Thienoazepine, immunoconjugate, and their uses

Abstract

The present invention provides immunoconjugates of formula (I) comprising an antibody linked by conjugation to one or more thienoazepine derivatives. The present invention also provides thienoazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions are suitable substrates for forming immunoconjugates via linkers or linking moieties. The present invention further provides the above immunoconjugates for use in methods for treating cancer.

Description

Technical Field

[0001] Cross - reference to related applications This non - provisional application claims the benefit of priority of U.S. Provisional Application No. 62 / 926,333, filed Oct. 25, 2019, and U.S. Provisional Application No. 62 / 984,184, filed Mar. 2, 2020, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a Sequence Listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Oct. 5, 2020, is named 17019_006WO1_SL.txt and is 299,288 bytes in size.

[0003] The present invention generally relates to immunoconjugates comprising an antibody conjugated to one or more thienoazepine molecules.

Background Art

[0004] There is a need for new compositions and methods for delivering antibodies and immune adjuvants in order to reach inaccessible tumors and / or to expand the treatment options for cancer patients and other subjects. The present invention provides such compositions and methods.

Summary of the Invention

[0005] The present disclosure generally targets immunoconjugates comprising an antibody linked by conjugation to one or more thienoazepine derivatives. The present invention further relates to a thienoazepine derivative intermediate composition comprising a reactive functional group. Such an intermediate composition is a suitable substrate for the formation of an immunoconjugate in which the antibody can covalently bind to a thienoazepine (TAZ) moiety having the following formula by a linker L:

Chemical formula

[0006] The present invention further relates to the use of such immunoconjugates in the treatment of diseases, specifically cancer.

[0007] One aspect of the present invention is an immunoconjugate comprising an antibody covalently bound to a linker covalently bound to one or more thienoazepine moieties.

[0008] Another aspect of the present invention is a thienoazepine-linker compound.

[0009] Another aspect of the present invention is a method for treating cancer, comprising administering a therapeutically effective amount of an immunoconjugate comprising an antibody linked by conjugation to one or more thienoazepine moieties.

[0010] Another aspect of the present invention is the use of an immunoconjugate comprising an antibody linked by conjugation to one or more thienoazepine moieties for treating cancer.

[0011] Another aspect of the present invention is a method for preparing an immunoconjugate by conjugation of one or more thienoazepine moieties with an antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

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Mode for Carrying Out the Invention

[0013] Specific embodiments of the present invention will now be referred to in more detail, examples of which are illustrated in the accompanying structures and formulas. The present invention is described in combination with the recited embodiments, but it should be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all alternative forms, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0014] Those skilled in the art will understand many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0015] Definitions The term "immunoconjugate" refers to an antibody construct covalently bound to an adjuvant moiety via a linker. The term "adjuvant" refers to a substance capable of inducing an immune response in a subject exposed to the adjuvant. The phrase "adjuvant moiety" refers to an adjuvant covalently bound to an antibody construct, for example via a linker, as described herein. The adjuvant moiety can induce an immune response while bound to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0016] "Adjuvant" refers to a substance that can induce an immune response in a subject exposed to the adjuvant. The term "adjuvant moiety" refers to an adjuvant that is covalently attached to an antibody construct, for example via a linker, as described herein. The adjuvant moiety can induce an immune response while bound to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0017] The terms "Toll-like receptor" and "TLR" refer to any member of a family of highly conserved mammalian proteins that recognize pathogen-associated molecular patterns and act as important signaling elements in innate immunity. TLR polypeptides share a characteristic structure that includes an extracellular domain with leucine-rich repeats, a transmembrane domain, and an intracellular domain involved in TLR signaling.

[0018] The terms "Toll-like receptor 7" and "TLR7" refer to a nucleic acid or polypeptide that shares at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a published TLR7 sequence, e.g., GenBank accession number AAZ99026 for the human TLR7 polypeptide, or GenBank accession number AAK62676 for the mouse TLR7 polypeptide.

[0019] The terms "Toll-like receptor 8" and "TLR8" refer to a nucleic acid or polypeptide that shares at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a published TLR7 sequence, e.g., GenBank accession number AAZ95441 for the human TLR8 polypeptide, or GenBank accession number AAK62677 for the mouse TLR8 polypeptide.

[0020] A "TLR agonist" is a substance that binds directly or indirectly to a TLR (e.g., TLR7 and / or TLR8) and induces TLR signaling. Any detectable difference in TLR signaling can indicate that the agonist stimulates or activates the TLR. The difference in signaling can be manifested, for example, as a change in the expression of target genes, a change in the phosphorylation of signaling components, a change in the intracellular localization of downstream elements such as nuclear factor κB (NF-κB), a change in the association of a specific component (such as interleukin-1 receptor-associated kinase (IRAK)) with other proteins or intracellular structures, or a change in the biochemical activity of a component such as a kinase (such as mitogen-activated protein kinase (MAPK)).

[0021] An "antibody" refers to a polypeptide containing an antigen-binding region (including complementarity-determining regions (CDRs)) from an immunoglobulin gene or a fragment thereof. The term "antibody" specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, and each pair has one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa) connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains are categorized by size and function, for example, variable domains or regions on the light and heavy chains (V L and V H ) as well as constant domains or regions on the light and heavy chains (C L and C H) are classified into. The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids, called a paratope, which is mainly involved in antigen recognition, that is, an antigen-binding domain. The light chain is classified into either κ or λ. The heavy chain is classified as γ, μ, α, δ, or ε, and these heavy chains define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of approximately 150 kDa composed of four peptide chains. IgG antibodies contain two identical class γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, and thus contain a quaternary structure of a tetramer. The two heavy chains are linked to each other and to the light chains by disulfide bonds. The resulting tetramer has two identical halves that come together to form a Y shape. Each end of the fork contains an identical antigen-binding domain. There are four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) in humans, named in order of their abundance in serum (i.e., IgG1 is the most abundant). Typically, the antigen-binding domain of an antibody is most important in the specificity and affinity of binding to cancer cells.

