

Chemical formula

[0350]

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

[0351]

Chemical formula

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

[0353] [Chemical formula] and each

[0354] [Chemical formula] indicates the attachment point to the remainder 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 include natural and / or non-natural amino acid residues. In some examples of formula (VI), when W 6 , if present, is a tripeptide residue. In some such examples, W 6 is

[0356] [Chemical formula] and each

[0357] [Chemical formula] indicates the attachment point to the remainder of the formula. In some examples of formula (VI), when W 6 , if present, is a dipeptide residue. In some such examples, when W 6 , if present,

[0358] [Chemical formula] and each

[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]

Chem.

[0366]

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

[0367]

Chem.

[0368]

Chem.

[0369]

Chem.

[0370]

Chem.

[0371]

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

[0372]

Chem.

[0373]

Chem.

[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 the antibody is conjugated to the linker precursor, for convenience, the conjugate is, in some or any embodiment, as follows:

[0380]

Chem.

[0381]

Chem.

[0382]

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 antigen-binding fragments 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 30, 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] Antibody-drug conjugates in which the antibody is selected from various therapeutic antibodies that are approved for use, are in clinical trials, or are under development for clinical use are also contemplated within the scope of the embodiments presented herein. Such therapeutic antibodies include, but are not limited to, rituximab (Rituxan®, IDEC / Genentech / Roche) (see, e.g., U.S. Patent No. 5,736,137), a chimeric anti-CD20 antibody approved for the treatment of non-Hodgkin's lymphoma, HuMax-CD20, an anti-CD20 currently being developed by Genmab, the anti-CD20 antibody described in U.S. Patent 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. Patent No. 5,677,171), a humanized anti-Her2 / neu antibody approved for the treatment of breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®, currently being developed by Genentech); anti-Her2 antibodies (U.S. Patent No. 4,753,894); cetuximab (Erbitux®, Imclone), a chimeric anti-EGFR antibody in clinical trials for various cancers (U.S. Patent No. 4,943,533; PCT Publication No. WO96 / 40210); ABX-EGF currently being developed by Abgenix-Immunex-Amgen (U.S. Patent No. 6,235,883); HuMax-EGFr currently being developed by Genmab (U.S. Patent No. 7,247,301); 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy, et al. (1987) Arch. Biochem. Biophys. 252(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. 5,891,996, 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, IDEC / ScheringIbritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed by AG, Gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Celltech / Wyeth, anti-LFA-3 developed by BiogenAlefacept (Amevive®), an Fc fusion protein, abciximab (ReoPro®) developed by Centocor / Lilly, basiliximab (Simulect®) developed by Novartis, palivizumab (Synagis®) developed by Medimmune, infliximab (Remicade®), an anti-TNF alpha antibody developed by Centocor, adalimumab (Humira®), an anti-TNF alpha antibody developed by Abbott, Humicade®), an anti-TNF alpha antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, etanercept (Enbrel®), a p75 TNF receptor Fc fusion protein developed by Immunex / Amgen, yenepcept, a p55 TNF receptor Fc fusion protein 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, Thioplatin (AS1407), developed by Antisoma, Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody developed by Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody developed by Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody developed by Biogen, Cambridge AntibodyCAT-152, an anti-TGF-β antibody developed by Technology; ABT874 (J695), an anti-IL-12p40 antibody developed by Abbott; CAT-192, an anti-TGFβ1 antibody developed by Cambridge Antibody Technology and Genzyme; CAT-213, an anti-eotaxin 1 antibody developed by Cambridge Antibody Technology; LymphoStat-B®, an anti-Blys antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; TRAIL-R1 mAb, an anti-TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; Avastin® (bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody developed by Genentech; anti-HER receptor family antibodies developed by Genentech; anti-tissue factor (ATF), an anti-tissue factor antibody 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 GcoSciences; HuMax-Lymphoma, developed by Genmab and Amgen; HuMax-TAC, developed by Genmab; IDECIDEC-131, an anti-CD40L antibody developed by IDEC Pharmaceuticals; IDEC-151 (Crenoliximab), 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; BEC2, an anti-idiotype antibody developed by Imclone; 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 (registered trademark) (Iabetuzumab), an anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics; LymphoCide (registered trademark) (Epratuzumab), an anti-CD22 antibody developed by Immunomedics; 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; MedareX and Immuno-DesignedOsidem® (IDM-1), an anti-Her2 antibody developed by 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; an anti-α5β1 integrin developed by Protein Design Labs; an anti-IL-12 developed by Protein 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, therapeutic agents include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO95 (alphaV integrin, Centocor); MEDI-522 (alphaVβ3 integrin, Medimmune); volociximab (alphaVβ1 integrin, Biogen / PDL); human mAb216 (B cell glyco Glycosolated epitope, NCl); BiTE MT103 (bispecific CD19×CD3, Medimmune); 4G7×H22 (bispecific B cell×Fc gamma R1, 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 (registered trademark) (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 Genetics); SGN-33 (Lintuzumab) (CD33, Seattle Genetics); Zanilimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis); SGN-40 (CD40, Seattle Genetics); 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 (Ticilimumab, 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 hormone-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-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone) are mentioned.;

[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 an enzyme-activated prodrug, 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 plasminogen 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-herpes simplex virus (HSV), anti-T cell receptor:CD3 complex / anti-influenza, 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 HER2 / 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, such as anti-rabbit IgG / anti-ferritin, anti-horseradish peroxidase (HRP) / anti-hormone, anti-somatostatin / anti-substance P, anti-HRP / anti-FITC, anti-CEA / anti-β-galactosidase (see Nolan, O et R. O'Kennedy, Biochim Biophys Acta. 1990 Aug. 1; 1040(1):1-11, incorporated herein by reference). Examples of trispecific antibodies include anti-CD3 / anti-CD4 / anti-CD37, anti-CD3 / anti-CD5 / anti-CD37, and anti-CD3 / anti-CD8 / anti-CD37.

[0388]

[0235] In any of the above embodiments and embodiments where the antibody conjugate has the structure according to formulas (V) and (VI), the structure enclosed in square brackets may be covalently bound to one or more non-natural amino acids of the antibody, and the one or more non-natural amino acids are located at sites independently selected from the group consisting of HC-F241, HC-F404, HC-Y180, and LC-K42 according to the Kabat numbering scheme or the EU numbering scheme of Kabat, and combinations thereof. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the site HC-F404 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the site HC-Y180 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the site HC-F241 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the site LC-K42 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the sites HC-F404 and HC-Y180 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the sites HC-F241, HC-F404, and HC-Y180 of the antibody. In some embodiments, at least one structure enclosed in square brackets is covalently bound to a non-natural amino acid at the site HC-F404 of the antibody, and at least one structure enclosed in square brackets is covalently bound to a non-natural amino acid at the site HC-Y180 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the sites HC-Y180 and LC-K42 of the antibody. In some embodiments, the structure enclosed in square brackets is covalently bound to one or more non-natural amino acids at the sites 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 can have an additional payload selected from the group consisting of: labels, dyes, polymers, water-soluble polymers, polyethylene glycol, derivatives of polyethylene glycol, photocrosslinkers, cytotoxic compounds, radionuclides, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, 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 noncovalently with other molecules, photocaging 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. 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 to provide the polypeptides with new biological properties. 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 attached to natural or modified amino acids. The molecular weight of the polymer can be in a wide range, including but not limited to, from about 100 Da to about 100,000 Da or greater.