[0022] "Antibody construct" refers to an antibody or fusion protein that includes (i) an antigen-binding domain and (ii) an Fc domain.

[0023] In some embodiments, the binder is an antigen-binding antibody "fragment", which is a construct that includes at least the antigen-binding region of the antibody, either alone or together with other components that make up the antigen-binding construct. Many different types of antibody "fragments" are known in the art, for example, (i) a monovalent fragment consisting of the V L , V H , C L , and CH1 domains, the Fab fragment, (ii) the F(ab’)2 fragment, a bivalent fragment that includes two Fab fragments linked by a disulfide bridge in the hinge region, (iii) a single group of V L and V HFv fragments composed of domains, (iv) Fab’ fragments resulting from cleavage of the disulfide bridges of F(ab’)2 fragments using mild reducing conditions, (v) disulfide-stabilized Fv fragments (dsFv), and (vi) two domains of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (i.e., V L and V H ), including single-chain Fv (scFv), which is a monovalent molecule.

[0024] An antibody or antibody fragment can be part of a larger construct, such as a conjugate of an antibody fragment to an additional region or a fusion construct. For example, in some embodiments, an antibody fragment can be fused to an Fc region as described herein. In other embodiments, an antibody fragment (e.g., Fab or scFv) can be part of a chimeric antigen receptor or chimeric T cell receptor by fusing, for example, to a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., a hinge region)) and any intercellular signaling domain. For example, an antibody fragment can be fused to the gamma and / or delta chains of a T cell receptor to provide a T cell receptor-like construct that binds to PD-L1. In yet another embodiment, an antibody fragment is part of a bispecific T cell engager (BiTE) that includes a CD1 or CD3 binding domain and a linker.

[0025] "Epitope" means any antigenic determinant or epitope determinant of an antigen to which an antigen-binding domain binds (i.e., at the paratope of the antigen-binding domain). Antigenic determinants usually consist of chemically active surface moieties of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0026] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three major classes of Fc receptors: (1) FcγRs that bind IgG, (2) FcαRs that bind IgA, and (3) FcεRs that bind IgE. The FcγR family includes several members such as FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). Fcγ receptors have different affinities for IgG and also different affinities for IgG subclasses (such as IgG1, IgG2, IgG3, and IgG4).

[0027] The "identity" of a nucleic acid or amino acid sequence referred to in this specification can be determined by comparing the nucleic acid or amino acid sequence of interest with a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., identical) between the optimally aligned sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the longer of the sequence of interest or the reference sequence). Alignment of sequences and calculation of percent identity can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, etc.) and FASTA programs (e.g., FASTA 3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7):951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). The percent identity of a sequence (%) is, for example, 100×[(same positions) / minimum(TG A ,TG B)] can also be calculated, where TG A and TG B are the total number of amino acid residues and the number of internal gap positions of the peptide sequences A and B of the alignment that minimizes TG A and TG B For example, see Russell et al., J. Mol Biol., 244:332-350 (1994).

[0028] The binding agent includes Ig heavy and light chain variable region polypeptides that together form an antigen-binding site. Each of the heavy and light chain variable regions is a polypeptide that includes three complementarity-determining regions (CDR1, CDR2, and CDR3) connected by framework regions. The binding agent can be any of various types of binding agents known in the art that include Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment", or a T cell receptor.

[0029] "Biosimilar" refers to an approved antibody construct having activity characteristics similar to those of previously approved PD-L1 targeting antibody constructs such as atezolizumab (TECENTRIQ™, Genentech, Inc.), durvalumab (IMFINZI™, AstraZeneca), and avelumab (BAVENCIO™, EMD Serono, Pfizer); previously approved HER2 targeting antibody constructs such as trastuzumab (HERCEPTIN™, Genentech, Inc.), and pertuzumab (PERJETA™, Genentech, Inc.), or an approved antibody construct such as labetuzumab (CEA-CIDE™, MN-14, hMN14, Immunomedics) (CAS registration number 219649-07-7) that targets CEA.

[0030] "BioBetter" refers to an approved antibody construct that is an improvement over previously approved antibody constructs such as atezolizumab, durvalumab, abemaciclib, trastuzumab, pertuzumab, and lapatinib. A BioBetter can have one or more modifications (e.g., an altered glycan profile, or a unique epitope) relative to a previously approved antibody construct.

[0031] "Amino acid" refers to any monomer unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring α-amino acids and their stereoisomers, as well as non-natural (not naturally occurring) amino acids and their stereoisomers. The "stereoisomers" of a given amino acid refer to isomers that have the same molecular formula and intramolecular bonds, but differ in the three-dimensional arrangement of the bonds and atoms (e.g., L-amino acids and the corresponding D-amino acids). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glycation) or deglycosylated. Amino acids may be represented herein by either the widely known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0032] Naturally occurring amino acids include those encoded by the genetic code and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0033] Naturally occurring amino acids include those formed in proteins by post-translational modification, such as citrulline (Cit).

[0034] Non-natural (not naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in either the L- or D-configuration that function similarly to naturally occurring amino acids. For example, an "amino acid analog" is a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to hydrogen, a carboxyl group, and an amino group), but has a modified side chain group or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure different from the general chemical structure of an amino acid, but functions in a similar manner to a naturally occurring amino acid.

[0035] "Linker" refers to a functional group that covalently attaches two or more parts of a compound or material. For example, the linking moiety can function to covalently attach an adjuvant moiety to an antibody construct in an immunoconjugate.

[0036] "Linking moiety" refers to a functional group that covalently attaches two or more parts of a compound or material. For example, the linking moiety can function to covalently attach an adjuvant moiety to an antibody in an immunoconjugate. Useful linkages for connecting a linking moiety to a protein and other materials include, but are not limited to, amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, and thiourea.

[0037] "Divalent" refers to a chemical moiety containing two attachment points for linking two functional groups; a polyvalent linking moiety can have additional attachment points for linking further functional groups. A divalent radical can be denoted by the suffix "diyl". For example, divalent linking moieties include divalent polymer moieties such as divalent poly(ethylene glycol), divalent cycloalkyl, divalent heterocycloalkyl, divalent aryl, and divalent heteroaryl groups. A "divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group" refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group having two attachment points for covalently linking two moieties in a molecule or material. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be substituted or unsubstituted. The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amide, acyl, nitro, cyano, and alkoxy.

[0038] Wavy line

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[0039] "Alkyl" refers to a straight-chain (linear) or branched-chain saturated aliphatic radical having the indicated number of carbon atoms. Alkyl can include any number of carbons, for example, from 1 to 12. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, 1-heptyl, 1-octyl, etc., but are not limited thereto. The alkyl group can be substituted or unsubstituted. The "substituted alkyl" group can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amide, acyl, nitro, cyano, and alkoxy.

[0040] The term "alkyl diyl" refers to a divalent alkyl radical. Examples of alkyl diyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), etc. The alkyl diyl group may also be referred to as an "alkylene" group.

[0041] "Alkenyl" refers to a straight-chain (linear) or branched-chain unsaturated aliphatic radical having at least one carbon-carbon double bond sp2 and the indicated number of carbon atoms. Alkenyl may contain 2 to about 12 or more carbon atoms. The alkenyl group is a radical having "cis" and "trans" orientations, or "E" and "Z" orientations. Examples include, but are not limited to, ethenyl or vinyl (-CH=CH2), allyl (-CH2CH=CH2), butenyl, pentenyl, and their isomers. The alkenyl group may be substituted or unsubstituted. The "substituted alkenyl" group may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amide, acyl, nitro, cyano, and alkoxy.

[0042] The term "alkenylene" or "alkenyl diyl" refers to a straight-chain or branched-chain divalent hydrocarbon radical. Examples include, but are not limited to, ethenylene or vinylene (-CH=CH-), allylene (-CH2CH=CH-), etc.

[0043] "Alkynyl" refers to a straight-chain (linear) or branched-chain unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon triple bond sp. Alkynyl can contain from 2 to about 12 or more carbon atoms. For example, C2-C6 alkynyl includes, but is not limited to, ethynyl (-C≡CH), propynyl (propargyl, -CH2C≡CH), butynyl, pentynyl, hexynyl, and their isomers. The alkynyl group can be either substituted or unsubstituted. The "substituted alkynyl" group can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amide, acyl, nitro, cyano, and alkoxy.

[0044] The term "alkynylene" or "alkynyldiyl" refers to a divalent alkynyl radical.

[0045] "Carbocycle", "carbocyclic", "cyclic carbon", and "cycloalkyl" refer to a saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing 3 to 12 ring atoms, or the indicated number of atoms. Saturated monocyclic carbocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocyclic rings include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. The carbocyclic group can be partially unsaturated and can have one or more double or triple bonds in the ring. Representative carbocyclic groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-, and 1,5-isomers), norbornene, and norbornadiene.

[0046] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0047] "Aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms (C6-C 20 ) derived by removing one hydrogen atom from a single carbon atom of the parent aromatic ring system. An aryl group can be monocyclic, condensed to form a bicyclic or tricyclic group, or linked by bonds to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl having a methylene linking group. Some aryl groups, such as phenyl, naphthalene, or biphenyl, have 6 to 12 ring members. Other aryl groups, such as phenyl and naphthyl, have 6 to 10 ring members.

[0048] The term "arylene" or "aryldiyl" means a divalent aromatic hydrocarbon radical of 6 to 20 carbon atoms (C6-C 20 ) derived by removing two hydrogen atoms from two carbon atoms of the parent aromatic ring system. Some aryldiyl groups are represented as "Ar" in an exemplary structure. Aryldiyl includes bicyclic radicals containing a saturated ring, a partially unsaturated ring, or an aromatic ring condensed to an aromatic cyclic carbon. General aryldiyl groups include, but are not limited to, benzene (phenyldiyl), substituted benzene, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, etc. The aryldiyl group is also called "arylene" and is optionally substituted with one or more substituents described herein.

[0049] The terms "heterocyclic ring", "heterocyclyl", and "heterocycle" are used interchangeably herein and refer to a carbocyclic radical of 3 to about 20 ring atoms that is saturated or partially unsaturated (i.e., has one or more double and / or triple bonds within the ring), where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally and independently substituted with one or more of the substituents described below. A heterocyclic ring is a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S), or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocyclic rings are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl" includes radicals in which a heterocyclic radical is fused to a saturated ring, a partially unsaturated ring, or an aromatic carbocyclic or heterocyclic ring.Examples of heterocycles include, but are not limited to, morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridylurea. Spiroheterocyclyl moieties are also included within the scope of this definition. Examples of spiroheterocyclyl moieties include azaspiro[2.5]octanyl and azaspiro[2.4]heptanyl. Examples of heterocyclic groups in which two ring atoms are substituted with an oxo (=O) moiety are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally and independently substituted with one or more substituents described herein.

[0050] The term "heterocyclyl diyl" refers to a divalent, saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms, where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally and independently substituted with one or more substituents as described. Examples of 5- and 6-membered heterocyclyl diyl include morpholinyl diyl, piperidinyl diyl, piperazinyl diyl, pyrrolidinyl diyl, dioxanyl diyl, thiomorpholinyl diyl, and S-dioxothiomorpholinyl diyl.

[0051] The term "heteroaryl" refers to a monovalent aromatic radical of 5-, 6-, or 7-membered rings, including a 5- to 20-atom fused ring system (at least one of which is aromatic) independently containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally and independently substituted with one or more substituents described herein.

[0052] The term "heteroaryldiyl" refers to a divalent aromatic radical of a 5-, 6-, or 7-membered ring, independently containing a 5- to 20-atom fused ring system containing one or more heteroatoms selected from nitrogen, oxygen, and sulfur, at least one of which is aromatic. Examples of 5- and 6-membered heteroaryldiyl include pyridyldiyl, imidazolydiyl, pyrimidinyl-diyl, pyrazolydiyl, triazolydiyl, pyrazinyl-diyl, tetrazolydiyl, furyl-diyl, thienyldiyl, isoxazolydiyl-diyl, thiazolydiyl, oxadiazolydiyl, oxazolydiyl, isothiazolydiyl, and pyrrolyldiyl.