[0391]

[0238] The selected polymer may be water-soluble such 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, when optimizing such parameters, the branching of the polymer should be taken into account. In general, the higher the molecular weight (or the more branched), the higher the polymer:protein ratio.

[0393]

[0240] The water-soluble polymer may have any structural form including, but not limited to, linear, dendritic, or branched forms. 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 encompass any polyethylene glycol molecule regardless of its size or end modification and can be represented as being linked to a polypeptide by the formula: X’O-(CH2CH2O) n -CH2CH2-Y, where n is from 2 to 10,000, X is an end modification including, but not limited to, H or C 1~4 alkyl, and Y is the attachment point to the polypeptide.

[0395]

[0242] In one case, the PEG has one end terminated with hydroxy or methoxy, i.e., X is H or CH3 (「methoxy PEG」). Alternatively, the 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 groups that are inert to the 20 common amino acids but specifically react with complementary functional groups present in non-natural coded 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 coded amino acid. For example, Y may be an amide linkage, a carbamate linkage, or a urea linkage with an amine group (including but not limited to the epsilon amine of lysine or the N-terminus) of the polypeptide. Alternatively, Y may be a maleimide linkage 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 generally not 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 coded amino acid such as the modified amino acids described herein to form a similar product. In some embodiments, a strong nucleophile (including but not limited to hydrazine, hydrazide, hydroxylamine, semicarbazide) may be reacted with an aldehyde group or a ketone group present in a non-natural coded amino acid to form a hydrazone, oxime, or semicarbazone, and if necessary, they can be further reduced by treatment with an appropriate reducing agent in some cases.Alternatively, a strong nucleophile can be incorporated into the polypeptide via a non-natural coded amino acid and used to preferentially react with a ketone or aldehyde group present in the water-soluble polymer.

[0396]

[0243] PEG of any molecular weight, including but not limited to, having a molecular weight of about 100 Daltons (Da) to 100,000 Da, or greater if desired (optionally including 0.1 to 50 kDa or 10 to 40 kDa but not limited thereto), can actually be used as desired. Branched-chain PEGs can also be used, including but not limited to, those containing PEG molecules having an MW in the range of 1 to 100 kDa (including 1 to 50 kDa or 5 to 20 kDa but not limited thereto) per chain. 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.

[0397]

[0244] Generally, at least one end of the PEG molecule is available for reaction with an antibody. For example, a PEG derivative retaining an alkyne moiety and an azide moiety for reacting with an amino acid side chain can be used to attach PEG to a non-natural encoded amino acid as described herein. When the non-natural encoded amino acid contains an azide, the PEG will typically contain either an alkyne moiety for effecting the formation of a [3+2] cycloaddition product, or an activated PEG species (i.e., an ester, carbonate) containing a phosphine group for effecting the formation of an amide linkage. Alternatively, when the non-natural encoded amino acid contains an alkyne, the PEG will typically contain an azide moiety for effecting the formation of a [3+2] Huisgen cycloaddition product. When the non-natural 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) for effecting the formation of a hydrazone linkage, an oxime linkage, and a semicarbazone linkage, respectively. In other alternative methods, the reverse orientation of the reactive groups described herein can be used. That is, the azide moiety of the non-natural encoded amino acid may be reacted with a PEG derivative containing an alkyne.

[0398]

[0245] In some embodiments, the polypeptide variant having the PEG derivative contains a chemical functionality that is reactive with a chemical functionality present in the side chain of the non-natural encoded amino acid.

[0399]

[0246] In certain embodiments, the payload is an azide - containing or acetylene - containing polymer that includes a water - soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da. The polymer backbone of the water - soluble polymer may be poly(ethylene glycol). However, a wide variety of water - soluble polymers are suitable for use, including, but not limited to, poly(ethylene) glycol as well as other related polymers including poly(dextran) and poly(propylene glycol), and it should be understood that the use of the term PEG or poly(ethylene glycol) is 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 polymer 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 may 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] Yet 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 cleavable 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-. It is understood by those skilled in the art 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, polymer backbones of water-soluble polymers having from 2 to about 300 termini are particularly suitable. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols) such as poly(propylene glycol) (“PPG”), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, and mixtures thereof. The molecular weight of each chain of the polymer backbone may vary, but is typically in the range of about 800 Da to about 100,000 Da, and in many cases 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 in no way exhaustive and is merely illustrative, and that all polymer materials having the qualities described herein are contemplated to be suitable for use.

[0406]

[0253] In some embodiments, the polymer derivative is “multifunctional,” which means that the polymer backbone has at least 2 termini, and perhaps as many as about 300 termini, that are functionalized or activated with functional groups. Examples of multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each of which is 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 that is apparent to those skilled in the art.

[0408]

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

[0409]

[0256] Useful linkers include those described herein. In certain embodiments, the linker is any divalent or polyvalent linker known to those of skill 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.

[0411]

[0258] As is 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: 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 end of a polymer 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 end of a polymer, 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. 4,618,492, 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 containing 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. It is expected that one skilled in the art can 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.

[0414]

[0261] The linker may have a wide range of molecular weights or molecular lengths. Linkers with larger or smaller molecular weights can be used to provide a desired spatial relationship or conformation between the polypeptide and the conjugate entity. Linkers with longer or shorter molecular lengths can also be used to provide a desired space or flexibility between the polypeptide and the conjugate entity. Similarly, linkers with a specific shape or conformation can be used to impart a specific shape or conformation to the polypeptide or conjugate 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 conjugate entity can provide new, modulated, or desired properties to the molecule.

[0415]

[0262] In some embodiments, provided herein is a water-soluble bifunctional linker having a dumbbell structure comprising a) an azide, alkyne, hydrazine, hydrazide, hydroxylamine, or carbonyl-containing moiety at at least a first end of a polymer backbone; 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 dendrimeric structure.

[0416] In some embodiments, the linker is derived from a linker precursor selected from the group consisting of: 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-iodoacetyl)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] 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 a protease. 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 (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 the 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 acetal. In some embodiments, substituted and unsubstituted 4-aminobutyric acid amide (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 the drug to the α-carbon of the glycine residue is another example of a self-cleaving spacer that can be useful for conjugates (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

[0419]

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

[0420]

[0267] In certain embodiments, the payload may be linked to a linker, sometimes referred to herein as a 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 known to those skilled in the art or as shown herein.

[0421]

[0268] Linker precursors can be prepared as described in the Examples section herein and / or by standard techniques, or obtained from commercial sources. See, for example, WO2019 / 055931, WO2019 / 055909, WO2017 / 132617, WO2017 / 132615, each of which is incorporated by reference in its entirety.

[0422]

[0269] For example, additional linkers such as linker precursors (A)-(H) and (J)-(M) 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]

Chemical formula

[0271] PA is a residue of a compound of formula (I-P), (I), (II), or (III), where 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 , -O-CH2-(phenyl)-CH2-NH- of -NH-, or is attached to the rest of the molecule via the nitrogen of ring B. In one embodiment, herein is provided a linker payload compound of formula (IV-P):

[0426]

Chemical formula

[0427]

Chemical formula

[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] [Chemical formula] (wherein B' is a spiroheterocycloalkyl containing 1, 2, 3, or 4 heteroatoms independently selected from N, S, and O); or

[0433] [Chemical formula] (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 present in the R 3 ' ring and is bonded to W 1 ; or R 3 ' is -O-CH2-(phenyl)-CH2-NH-, where NH is bonded to W 1 ).

[0434]

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

[0435] [Chemical formula] where the subscript d is an integer selected from 1 to 10, and each

[0436] [Chemical formula] indicates the attachment point to the rest of the formula.

[0437]

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

[0438] [Chemical formula] where each

[0439] [Chemical formula] indicates the attachment point to the rest of the formula.

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

[0447]

Chem.

[0448] [Chemical] indicates the attachment point to the rest of the formula.

[0449]

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

[0450] [Chemical] is, and each

[0451] [Chemical] indicates the attachment point to the rest of the formula.

[0452]

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

[0453] [Chemical] is,

[0454] [Chemical] indicates the attachment point to the rest of the formula.

[0455]

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

[0456] [Chemical] is, where the subscript b is an integer selected from 1 to 10,

[0457] [Chemical] indicates the attachment point to the rest of the formula.

[0458]

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

[0459]

Chem.

[0460]

Chem.

[0461]

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

[0462]

Chem.

[0463]

Chem.

[0464] 5. Antibody Specificity

[0284] The conjugate comprises 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 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 comprises 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 comprises 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 single-chain variable fragment (scFv) fragment. In some aspects, the antibody fragment is an scFv-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 (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of carbohydrate moieties to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates potential glycosylation sites.

[0473]

[0293] "O-linked" glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino 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 an antibody can be achieved by altering the amino acid sequence such that one or more of the above-described 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 (where applicable) the sequence of the antibody.

[0475] 7. Modified Amino Acids

[0295] If the antibody conjugate contains a modified amino acid, the modified amino acid may 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 a 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.

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

[0480]

Chem.

[0481]

Chem.

[0482]

[0298] In certain embodiments, an amino acid residue is of 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 unnatural amino acids can be incorporated into the polypeptide chain in exactly the same manner as when natural amino acids are incorporated into the same polypeptide chain. In certain embodiments, the unnatural 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 its 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 R 1 NR 2z R 3z 、R 1z C(=O)R 2z 、R 1z C(=O)OR 2z 、R 1z N3、R 1z C(≡CH) selected from the group consisting of. 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 unnatural coded amino acid contains a side chain functional group that efficiently and selectively reacts with functional groups (including, but not limited to, azide group, ketone group, aldehyde group, and aminooxy group) not found in the 20 common amino acids to form a stable conjugate. For example, an antigen-binding polypeptide containing an unnatural coded amino acid with an azide functional group is reacted with a polymer (including, but not limited to, poly(ethylene glycol)) or, alternatively, with a second polypeptide containing an alkyne moiety to form a stable conjugate, resulting in a selective reaction of the azide functional group and the alkyne functional group and forming a Huisgen [3+2] cycloaddition product.

[0487]

[0302] Representative unnatural coded amino acids that may be suitable for use in the present invention and useful for reaction with water-soluble polymers include, but are not limited to, those having carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactive groups. In some embodiments, the unnatural coded amino acid contains 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 that is not normally found in nature, including, but not limited to, alkenes, oximes, thioethers, and amides. Further, examples of such amino acids include saccharides not normally found in naturally occurring proteins, such as 2-deoxy-glucose and 2-deoxygalactose.

[0488]

[0303] Many of the unnatural coded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St. Louis, Missouri, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), or Peptech (Burlington, Massachusetts, USA). Those that are 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 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). See also U.S. Patent Application Publication Nos. 2003 / 0082575 and 2003 / 0108885. These documents are incorporated by reference. In addition to unnatural amino acids containing unnatural side chains, unnatural amino acids that may be suitable for use in the present invention are optionally, but not limited to, the structures of Formulas II and III:

[0489]

Chemical Formula

[0490]

[0304] Many unnatural amino acids are based on natural amino acids such as 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 20It includes linear or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, nitro groups, or alkynyl groups, etc. In addition, polysubstituted aryl rings are also contemplated. 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. Exemplary 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 groups, methoxy groups, methyl groups, allyl groups, aldehydes, azides, iodine, bromine, keto groups (including but not limited to acetyl groups), benzoyl, or alkynyl groups, 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 non-natural amino acids suitable for use in the present invention are commercially available, for example, from Sigma (USA) or Aldrich (Milwaukee, Wisconsin, USA). Those that are 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 non-natural 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, filed December 22, 2003, entitled "Protein Arrays", 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, among other things, nucleophilic addition or aldol condensation reactions.

[0493]

[0307] Representative carbonyl - containing amino acids are

[0494]

Chemical formula

[0495]

[0308] In one example, a non-naturally encoded 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 involve 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 involves 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, e.g., U.S. Patent No. 6,423,685. This document is incorporated herein by reference.

[0496]

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

[0497]

[0310] Non-natural encoded amino acids containing nucleophilic groups such as hydrazine, hydrazide, or semicarbazide react with various electrophilic groups to enable the formation of conjugates (including but not limited to PEG or other water-soluble polymers).

[0498]

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

[0499]

Chemical formula

[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 encoded 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). Due to their unique reactivity, 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.

[0503]

[0315] Unnatural encoded amino acids containing an aminooxy (also called hydroxylamine) group can react with various electrophilic groups to form conjugates (including, but not limited to, PEG or other water-soluble polymers). 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]

Chem.

[0506]

[0317] Aminooxy-containing amino acids can be prepared from readily available amino acid precursors (homoserine, 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.

[0507]

[0318] Due to their unique reactivity, azide and alkyne functional groups are extremely useful for the selective modification of polypeptides and other biological molecules. Organic azides, especially aliphatic azides, and alkynes are generally stable towards common reactive chemical conditions. In particular, both azide and alkyne functional groups are inert towards the side chains (i.e., R groups) of the 20 common amino acids found in naturally occurring polypeptides. However, upon close proximity, the "spring-loaded" nature of the azide and alkyne groups is exploited, and they react selectively and efficiently via the Huisgen [3+2] cycloaddition reaction to generate the corresponding triazoles. 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 a nucleophilic substitution (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). By incorporating unnatural coded amino acids that retain azide-containing side chains and alkyne-containing side chains, the resulting polypeptide can be selectively modified at the positions of the unnatural coded 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 in the form of a 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 a Huisgen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the antigen-binding polypeptide contains an unnatural coded 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] Further, the azido functional group can selectively react with a water-soluble polymer containing an aryl ester and appropriately functionalized 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 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 aryl ester and phosphine moieties are

[0512]

Chemical formula

[0513]

[0323] Also, the azide functional group can selectively react with a water-soluble polymer containing a thioester and appropriately functionalized 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 form the corresponding amide. Representative water-soluble polymers containing thioester and phosphine moieties are

[0514]

Chem.

[0515]

[0324] Representative alkyne-containing amino acids are

[0516]

Chem.

[0517]

[0325] Alkynyl-containing amino acids are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, Massachusetts). Alternatively, alkynyl-containing amino acids can be prepared according to standard methods. For example, for example, p-propargyloxyphenylalanine can be synthesized as described in Dieters, 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-azidophenylalanine 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 a suitable leaving group (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] Beta-substituted aminothiol functional groups are extremely useful for the selective modification of polypeptides and other biological molecules containing aldehyde groups through the formation of thiazolidines due to their unique reactivity. 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 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 through the formation of thiazolidines.