[0053] A heterocyclic or heteroaryl group can be bonded, if possible, by carbon (carbon bond) or nitrogen (nitrogen bond). By way of example, but not limitation, a heterocyclic or heteroaryl bonded by carbon is bonded at the 2, 3, 4, 5, or 6 position of pyridine, the 3, 4, 5, or 6 position of pyridazine, the 2, 4, 5, or 6 position of pyrimidine, the 2, 3, 5, or 6 position of pyrazine, the 2, 3, 4, or 5 position of furan, tetrahydrofuran, thiophene, thiphene, pyrrole or tetrahydropyrrole, the 2, 4, or 5 position of oxazole, imidazole or thiazole, the 3, 4, or 5 position of isoxazole, pyrazole, or isothiazole, the 2 or 3 position of aziridine, the 2, 3, or 4 position of azetidine, the 2, 3, 4, 5, 6, 7, or 8 position of quinoline, or the 1, 3, 4, 5, 6, 7, or 8 position of isoquinoline.

[0054] By way of example, but not limitation, a heterocyclic or heteroaryl bonded by nitrogen is bonded at the 1 position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, isoindole, or isoindoline, the 4 position of morpholine, and the 9 position of carbazole, or β-carboline.

[0055] The terms "halo" and "halogen", by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0056] The term "carbonyl", by itself or as part of another substituent, refers to C(=O) or -C(=O)-, i.e., the carbon atom is double-bonded to oxygen and is bonded to the other two groups of the moiety having the carbonyl.

[0057] As used herein, the phrase "quaternary ammonium salt" refers to a tertiary amine quaternized with an alkyl substituent (e.g., a C1-C4 alkyl such as methyl, ethyl, propyl, or butyl).

[0058] The terms "treat", "treatment", and "treating" refer to alleviation; remission; reduction of symptoms, or making symptoms, injuries, conditions, or states more tolerable to the patient; decreasing the rate of progression of symptoms; reducing the frequency or duration of a symptom or condition; or, in some circumstances, preventing the onset of a symptom, and refer to signs of success in the treatment or improvement of an injury, condition, state (e.g., cancer), or symptom (e.g., cognitive impairment) that include any objective or subjective parameter. Treatment or improvement of a symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0059] The terms "cancer", "neoplasm", and "tumor" are used herein to refer to cells that exhibit an autonomous, unregulated growth such that they display an abnormal growth phenotype characterized by a significant loss of control over cell proliferation relative to cell growth. Cells that are the subject of detection, analysis, and / or treatment in the context of the present invention include cancer cells (e.g., cancer cells from an individual having cancer), malignant cancer cells, pre-metastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Substantially all tissue cancers are known. The phrase "cancer burden" refers to the amount of cancer cells or cancer volume in a subject. Thus, reducing the cancer burden refers to reducing the number of cancer cells or the cancer cell volume in the subject. As used herein, the term "cancer cell" refers to any cell that is a cancer cell (e.g., derived from any cancer for which an individual can be treated, e.g., isolated from an individual having cancer) or is derived from a cancer cell, e.g., is a clone of a cancer cell. For example, a cancer cell can be derived from an established cancer cell line, can be a primary cell isolated from an individual having cancer, can be a progeny cell from a primary cell isolated from an individual having cancer, and the like. In some embodiments, the term can also refer to a portion of a cancer cell, such as an intracellular portion, a cell membrane portion, or a cell lysate of a cancer cell. Many types of cancer are known to those of skill in the art and include solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, as well as circulating cancers such as leukemias.

[0060] As used herein, the term "cancer" includes any form of cancer including solid tumor cancers (e.g., skin, lung, prostate, breast, stomach, bladder, colon, ovary, pancreas, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine) and liquid cancers (e.g., blood cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including, but not limited to, both primary tumors and metastatic tumors, such as minimal residual disease, by way of example.

[0061] "PD-L1 expression" refers to cells having the PD-L1 receptor on the surface of the cell. As used herein, "PD-L1 overexpression" refers to cells having more PD-L1 receptors as compared to the corresponding non-cancerous cells.

[0062] "HER2" refers to the protein human epidermal growth factor receptor 2.

[0063] "HER2 expression" refers to cells having the HER2 receptor on the surface of the cell. For example, a cell may have from about 20,000 to about 50,000 HER2 receptors on the surface of the cell. As used herein, "HER2 overexpression" refers to cells having more than about 50,000 HER2 receptors. For example, a cell may have 2, 5, 10, 100, 1,000, 10,000, 100,000, or 10,000,000 times the number of HER2 receptors as compared to the corresponding non-cancerous cells (e.g., about 1 million or 2 million HER2 receptors). HER2 is estimated to be overexpressed in about 25% to about 30% of breast cancers.

[0064] The "pathology" of cancer includes all phenomena that impair the health of a patient. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, interference with the normal function of adjacent cells, release of cytokines or other secreted products at abnormal levels, suppression or exacerbation of inflammatory or immune responses, neoplasms, pre-malignant tumors, malignant tumors, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0065] As used herein, the terms "cancer recurrence" and "tumor recurrence," and grammatical variations thereof, refer to further growth of neoplastic or cancerous cells after diagnosis of cancer. In particular, recurrence can occur when further cancerous cell growth occurs in cancerous tissue. Similarly, "tumor spread" occurs when the cells of a tumor spread to local or distant tissues or organs, and thus tumor spread encompasses metastasis of the tumor. "Tumor invasion" occurs when tumor growth spreads locally and impairs the function of the tissues involved by compression, destruction, or suppression of normal organ function.

[0066] As used herein, the term "metastasis" refers to the growth of a cancerous tumor in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process such as the detachment of cancer cells from the original tumor site and the movement and / or invasion of cancer cells to other parts of the body.