[0522]

[0329] Specific examples of useful unnatural amino acids include, but are not limited to, the following: 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 unnatural amino acid is selected from p-acetyl-phenylalanine, p-ethynyl-phenylalanine, p-propynyloxyphenylalanine, p-azido-methyl-phenylalanine, and p-azido-phenylalanine. One particularly useful unnatural amino acid is p-azidophenylalanine. This amino acid residue is known to those skilled in the art to facilitate, for example, a Huisgen [3+2] cycloaddition reaction (so-called "click" chemical reaction) with a compound bearing an alkynyl group. This reaction enables those skilled in the art to readily and rapidly conjugate an antibody at a site-specific position of the unnatural amino acid.

[0524]

[0331] In certain embodiments, the first reactive group is an alkynyl moiety (including, but not limited to, the unnatural amino acid p-propargyloxyphenylalanine, and the propargyl group may also be referred to as an acetylene moiety), 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, 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 1~10 alkylene or C 1~10 heteroalkylene.

[0526]

[0333] The unnatural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one functional group in the side chain of the unnatural amino acid has at least one characteristic 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 stability equivalent to that of the naturally occurring amino acids or under typical physiological conditions, more preferably such incorporation can be effected 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 the biological properties is the purpose of the modification / transformation), or the conversion can be effected 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 masking oxime, or a protecting or masking group that can be converted to an oxime group after deprotection of the protecting group or de-masking of the masking group. Also, the unnatural amino acid may contain a protecting or masking carbonyl group or dicarbonyl group, which can be converted to a carbonyl group or dicarbonyl group after deprotection of the protecting group or de-masking of the masking group, thereby being available for reaction with hydroxylamine or an oxime to form an oxime group.

[0527]

[0334] In further embodiments, 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 a photoactivatable crosslinker, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids having a novel functional group, amino acids that interact covalently or non-covalently with other molecules, photocaged and / or photoisomerizable amino acids, amino acids containing biotin or a biotin analog, 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 cleavable and / or photocleavable amino acids, including, but not limited to, polyethers, or amino acids having an extended side chain compared to natural amino acids, including 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 an amino acid, and amino acids containing one or more toxic moieties.

[0528]

[0335] In some embodiments, the non-natural amino acid includes 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 into antibodies via 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 photo-crosslinking 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, for example, an isotope-labeled methyl group and used as a probe for local structure and dynamics using, for example, nuclear magnetic resonance and vibrational spectroscopy.

[0530]

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

[0531]

Chemical formula

[0532]

Chemical formula

[0533] In certain embodiments, the compound of formula (AA) is stable in an aqueous solution for at least one month under weakly acidic conditions. 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, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of the compound of formula (AA), R1 is resin, amino acid, polypeptide, or 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 resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of the compound of formula (AA), R2 is 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]

Chemical formula

[0538]

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

[0539]

Chemical formula

[0540]

Chemical formula

[0541]

Chemical formula

[0542]

Chemical formula

[0543]

Chemical formula

[0544]

Chemical formula

[0545]

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

[0546]

Chem.

[0547]

Chem.

[0548]

Chem.

[0549]

[0343] In certain embodiments, Ar is

[0550]

Chem.

[0551]

[0344] In certain embodiments, Ar is

[0552]

Chem.

[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]

Chemical formula

[0562]

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

[0563]

Chemical formula

[0564]

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

[0565]

Chem.

[0566]

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

[0567]

Chem.

[0568]

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

[0569]

Chem.

[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 compounds 30, 53, 56, 59, 60, 61, and 62 above. 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 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) 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, for use in the treatment and / or prevention of cancer (e.g., pancreatic cancer, multiple myeloma); (c) Processes for the preparation of compounds of formula I and / or II and / or III and / or V and / or VI as described herein, which are described in more detail elsewhere in this specification and / or in the Examples section; (d) Pharmaceutical formulations comprising a compound of formula I and / or II and / or III and / or V and / or VI as described herein, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent; (e) A pharmaceutical formulation comprising a compound of formula I and / or II and / or III and / or V and / or VI, or a pharmaceutically acceptable salt thereof, as described herein, together with one or more other effective anti-cancer agents, optionally in a pharmaceutically acceptable carrier or diluent; (f) Use of a compound of formula I and / or II and / or III and / or V and / or VI, or a pharmaceutical composition 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 a compound of formula I and / or II and / or III and / or V and / or VI, a pharmaceutically acceptable salt or composition thereof, as described herein; or (g) A method for treating cancer and / or inflammatory conditions comprising administering a compound of formula I and / or II and / or III and / or V and / or VI, a pharmaceutically acceptable salt or composition thereof, as described herein, in combination with and / or alternately with one or more effective anti-cancer agents is provided.

[0578] Optically active compounds

[0359] It is understood that the compounds provided herein have several chiral centers and may exist in optically active and racemic forms and may be isolated. Some compounds may exhibit polymorphism. Any racemic, optically active, diastereomeric, polymorphic, or stereoisomeric form of the compounds provided herein having the useful properties described herein, or mixtures thereof, 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, with the L enantiomer being the naturally occurring configuration) and can exist as distinct enantiomers.

[0580]

[0361] Examples of methods for obtaining optically active materials are known in the art and include, at least, the following. i) Physical separation of crystals - a technique for manually separating macroscopic crystals of individual enantiomers. This technique can be used when crystals of the individual 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 racemic solution, 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 produce asymmetry (i.e., chirality) in the product, which can be achieved using a chiral catalyst or a chiral auxiliary; vi) Diastereomer separation - a technique for reacting a racemic compound with an enantiomerically pure reagent (a chiral auxiliary) that converts the individual enantiomers into diastereomers. The resulting diastereomers are then separated by chromatography or crystallization based on the more distinct structural differences, and the chiral auxiliary is later removed to obtain the desired enantiomer. vii) Primary and secondary asymmetric transformations - The technique where diastereomers from a racemate equilibrate to become predominant in a solution of diastereomers from the desired enantiomer, or where preferential crystallization of diastereomers from the desired enantiomer disrupts the equilibrium and ultimately, in principle, all the material is converted to 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 impaired during the synthesis process; x) Chiral liquid chromatography - A technique for separating the 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 the enantiomers by different interactions with a column containing a fixed non-racemic chiral adsorption phase in a gaseous mobile phase; xii) Extraction with a chiral solvent - A technique for separating enantiomers by preferentially dissolving one enantiomer in a specific chiral solvent; xiii) Transport across a chiral membrane - A technique where a racemate is placed in contact with a thin film barrier. The barrier typically separates two miscible fluids containing the racemate, and a driving force such as a concentration or pressure difference causes preferential transport across the membrane barrier. The separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through.

[0581]

[0362] In some embodiments, provided herein are compositions of compounds of formula (I-P) and / or formula I and / or II and / or III and / or V and / or VI that substantially do not contain the designated enantiomers of the compounds. In certain embodiments, in the methods and compounds of the present invention, the compounds substantially do not contain enantiomers. In some embodiments, a composition comprising a compound comprises at least 85 wt%, 90 wt%, 95 wt%, 98 wt%, and 99 wt% to 100 wt% of the compound, with the balance containing other chemical species or enantiomers.

[0582] Isotope-enriched compound

[0583]

[0363] Also provided herein are isotope-enriched compounds, e.g., but not limited to, isotope-enriched compounds of formula (I-P) and / or formula I and / or II and / or III and / or V and / or VI.

[0584]

[0364] Isotope enrichment of a medicament (e.g., deuteration) to improve pharmacokinetics (“PK”), pharmacodynamic effects (“PD”), and toxicity profiles has been demonstrated in the past for several classes of drugs. See, e.g., 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) to reduce or eliminate unwanted metabolites, (2) to extend the half-life of the parent drug, (3) to reduce the number of administrations required to produce the desired effect, (4) to reduce the amount required to produce the desired effect, (5) to increase the formation of any active metabolites if formed, and / or (6) to reduce the production of harmful metabolites in a particular tissue, and / or for combination therapy, whether or not the combination therapy is intended, a drug with improved efficacy and / or a drug 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, when a C-H bond is broken during the rate-determining step of a chemical reaction (i.e., the step with the highest energy of the transition state), replacing the 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 the 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 hydrogen is replaced by deuterium. The DKIE can range from about 1 (no isotope effect) to very large numbers, such as 50 or more, meaning that replacing hydrogen with deuterium can make the reaction 50 times or more slower. The high DKIE values are partly seen as 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 there can be cases where the 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 considerably 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 occurs naturally in the environment at the lowest concentrations and is most commonly seen as T2O. Tritium decomposes slowly (half-life = 12.3 years) and emits low-energy beta particles that cannot penetrate the outer layer of human skin. The main danger associated with this isotope is internal exposure, but a large amount must be ingested to cause a significant health risk. Compared with 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, substitution of 13 C or 14 C, substitution of sulfur with 33 S, 34 S, 36 S, substitution of nitrogen with 15 N, and substitution of oxygen with 17 O or 18 O results in a similar kinetic isotope effect.

[0589]

[0369] For example, DKIE was likely used to reduce the hepatotoxicity of halothane by restricting the generation of reactive species such as trifluoroacetyl chloride. 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 posits 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 stereostructures. 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 the animal body, various enzymes are expressed for the purpose of eliminating foreign substances such as therapeutic agents from its circulatory system. Examples of such enzymes include cytochrome P450 enzymes ("CYP"), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases, which 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 oxidizing 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 stable or unstable under physiological conditions and can have pharmacokinetic profiles, pharmacodynamic profiles, and acute and long-term toxicity profiles that are substantially different from those of the parent compound. In many drugs, such oxidation is rapid. Therefore, with these drugs, it is often necessary to administer them multiple times or increase the daily dose.

[0591]

[0371] Thus, isotopic enrichment at specific positions of the compounds provided herein can result in detectable KIEs that affect the pharmacokinetic, pharmacological, and / or toxicological profiles of the compounds provided herein compared to similar compounds having a natural isotopic 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 provides 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 amine R 5React 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 a suitably 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 compounds of 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 can be obtained, for example, using the hybridoma method first described by Kohler et al., Nature, 1975, 256:495-497 (which is incorporated herein by reference in its entirety) and / or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567, which is incorporated herein by reference in its entirety). 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 herein by reference in its entirety.

[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 rd ed. (1986) Academic Press, San Diego, CA, which is incorporated herein by reference in its entirety.

[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 the 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 such substances prevent the growth of HGPRT-deficient cells.

[0599]

[0377] Useful myeloma cells efficiently fuse, support stable high-level production of antibodies by the 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 mouse myeloma lines such as MOP-21 and MC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, California), and 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.

[0600]

[0378] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or biological activity have been 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 genes encoding the heavy and light chains of a monoclonal antibody). Thus, hybridoma cells can serve as a useful source of DNA encoding antibodies having desired properties. Once isolated, the DNA is placed in an expression vector and then transfected into host cells such as bacteria (e.g., Escherichia coli), yeast (e.g., Saccharomyces or Pichia sp.), COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce antibodies unless transfected, to produce monoclonal antibodies.

[0602] 9.3. Humanized Antibodies [

[0380] ] Humanized antibodies can be generated by replacing most or all of the structural portions of non-human monoclonal antibodies with corresponding human antibody sequences. As a result, hybrid molecules are generated in which only the antigen-specific variable portions, i.e., the CDRs, are composed of non-human sequences. Methods for obtaining humanized antibodies include, for example, those described in the following references: Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc. Nat. Acad. Sci. U.S.A., 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. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370. Each of these references is incorporated herein by reference in its entirety.

[0603] 9.4. Human Antibodies

[0381] Human antibodies can be generated by a variety of techniques known in the art, for example, by using transgenic animals (e.g., humanized mice). For example, see Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993, 362:255-258; Bruggermann et al., Year in Immuno, 1993, 7:33; and U.S. Pat. Nos. 5,591,669, 5,589,369, and 5,545,807. Each of these references is incorporated by reference in its entirety. Also, human antibodies can be derived from phage display libraries (e.g., see Hoogenboom et al., J. Mol. Biol., 1991, 227: 381-388; Marks et al., J. Mol. Biol., 1991, 222: 581-597; and U.S. Pat. Nos. 5,565,332 and 5,573,905. Each of these references is incorporated by reference in its entirety). Also, human antibodies can be generated by in vitro activated B cells (e.g., see U.S. Pat. Nos. 5,567,610 and 5,229,275. Each of these references is incorporated by reference in its entirety). Also, human antibodies can be derived from yeast-based libraries (e.g., see U.S. Pat. No. 8,691,730. This reference is incorporated by reference in its entirety).

[0604] 9.5. Conjugation

[0382] Antibody conjugates can be prepared by standard techniques. In certain embodiments, the antibody is contacted with a payload precursor under conditions suitable to form a bond between the antibody and the payload to form an antibody-payload conjugate. In certain embodiments, the antibody is contacted with a linker precursor under conditions suitable to form a bond between the antibody and the linker. The resulting antibody-linker is contacted with a payload precursor under conditions suitable to form a bond between the antibody-linker and the payload to form an antibody-linker-payload conjugate. In certain embodiments, the payload precursor is contacted with the linker precursor under conditions suitable to form a bond between the payload and the linker. The resulting payload-linker is contacted with an antibody under conditions suitable to form a bond between the payload-linker and the antibody to form an antibody-linker-payload conjugate. Suitable linkers for preparing antibody conjugates are disclosed herein, and representative conditions for conjugation are described in the examples below.

[0605]

[0383] In some embodiments, the conjugate is prepared by contacting an antibody disclosed herein with a linker precursor having any of the structures of (A)-(H) and (J)-(M).

[0606]

Chemical formula

[0607]

Chemical formula

[0608]

Chemical formula

[0609]

Chemical formula

[0610]

Chemical formula

[0611]

Chemical formula

[0612]

Chemical formula

[0613]

Chemical formula

[0614]

Chemical formula

[0615]

Chemical formula

[0616]

Chemical formula

[0617]

Chemical formula

[0618] 10. Vectors, Host Cells, and Recombinant Methods

[0384] Embodiments also relate to providing an isolated nucleic acid encoding an antibody, a vector and a host cell comprising the nucleic acid, and recombinant techniques for producing the antibody.