[0067] The phrases "effective amount" and "therapeutically effective amount" refer to the dosage or amount of a substance, such as an immunoconjugate, that produces a therapeutic effect when administered. The exact dosage will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11 th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22 ndSee Edition, (Pharmaceutical Press, London, 2012). In the case of cancer, a therapeutically effective amount of the immunoconjugate can reduce the number of cancer cells, reduce tumor size, inhibit cancer cell infiltration into peripheral organs (i.e., a certain degree of deceleration and preferably stop), inhibit tumor metastasis (i.e., a certain degree of deceleration and preferably stop), inhibit tumor growth to a certain extent, and / or reduce one or more of the symptoms associated with cancer to a certain extent. To the extent that the immunoconjugate can prevent the growth of existing cancer cells and / or kill them, the immunoconjugate can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by evaluation of the time to disease progression (TTP) and / or determination of the response rate (RR).

[0068] "Recipient", "individual", "subject", "host", and "patient" are used interchangeably and refer to any mammalian subject (e.g., human) for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is a human.

[0069] The term "synergistic adjuvant" or "synergistic combination" in the context of the present invention includes combinations of two immunomodulators such as receptor agonists, cytokines, and adjuvant polypeptides, which, when combined, elicit a synergistic effect on immunity compared to administration of any of them alone. In particular, the immunoconjugates disclosed herein include a synergistic combination of the claimed adjuvant and antibody construct. These synergistic combinations upon administration elicit a greater effect on immunity, for example, compared to when the antibody construct or adjuvant is administered in the absence of the other moieties. Further, the amount of the immunoconjugate (measured by the total number of antibody constructs or the total number of adjuvants administered as part of the immunoconjugate) can be administered in a reduced amount compared to when either the antibody construct or adjuvant is administered alone.

[0070] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or implantation of a slow release device such as a small osmotic pump, for example, to a subject.

[0071] The terms "about" and "approximately" as used herein to modify a numerical value indicate the proximity range surrounding the numerical value. Thus, if "X" is a value, "about X" or "approximately X" indicates a value from 0.9X to 1.1X, such as a value from 0.95X to 1.05X or from 0.99X to 1.01X. Reference to "about X" or "approximately X" specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "approximately X" are intended to teach and provide written description support for claim limitations such as "0.98X", for example.

[0072] antibody The immunoconjugates of the present invention contain an antibody. Included within the scope of embodiments of the present invention are functional variants of the antibody constructs or antigen-binding domains described herein. As used herein, the term "functional variant" refers to an antibody construct having an antigen-binding domain that has substantial or significant sequence identity or similarity to a parental antibody construct or antigen-binding domain, and this functional variant retains the biological activity of the antibody construct or antigen-binding domain of which it is a variant. Functional variants include, for example, antibody constructs or antigen-binding domains (parental antibody constructs or antigen-binding domains) described herein that retain the ability to recognize target cells that express, to the same extent, to a comparable extent, or to a higher extent, PD-L1, HER2, or CEA as the parental antibody construct or antigen-binding domain.

[0073] With respect to an antibody construct or antigen-binding domain, a functional variant can have an amino acid sequence that is, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more, identical to the antibody construct or antigen-binding domain.

[0074] A functional variant can include, for example, the amino acid sequence of a parental antibody construct or antigen-binding domain having at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can include the amino acid sequence of a parental antibody construct or antigen-binding domain having at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. A non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parental antibody construct or antigen-binding domain.

[0075] Antibodies comprising the immunoconjugates of the present invention include Fc engineered variants. In some embodiments, mutations in the Fc region that result in modulation of binding to one or more Fc receptors are the following mutations: SD (S239D), SDIE (S239D / I332E), SE (S267E), SELF (S267E / L328F), SDIE (S239D / I332E), SDIEAL (S239D / I332E / A330L), GA (G236A), ALIE (A330L / I332E), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328, and A330. Additional Fc region modifications for modulating Fc receptor binding are described, for example, in U.S. Patent Application Publication No. 2016 / 0145350 and U.S. Patents Nos. 7,416,726 and 5,624,821, which are incorporated herein by reference in their entireties.

[0076] Antibodies comprising the immunoconjugates of the present invention include glycan variants such as afucosylation. In some embodiments, the Fc region of the binder is modified to have an altered glycosylation pattern of the Fc region as compared to the native unmodified Fc region.

[0077] The amino acid substitutions of the antibody constructs or antigen-binding domains of the present invention are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain specific physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions include substituting an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substituting an amino acid having a nonpolar side chain with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substituting a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substituting an uncharged amino acid having a polar side chain with another uncharged amino acid having a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substituting an amino acid having a beta-branched side chain with another amino acid having a beta-branched side chain (e.g., Ile, Thr, and Val), substituting an amino acid having an aromatic side chain with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr), and the like.

[0078] The antibody construct or antigen-binding domain can consist essentially of the specific amino acid sequence or sequences described herein such that other components, such as other amino acids, do not substantially alter the biological activity of the antibody construct or antigen-binding domain functional variant.

[0079] In some embodiments, the antibody in the immune complex comprises a modified Fc region, and the modification regulates the binding of the Fc region to one or more Fc receptors.

[0080] In some embodiments, the antibody in the immune complex (e.g., an antibody conjugated to at least two adjuvant moieties) comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) within the Fc region, thereby modulating (e.g., increasing or decreasing) the binding to one or more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and / or FcγRIIIB (CD16b)) as compared to a native antibody having no mutations in the Fc region. In some embodiments, the antibody in the immune complex comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) within the Fc region that decrease the binding of the Fc region of the antibody to FcγRIIB. In some embodiments, the antibody in the immune complex comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) within the Fc region that decrease the binding of the antibody to FcγRIIB while maintaining the same binding or increasing the binding to FcγRI (CD64), FcγRIIA (CD32A), and / or FcRγIIIA (CD16a) as compared to a native antibody having no mutations in the Fc region. In some embodiments, the antibody in the immune complex comprises one or more modifications within the Fc region that increase the binding of the Fc region of the antibody to FcγRIIB.