[0619]

[0385] To recombinantly produce an antibody, a nucleic acid encoding the antibody may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of DNA) or expression. In some embodiments, the nucleic acid can be produced by homologous recombination as described, for example, in U.S. Patent No. 5,204,244. This document is incorporated herein by reference in its entirety.

[0620]

[0386] Many different vectors are known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence, as described, for example, in U.S. Patent No. 5,534,615. This document is incorporated herein by reference in its entirety.

[0621]

[0387] Exemplary examples of suitable host cells are provided below. Such host cells are not intended to be limiting.

[0622] Suitable host cells include any prokaryotic (e.g., bacterial) cell, lower eukaryotic (e.g., yeast) cell, or higher eukaryotic (e.g., mammalian) cell. Suitable prokaryotes include eubacteria such as gram-negative or gram-positive organisms, for example, Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescens), Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa), and Enterobacteriaceae such as Streptomyces. One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli X1776, and E. coli W3110 are also suitable.

[0623] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning hosts or expression hosts for vectors encoding antibodies. Saccharomyces cerevisiae, i.e., common baker's yeast, is a widely used lower eukaryotic host microorganism. However, many other genera, species, and strains are available and useful, such as Spodoptera frugiperda (e.g., SF9), Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S. occidentalis), and filamentous fungi such as, for example, Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A. niger).

[0624]

[0390] Useful mammalian host cells include COS-7 cells, HEK293 cells, baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO), mouse Sertoli cells, and African green monkey kidney cells (VERO-76), among others.

[0625]

[0391] The host cells used for producing the antibodies of the present invention can be cultured in various media. For example, commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle Medium (DMEM) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz., 1979, 58:44; Barnes et al., Anal. Biochem., 1980, 102:255; and U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469, or WO90 / 03430 and WO87 / 00195 can be used. Each of the above-mentioned documents is incorporated by reference in its entirety.

[0626]

[0392] Any of these media may be supplemented, if necessary, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Also, any other necessary supplements may be included at appropriate concentrations, and appropriate concentrations will be known to those skilled in the art.

[0627]

[0393] Culture conditions such as temperature and pH are those previously used with the host cells selected for expression and will be apparent to those skilled in the art.

[0628]

[0394] When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. When producing the antibody intracellularly, as a first step, particulate debris is removed, regardless of whether it is associated with the host cell or the lysed fragment, for example, by centrifugation or ultrafiltration. For example, Carter et al. (Bio / Technology, 1992, 10:163-167) describe procedures for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. The cell debris can be removed by centrifugation.

[0629]

[0395] In some embodiments, the antibody is produced in a cell-free system. In some aspects, the cell-free system is an in vitro transcription and translation system as described in Yin et al., mAbs, 2012, 4:217-225. This document is incorporated by reference in its entirety. In some aspects, in the cell-free system, cell-free extracts from eukaryotic cells or prokaryotic cells are used. In some aspects, the prokaryotic cell is E. coli. Cell-free expression of the antibody can be useful, for example, when the antibody accumulates intracellularly as insoluble aggregates or when the yield from periplasmic expression is low. Antibodies produced in a cell-free system may be non-glycosylated depending on the source of the cells.

[0630]

[0396] When the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore® Pellcon® ultrafiltration unit. Any of the above-described steps may include a protease inhibitor such as PMSF to inhibit proteolysis and may include an antibiotic to prevent the growth of accidental contaminants.

[0631]

[0397] Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and affinity chromatography is a particularly useful purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used for the purification of antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth., 1983, 62:1-13. This reference is incorporated herein by reference in its entirety). Protein G is useful for all mouse isotypes and human γ3 (Guss et al., EMBO J., 1986, 5:1567-1575. This reference is incorporated herein by reference in its entirety).

[0632]

[0398] The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mec...

Claims

1. A compound of formula (I): 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein R １ａ 、R １ｂ 、R ２ａ 、and R ２ｂ is, in each occurrence, hydrogen, Ring A is monocyclic aryl, Ring B is 1 to 2 R ３ wherein said heterocycloalkyl contains one N and is substituted with R ３ is independently, at each occurrence, -N(R ３ａ ) ２ , -OR ３ｂ , -C(R ３ｃ ) ２ N.H. ２ , C １～６ alkyl, or heterocycloalkyl, or two R ３ together with the carbon atom to which they are attached form a spiroheterocycloalkyl, R ３ The heterocycloalkyl and spiroheterocycloalkyl of the formula (I) contain 1 or 2 heteroatoms independently selected from N 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- to 3-membered R ３ substituted 5- to 6-membered N-linked heteroaryl, where the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N and O, and R ３ is, in each occurrence, independently, -C(R ３ ), NH ３ｃ ), heteroaryl, or partially saturated heteroaryl, or two R ２ NH ２ bonded to the same carbon form, together with the carbon atom to which they are bonded, a spiroheterocycloalkyl, where the spiroheterocycloalkyl, the heteroaryl, and the partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 N atoms and are optionally substituted with 1 to 2 C ３ alkyl, １～３ or or, Ring B is unsubstituted 2,5-diazabicyclo[2.2.2]octanyl or 3,9-diazabicyclo[3.3.2]decanyl, 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, R ４ is C １～６ alkyl, and R ５ is hydroxy, or 【Chemical Formula 3】 One R selected from ５ａ C optionally substituted by a １～６ group which is alkyl, a compound, or a pharmaceutically acceptable salt thereof.

2. Ring A is monocyclic aryl, Ring B is 1 to 2 R ３ is a 4-membered N-linked heterocycloalkyl substituted with ３ is independently, at each occurrence, -N(R ３ａ ) ２ or heterocycloalkyl, or two R ３ together with the carbon atom to which they are attached form a spiroheterocycloalkyl, wherein said heterocycloalkyl and said spiroheterocycloalkyl contain 1 or 2 heteroatoms selected from N and O, and optionally 1 to 2 C １～３ further substituted with alkyl; or, Ring B is a 5- to 6-membered N-linked heterocycloalkyl substituted with 1 to 3 Rs ３ wherein each occurrence of R is, independently, a 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 spiroheterocycloalkyl and the partially saturated heteroaryl contain 1, 2, 3, or 4 N atoms and are optionally further substituted with 1 to 2 C ３ alkyls, ３ and R is a 5- to 6-membered N-linked heterocycloalkyl substituted with 1 to 3 Rs １～３ ​ R ５ is hydroxy or [Chemical Formula 4] C optionally replaced by １～６ The compound according to claim 1, which is alkyl.

3. A compound of formula (II): 【Chemical Formula 5】 or a pharmaceutically acceptable salt thereof, wherein Ring A is a 6-membered aryl ring, and Y １ , Y ２ , Y ３ , and Y ４ is C The compound according to claim 1.

4. The compound according to claim 1, wherein Ring A is a phenyl ring.

5. In ring A, at least one -OR ４ is a group [Chemical Formula 6] is in the ortho position to, and each 【Chemical Formula 7】 represents a point of attachment to the rest of the formula, the compound according to claim 1.