[0081] In some embodiments, the engineered binding is provided by mutations in the Fc region of the antibody as compared to the native Fc region of the antibody. The mutations can be present in the CH2 domain, the CH3 domain, or a combination thereof. "Native Fc region" is synonymous with "wild-type Fc region" and includes an amino acid sequence that is identical to the amino acid sequence of the Fc region found in nature or an amino acid sequence that is identical to the amino acid sequence of the Fc region found in a native antibody (e.g., cetuximab). Native sequence human Fc regions include native sequence human IgG1 Fc region, native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as their native variants. Native sequence Fc includes various allotypes of Fcs (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0082] In some embodiments, the mutations in the Fc region that modulate binding to one or more Fc receptors can include one or more of the following mutations: SD (S239D), SDIE (S239D / I332E), SE (S267E), SELF (S267E / L328F), SDIE (S239D / I332E), SDIEAL (S239D / I332E / A330L), GA (G236A), ALIE (A330L / I332E), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E233, G237, P238, H268, P271, L328, and A330. Additional Fc region modifications for modulating Fc receptor binding are described, for example, in US2016 / 0145350 and US7416726 and US5624821, which are hereby incorporated by reference in their entireties.

[0083] In some embodiments, the Fc region of the antibody of the immune complex is modified to change the glycosylation pattern of the Fc region as compared to the native unmodified Fc region.

[0084] Human immunoglobulins are glycosylated at the Asn297 residue of the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of the core heptasaccharide N-acetylglucosamine 4 mannose 3 (GlcNAc4Man3). Removal of the heptasaccharide by endoglycosidase or PNGaseF is known to cause conformational changes in the antibody Fc region, significantly reducing antibody binding affinity for activating FcγRs and potentially reducing effector function. The core heptasaccharide is often modified with galactose, bisecting GlcNAc, fucose, or sialic acid, differentially affecting Fc binding to activating and inhibitory FcγRs. Furthermore, α2,6-sialylation has been shown to enhance anti-inflammatory activity in vivo, while defucosylation results in improved FcγRIIIa binding and a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Thus, specific glycosylation patterns can be used to control inflammatory effector functions.

[0085] In some embodiments, the modification to alter the glycosylation pattern is a mutation. For example, a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of controlling the immune response using antibodies that modulate FcγR regulatory signaling are described, for example, in U.S. Patent No. 7,416,726 and U.S. Patent Application Publication Nos. 2007 / 0014795 and 2008 / 0286819, which are hereby incorporated by reference in their entirety.

[0086] In some embodiments, the antibody of the immune complex is modified to include a modified Fab region having a non-natural glycosylation pattern. For example, hybridomas can be genetically modified to secrete defucosylated mAbs, desialylated mAbs, or deglycosylated Fc with specific mutations that can increase FcRγIIIa binding and effector function. In some embodiments, the antibody of the immune complex is modified to be defucosylated.

[0087] In some embodiments, the entire Fc region of an antibody in an immune complex is exchanged with a different Fc region, such that the Fab region of the antibody is attached to a non-natural Fc region. For example, the Fab region of cetuximab, which normally contains an IgG1 Fc region, can be attached to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which normally contains an IgG4 Fc region, can be attached to IgG1, IgG2, IgG3, IgA1, or IgG2. In some embodiments, an Fc-modified antibody having a non-natural Fc domain also includes one or more amino acid modifications, such as the S228P mutation within IgG4 Fc, that modulate the stability of the described Fc domain. In some embodiments, an Fc-modified antibody having a non-natural Fc domain also includes one or more amino acid modifications described herein that modulate Fc binding to FcRs.

[0088] In some embodiments, a modification that modulates the binding of the Fc region to an FcR does not change the binding of the Fab region of the antibody to its antigen, as compared to the native, unmodified antibody. In other embodiments, a modification that modulates the binding of the Fc region to an FcR also increases the binding of the Fab region of the antibody to its antigen, as compared to the native, unmodified antibody.

[0089] In an exemplary embodiment, an immunoconjugate of the invention includes an antibody construct that includes an antigen-binding domain that specifically recognizes and binds PD-L1.

[0090] Programmed Death-Ligand 1 (PD-L1, Cluster of Differentiation 274, CD274, B7 homolog 1, or B7-H1) belongs to the B7 protein superfamily and is a ligand for Programmed Cell Death Protein 1 (PD-1, PDCD1, Cluster of Differentiation 279, or CD279). PD-L1 may also interact with B7.1 (CD80), and such interaction is thought to inhibit T cell priming. The PD-L1 / PD-1 axis plays a major role in the suppression of adaptive immune responses. More specifically, it is thought that binding of PD-L1 to its receptor PD-1 transmits signals that inhibit T cell activation and proliferation. Agents that bind to PD-L1 and prevent the ligand from binding to the PD-1 receptor can prevent this immunosuppression and thus enhance the immune response when desired, such as in the treatment of cancer or infections. Since the PD-L1 / PD-1 pathway also contributes to the prevention of autoimmunity, agonist agents for PD-L1 or agents that deliver immunosuppressive payloads may be useful in the treatment of autoimmune diseases.

[0091] Several antibodies targeting PD-L1, including atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™), have been developed for the treatment of cancer. Nevertheless, there remains a continuing need for new PD-L1 binding agents, such as agents that bind to PD-L1 with high affinity and effectively prevent PD-L1 / PD-1 signaling, and agents that can deliver a therapeutic payload to PD-L1 expressing cells. Additionally, new PD-L1 binding agents are needed for the treatment of autoimmune diseases and infections.

[0092] Provided is a method of delivering a thienoazepine derivative payload to cells expressing PD-L1, comprising administering to a cell or a mammal comprising the cell an immunoconjugate comprising an anti-PD-L1 antibody covalently linked to a linker covalently linked to one or more thienoazepine moieties.

[0093] Methods for enhancing, reducing, or inhibiting the immune response of a mammal, and methods for treating a disease, disorder, or condition in a mammal responsive to PD-L1 inhibition, including administering to the mammal a PD-L1 immunoconjugate, are also provided.

[0094] The present invention provides a PD-L1 binder comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide.

[0095] The PD-L1 binder specifically binds to PD-L1. Due to the binding specificity of the agent, PD-L1-expressing cells can be targeted, for example, to deliver a therapeutic payload to such cells.

[0096] In some embodiments, the PD-L1 binder (type A or type B) binds to human PD-L1, e.g., the protein comprising SEQ ID NO: 307. However, binders that bind to any PD-L1 homolog or paralog are also included. In some embodiments, the PD-L1 protein comprises at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to SEQ ID NO: 307. In some embodiments, the binder binds to human PD-L1 and cynomolgus monkey PD-L1; or human, cynomolgus monkey, and mouse PD-L1. MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET SEQ ID NO: 307

[0097] In some embodiments, the PD-L1 binder binds to PD-L1 without substantially inhibiting or preventing PD-L1 from binding to its receptor, PD-1. However, in other embodiments, the PD-L1 binder can completely or partially block (inhibit or prevent) PD-L1 from binding to its receptor, PD-1, and thus can inhibit PD-L1 / PD-1 signaling using an antibody (e.g., for therapeutic purposes).

[0098] The antibody or antigen-binding antibody fragment may be monospecific for PD-L1 or may be bispecific or multispecific. For example, in a bivalent or multivalent antibody or antibody fragment, the binding domains may differ such that they target different epitopes of the same antigen or different antigens. Methods of constructing multivalent binding constructs are known in the art. Bispecific and multispecific antibodies are known in the art. Further, a peptide linker too short to allow pairing between V H and V L contains V L connected to V H respectively, whereby different V H -V LDimer, trimer, or tetramer of polypeptide chains that drive pairing between complementary domains on the polypeptide chain to generate multimeric molecules having two, three, or four functional antigen-binding sites, and diabodies, tribodies, or tetrabodies can be provided. Also, a bis-scFv fragment, which is a small scFv fragment having two different variable domains, can be generated to generate a bispecific bis-scFv fragment that can bind to two different epitopes. Fab dimers (Fab2) and Fab trimers (Fab3) can be generated using genetic engineering methods to create multispecific constructs based on Fab fragments.

[0099] The PD-L1 binder can also be an antibody conjugate. In this regard, the PD-L1 binder can be a conjugate of (1) an antibody, alternative scaffold, or fragment thereof, and (2) a protein or non-protein moiety. For example, the PD-L1 binder can be conjugated to a peptide, fluorescent molecule, chemotherapeutic agent or other cytotoxic payload, immunostimulatory agent or immunosuppressive agent.

[0100] The PD-L1 binder can be, or can be obtained from, a human antibody, non-human antibody, humanized antibody, or chimeric antibody, or the corresponding antibody fragment. A "chimeric" antibody is typically an antibody or fragment thereof that includes a human constant region and a non-human variable region. A "humanized" antibody is typically a monoclonal antibody that includes a human antibody scaffold but has amino acids or sequences of non-human origin in at least one CDR (e.g., 1, 2, 3, 4, 5, or all 6 CDRs).

[0101] PD-L1 binder-Type A This specification provides a PD-L1 binder comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. In some embodiments, the PD-L1 binder (Type A) comprises any one of the immunoglobulin heavy chain variable regions of SEQ ID NOs: 223 to 264, or at least its CDRs; and any one of the immunoglobulin light chain variable regions of SEQ ID NOs: 265 to 306 or at least its CDRs. In other embodiments, the PD-L1 binder (Type A) comprises an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 223 to 264, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 265 to 306. In still other embodiments, the PD-L1 binder (Type A), the immunoglobulin heavy chain variable region polypeptide comprises Complementary Determining Region 1 (HCDR1) comprising any one of SEQ ID NOs: 1 to 23, Complementary Determining Region 2 (HCDR2) comprising any one of SEQ ID NOs: 24 to 57, and Complementary Determining Region 3 (HCDR3) comprising any one of SEQ ID NOs: 58 to 95; and / or the immunoglobulin light chain variable region polypeptide comprises Complementary Determining Region 1 (LCDR1) comprising any one of SEQ ID NOs: 96 to 128, Complementary Determining Region 2 (LCDR2) comprising any one of SEQ ID NOs: 129 to 151, and Complementary Determining Region 3 (LCDR3) comprising any one of SEQ ID NOs: 152 to 155. Also provided are nucleic acids encoding the PD-L1 binder, or its individual heavy and light chains; vectors and cells comprising the nucleic acids; and compositions comprising the binder or nucleic acids.

[0102] Furthermore, in some embodiments, the PD-L1 binder (Type A) provided herein, when binding to PD-L1 on the cell surface, causes internalization of PD-L1 or the PD-L1 / PD-L1 binder complex. Without wishing to be bound by a particular theory or mechanism of action, the PD-L1 binder according to this embodiment is thought to cause PD-L1 internalization upon binding, remain bound to PD-L1 during internalization, and result in internalization of the binder together with PD-L1. Cellular internalization of PD-L1 and the bound PD-L1 binder can be determined by any suitable method, such as an assay of persistence on the cell surface and / or detection of internalized antibodies. In some embodiments, the PD-L1 binder internalizes strongly enough such that at least about 25% (e.g., at least about 35%, at least about 50%, at least about 75%, or at least about 90%) of the PD-L1 binder that binds to PD-L1 on the cell surface is internalized (e.g., using a surface persistence assay, no more than about 75%, about 65%, about 50%, about 25%, or about 10% of the PD-L1 binder molecules that bind to PD-L1 on the cell surface at the start of the assay remain bound at the end of the assay).

[0103] In one embodiment, the PD-L1 binder (Type A) comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, a sequence identical to SEQ ID NOs: 223-264, or at least its CDRs; and / or an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, a sequence identical to SEQ ID NOs: 265-306 or at least its CDRs.