6. A compound of formula (III): 【Chemical 8】 having the structure of, wherein R １ａ 、 R １ｂ 、 R ２ａ 、 and R ２ｂ is, in each occurrence, hydrogen, 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, or heterocycloalkyl, or two Rs １～６ bonded to the same carbon together with the carbon atom to which they are bonded form spiroheterocycloalkyl, wherein said heterocycloalkyl and said spiroheterocycloalkyl of R ３ contain 1 or 2 heteroatoms independently selected from N and O and are optionally substituted with 1 to 2 C ３ alkyl, or １～３ are unsubstituted or, Ring B is a 1-3 R ３ -substituted N-linked piperidinyl, piperazinyl, morpholinyl, or triazolyl ring, where R ３ is, in each occurrence, independently, -C(R ３ｃ ), ２ NH ２ , 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, where the spiroheterocycloalkyl, the heteroaryl, and the partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 N atoms and are optionally substituted with 1-2 C １～３ alkyl, 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 or 2 heteroatoms independently selected from N and O, and R ３ is, in each occurrence, independently, -C(R ３ｃ ), NH ２ , heteroaryl, or partially saturated heteroaryl, or two Rs ２ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are bonded, wherein said spiroheterocycloalkyl, said heteroaryl, and said partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 N atoms and are optionally substituted with 1 to 2 C ３ alkyls, 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 which, in each occurrence, is independently hydrogen, 【Chemical Formula 9】 , and -CH ２ -aryl-CH ２ NH ２ selected from, R ３ｃ is, in each occurrence, independently 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 hydroxy or 【Chemical Formula 10】 One R selected from ５ａ C optionally substituted with a １～６ alkyl, the compound according to claim 1.

7. R ４ The compound according to claim 1, wherein R is methyl, ethyl, propyl, or isopropyl.

8. R ４ The compound according to claim 1, wherein R is methyl.

9. R ５ is C １～６ alkyl optionally substituted with hydroxy, the compound according to claim 1.

10. R ５ is 【Chemical 11】 and each 【Chemical 12】 represents a point of attachment to the rest of the formula, the compound according to claim 9.

11. R ５ is 【Chemical Formula 13】 and each 【Chemical 14】 represents a point of attachment to the rest of the formula, the compound according to claim 1.

12. 【Fig. 15】 In which ring B is a 4-membered fully saturated heterocycloalkyl ring substituted with 1 to 2 Rs ３ or a 5- or 6-membered fully saturated heterocycloalkyl ring substituted with 1 to 3 Rs ３ The compound according to claim 1, which is such a ring

13. 【Fig. 16】 In which 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 ３ The compound according to claim 1, which is a 4-, 5- or 6-membered fully saturated heterocycloalkyl ring substituted with

14. 【Fig. 17】 is 【Chemical Formula 18】 and each 【Chemical 19】 represents a point of attachment to the rest of the formula, the compound according to claim 1.

15. 【Fig. 20-1】 【Chemical 20-2】 [[Chemical 20-3]] A compound selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

16. A pharmaceutical composition comprising the compound according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. Use of an effective amount of the compound according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 in the manufacture of a medicament for the treatment or prevention of a disease or condition.

18. Use according to claim 17 for the treatment or prevention of cancer.

19. A compound of formula (IV): 【Chemical 21】 or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein W １ is a single bond, does not exist, or the formula 【Chemical 22】 (wherein the subscript e is an integer selected from 1 to 10, and each 【Chemical 23】 is a divalent attachment group representing a point of attachment to the rest of the formula), X is absent or 【Chemical Formula 24】 is the subscript b is an integer selected from 1 to 10, R Ａ which, when present, is, independently at each occurrence, selected from C １～３ alkyl each RT, when present, is a release-inducing group of the formula 【Chemical 25】 (wherein 【Chemical 26】 represents a point of attachment to the rest of the formula), HP, when present, 【Chemical 27】 (wherein the subscript b is an integer selected from 1 to 10, [Chemical Formula 28] represents a point of attachment to the rest of the formula) is a hydrophilic group selected from HP1, when present, has the formula 【Chemical 29】 where the subscript b is an integer selected from 1 to 10, 【Chemical 30】 and indicates the point of attachment to the remainder of the formula) is a hydrophilic group, W ６ is a residue of a peptide or is absent, SG is absent or has the formula 【Chemical 31】 where the subscript d is an integer selected from 1 to 10, and each 【Chemical 32】 indicates the point of attachment to the remainder of the formula) is a divalent spacer group, R is hydrogen or has the formula 【Chemical 33】 , -N3, or -SH (where R201 is C1-6 alkyl, and each 【Chemical 34】 indicates the point of attachment to the remainder of the formula) is a terminal conjugate group, PA is a residue of formula (I): 【Chemical 35】 or a pharmaceutically acceptable salt thereof, wherein R １ａ 、R １ｂ 、R ２ａ 、and R ２ｂ is, in each occurrence, hydrogen, ring A is monocyclic aryl, Ring B is a 4-membered N-linked heterocycloalkyl substituted with 1 to 2 Rs ３ wherein the heterocycloalkyl contains 1 N atom, and R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ )(R ３ｃ )NH ２ , C ２ alkyl, or heterocycloalkyl, or two Rs １～６ bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are bonded, and said heterocycloalkyl and said spiroheterocycloalkyl of R ３ contain 1 or 2 heteroatoms independently selected from N 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 said heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N and O, and R ３ is, in each occurrence, independently, -C(R ３ )) ３ｃ NH ２ , heteroaryl, or partially saturated heteroaryl, or two Rs ２ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are bonded, wherein said spiroheterocycloalkyl, said heteroaryl, and said partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 N atoms and are optionally substituted with 1 to 2 C ３ alkyls, or １～３ is substituted with 1 to 3 Rs or ring B is unsubstituted 2,5-diazabicyclo[2.2.2]octanyl or 3,9-diazabicyclo[3.3.2]decanyl, 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 36】 (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, R ４ is C １～６ alkyl, and R ５ is hydroxy, and 【Chemical 37】 One R selected from ５ａ C optionally substituted with a １～６ group and being alkyl, 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 R, -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

20. Formula (IVa), (IVb), (IVc), (IVd), or (IVe): 【Chemical 38】 wherein B' is spiroheterocycloalkyl); or 【Chemical 39】 (wherein, R ３ ' is heterocycloalkyl or partially saturated heteroaryl, said heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N and O, said partially saturated heteroaryl contains 1, 2, 3, or 4 N atoms, 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 19, according to

21. where SG is 【Chemical 40】 and the subscript d is an integer selected from 1 to 10, and each 【Chemical 41】 indicates the point of attachment to the remainder of the formula, the compound according to claim 20.

22. where SG is 【Chemical Formula 42】 and each 【Chemical 43】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

23. W １ if present, 【Chemical 44】 and the subscript e is an integer selected from 1 to 10, and each 【Chemical 45】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

24. W １ if present, 【Chemical 46】 and each 【Chemical 47】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

25. W ６ The compound according to claim 19, wherein W, when present, is a tripeptide residue.

26. W ６ If present, 【Chemical Formula 48】 and each 【Chemical 49】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

27. W ６ The compound according to claim 19, wherein W, when present, is a dipeptide residue.

28. W ６ if present, 【Chemical Formula 50】 and each 【Chemical 51】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

29. where RT is 【Chemical Formula 52】 and 【Chemical 53】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

30. HP １ is 【Chemical 54】 and the subscript b is an integer selected from 1 to 10, 【Chemical Formula 55】 and indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

31. where R is 【Chemical 56】 , -N ３ , or -SH, and R ２０１ is C １～６ alkyl, and each 【Chemical 57】 and indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

32. where PA is 【Chemical Formula 58-1】 【Chemical Formula 58-2】 【Chemical Formula 58-3】 selected from the group consisting of, and each 【Chemical Formula 59】 indicates the point of attachment to the remainder of the formula, the compound according to claim 19.

33. 【Fig. 60-1】 【Chemical Formula 60-2】 The compound according to claim 19, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, selected from the group consisting of.

34. Formula (V): 【Chemical Formula 61】 An antibody-drug conjugate according to, or a pharmaceutically acceptable salt, stereoisomer, or tautomer 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): 【Chemical Formula 62】 In the formula, R １ａ 、R １ｂ 、R ２ａ 、and R ２ｂ is, in each occurrence, hydrogen, Ring A is a monocyclic aryl, Ring B is a 4-membered N-linked heterocycloalkyl substituted with 1 to 2 Rs ３ wherein the heterocycloalkyl contains one N, and R ３ is, in each occurrence, independently, -N(R ３ａ ), -OR ２ , -C(R ３ｂ )(R ３ｃ )NH ２ , C ２ alkyl, or heterocycloalkyl, or two Rs １～６ bonded to the same carbon form a spiroheterocycloalkyl together with the carbon atom to which they are bonded, and said heterocycloalkyl and said spiroheterocycloalkyl of R ３ contain 1 or 2 heteroatoms independently selected from N 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 1-3 R ３ substituted with 5- to 6-membered N-linked heteroaryl, where the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N and O, and R ３ is, in each occurrence, independently, -C(R ３ ), NH ３ｃ ), heteroaryl, or partially saturated heteroaryl, or two R ２ NH ２ bonded to the same carbon form a spiroheterocycloalkyl with the carbon atom to which they are bonded, where the spiroheterocycloalkyl, the heteroaryl, and the partially saturated heteroaryl of R ３ contain 1, 2, 3, or 4 N atoms and are optionally substituted with 1-2 C ３ alkyl, or １～３ ​ Or, Ring B is unsubstituted 2,5-diazabicyclo[2.2.2]octanyl, or 3,9-diazabicyclo[3.3.2]decanyl, 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 63】 (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, R ４ is C １～６ alkyl, and R ５ is hydroxy, and 【Chemical Formula 64】 One R selected from ５ａ C optionally substituted with a １～６ group and being alkyl, The subscript n is an integer selected from 1 to 30, an antibody-drug conjugate.

35. Formula (VI): 【Chemical Formula 65】 An antibody-drug conjugate according to claim 34, or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, wherein in the formula, Each W １ is, independently, a single bond, absent, or a group of the formula 【Chemical Formula 66】 (where the subscript e is an integer selected from 1 to 10, and each 【Chemical 67】 is a divalent attachment group indicating the point of attachment to the rest of the formula), Each X, in each occurrence, is independently either absent or 【Chemical Formula 68】 is, The subscript b is an integer from 1 to 10, Each R Ａ when present, in each occurrence, independently, C １～３ selected from alkyl, Each RT, when present, in each occurrence, is independently a release-inducing group of the formula 【Chemical Formula 69】 (where 【Chemical 70】 is a point of attachment to the rest of the formula), Each HP, when present, is independently 【Chemical Formula 71】 (where the subscript b is an integer selected from 1 to 10, 【Chemical Formula 72】 is a point of attachment to the rest of the formula), a hydrophilic group selected from HP 1, when present, is a hydrophilic group of the formula 【Chemical 73】 (where the subscript b is an integer selected from 1 to 10, 【Chemical 74】 is a point of attachment to the rest of the formula), 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 of the formula 【Chemical 75】 (where the subscript d is an integer selected from 1 to 10, and each 【Chemical 76】 is a point of attachment to the rest of the formula), Each R', in each occurrence, is independently 【Chemical 77】 (where R 201 is C 1-6 alkyl, and each 【Chemical 78】 is a point of attachment to the rest of the formula, 【Chemical Formula 79】 is a point of attachment to the antibody or an antigen-binding fragment thereof, 【Chemical 80】 is a point of attachment to the antibody or an antigen-binding fragment thereof via the sulfur atom of a cysteine residue), a divalent residue of a conjugated group selected from 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). Antibody-drug conjugate.

36. SG is 【Chemical 81】 where the subscript d is an integer selected from 1 to 10, and each 【Chemical 82】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

37. SG is 【Chemical 83】 and each 【Chemical 84】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

38. W １ if present, 【Chemical 85】 where the subscript e is an integer selected from 1 to 10, and each 【Chemical 86】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

39. W １ if present, 【Chemical 87】 and each ​ represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

40. W ６ The antibody-drug conjugate according to claim 35, wherein W, when present, is a tripeptide residue.

41. W ６ If present, 【Chemical 89】 and each 【Chemical Formula 90】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

42. W ６ The antibody-drug conjugate according to claim 35, wherein, when present, W is a dipeptide residue.

43. W ６ if present, 【Chemical Formula 91】 and each 【Chemical Formula 92】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

44. RT is 【Chemical Formula 93】 and 【Chemical Formula 94】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

45. HP is 【Chemical Formula 95】 where the subscript b is an integer selected from 1 to 10, and 【Chemical Formula 96】 represents an attachment point to the rest of the formula, the antibody-drug conjugate according to claim 35.

46. R’ is 【Chemical Formula 97】 and 【Chemical Formula 98】 represents an attachment point to the rest of the formula, and 【Chemical Formula 99】 represents an attachment point to the antibody or its antigen-binding fragment, the antibody-drug conjugate according to claim 35.

47. 【Fig. 100-1】 【Chemical Formula 100-2】 【Chemical 100-3】 or is selected from the group consisting of its pharmaceutically acceptable salts, stereoisomers, or tautomers, and each 【Chemical 101】 represents an attachment point to the rest of the formula; L is a linker, Ab is an antibody or its antigen-binding fragment, the antibody-drug conjugate according to claim 35.

48. 【Fig. 102】 or is selected from the group consisting of its pharmaceutically acceptable salts, stereoisomers, or tautomers, the antibody-drug conjugate according to claim 35.

49. The antibody or its antigen-binding fragment is selected from the group consisting of anti-BCMA, anti-Muc16, trastuzumab, sofituzumab, anti-GFP, and anti-FolRa, or their antigen-binding fragments, the antibody-drug conjugate according to claim 35.

50. The antibody or its antigen-binding fragment contains the Y180 para-azidomethyl-L-phenylalanine mutation, the F404 para-azidomethyl-L-phenylalanine mutation, or both, the antibody-drug conjugate according to claim 35.

51. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 35 to 50 and a pharmaceutically acceptable carrier.

52. Use of an effective amount of the antibody-drug conjugate according to any one of claims 35 to 50 or the pharmaceutical composition according to claim 51 in the manufacture of a medicament for the treatment or prevention of cancer, or an inflammatory disease or condition.

53. Use of an effective amount of the antibody conjugate according to any one of claims 35 to 50 or the pharmaceutical composition according to claim 51 in the manufacture of a medicament for the diagnosis of cancer, or an inflammatory disease or condition.

54. Use according to claim 52 in the manufacture of a medicament for the treatment or prevention of cancer.

55. Use according to claim 52 in the manufacture of a medicament for the treatment or prevention of an inflammatory disease or condition.

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