[0104] As a further example, the PD-L1 binder (Type A) can include the following: (1) The immunoglobulin heavy chain variable region of SEQ ID NO: 223, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 265, or at least its CDR; (2) The immunoglobulin heavy chain variable region of SEQ ID NO: 224, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 266, or at least its CDR; (3) The immunoglobulin heavy chain variable region of SEQ ID NO: 225, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 267, or at least its CDR; (4) The immunoglobulin heavy chain variable region of SEQ ID NO: 226, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 268, or at least its CDR; (5) The immunoglobulin heavy chain variable region of SEQ ID NO: 227, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 269, or at least its CDR; (6) The immunoglobulin heavy chain variable region of SEQ ID NO: 228, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 270, or at least its CDR; (7) The immunoglobulin heavy chain variable region of SEQ ID NO: 229, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 271, or at least its CDR; (8) The immunoglobulin heavy chain variable region of SEQ ID NO: 230, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 272, or at least its CDR;2 (9) The immunoglobulin heavy chain variable region of SEQ ID NO: 231, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 273, or at least its CDR; (10) The immunoglobulin heavy chain variable region of SEQ ID NO: 232, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 274, or at least its CDR; (11) The immunoglobulin heavy chain variable region of SEQ ID NO: 233, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 275, or at least its CDR; (12) The immunoglobulin heavy chain variable region of SEQ ID NO: 234, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 276, or at least its CDR; (13) The immunoglobulin heavy chain variable region of SEQ ID NO: 235, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 277, or at least its CDR; (14) The immunoglobulin heavy chain variable region of SEQ ID NO: 236, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 278, or at least its CDR; (15) The immunoglobulin heavy chain variable region of SEQ ID NO: 237, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 279, or at least its CDR; (16) The immunoglobulin heavy chain variable region of SEQ ID NO: 238, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 280, or at least its CDR; (17) The immunoglobulin heavy chain variable region of SEQ ID NO: 239, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDR; (18) The immunoglobulin heavy chain variable region of SEQ ID NO: 240, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 282, or at least its CDR; (19) The immunoglobulin heavy chain variable region of SEQ ID NO: 241, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 283, or at least its CDR; (20) The immunoglobulin heavy chain variable region of SEQ ID NO: 242, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 284, or at least its CDR; (21) The immunoglobulin heavy chain variable region of SEQ ID NO: 243, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 285, or at least its CDR; (22) The immunoglobulin heavy chain variable region of SEQ ID NO: 244, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 286, or at least its CDR; (23) The immunoglobulin heavy chain variable region of SEQ ID NO: 245, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 287, or at least its CDR; (24) The immunoglobulin heavy chain variable region of SEQ ID NO: 246, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 288, or at least its CDR; (25) The immunoglobulin heavy chain variable region of SEQ ID NO: 247, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 289, or at least its CDR; (26) The immunoglobulin heavy chain variable region of SEQ ID NO: 248, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 290, or at least its CDR; (27) The immunoglobulin heavy chain variable region of SEQ ID NO: 249, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 291, or at least its CDR; (28) The immunoglobulin heavy chain variable region of SEQ ID NO: 250, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 292, or at least its CDR; (29) The immunoglobulin heavy chain variable region of SEQ ID NO: 251, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 293, or at least its CDR; (30) The immunoglobulin heavy chain variable region of SEQ ID NO: 252, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 294, or at least its CDR; (31) The immunoglobulin heavy chain variable region of SEQ ID NO: 253, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 295, or at least its CDR; (32) The immunoglobulin heavy chain variable region of SEQ ID NO: 254, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 296, or at least its CDR; (33) The immunoglobulin heavy chain variable region of SEQ ID NO: 255, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 297, or at least its CDR; (34) The immunoglobulin heavy chain variable region of SEQ ID NO: 256, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 298, or at least its CDR; (35) The immunoglobulin heavy chain variable region of SEQ ID NO: 257, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 299, or at least its CDR; (36) The immunoglobulin heavy chain variable region of SEQ ID NO: 258, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 300, or at least its CDR; (37) The immunoglobulin heavy chain variable region of SEQ ID NO: 259, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 301, or at least its CDR; (38) The immunoglobulin heavy chain variable region of SEQ ID NO: 260, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 302, or at least its CDR; (39) The immunoglobulin heavy chain variable region of SEQ ID NO: 261, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 303, or at least its CDR; (40) The immunoglobulin heavy chain variable region of SEQ ID NO: 262, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 304, or at least its CDR; (41) The immunoglobulin heavy chain variable region of SEQ ID NO: 263, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 305, or at least its CDR; (42) The immunoglobulin heavy chain variable region of SEQ ID NO: 164, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 306, or at least its CDR; and / or (43) The immunoglobulin heavy chain variable region of FIGS. 4A - D and / or the immunoglobulin light chain variable region of FIGS. 4E - G, or at least its CDR.

[0105] The CDR of a given heavy or light chain Ig sequence can be determined according to any of various known Ig numbering schemes (e.g., Kabat, Chothia, Martin (extended Chothia), IGMT, AbM). In certain embodiments, the PD - L1 binder (type A) comprises one or more of the following CDRs: Any one of SEQ ID NOs: 1 - 23, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 1 - 23, and comprising or consisting of an HCDR1; Any one of SEQ ID NOs: 24 - 57, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 24 - 57, and comprising or consisting of an HCDR2; and Any one of SEQ ID NOs: 58 - 95, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 58 - 95, and comprising or consisting of an HCDR3; and / or the immunoglobulin light chain polypeptide is LCDR1 comprising or consisting of an array that is any one of SEQ ID NOs: 96 to 128, or at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 96 to 128; LCDR2 comprising or consisting of an array that is any one of SEQ ID NOs: 129 to 151, or at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 129 to 151; and LCDR3 comprising or consisting of an array that is any one of SEQ ID NOs: 152 to 155, or at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 152 to 155.

[0106] In certain embodiments, the binder (type A) comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, wherein (1) the immunoglobulin heavy chain polypeptide comprises HCDR1 comprising or consisting of SEQ ID NO: 1, HCDR2 comprising or consisting of SEQ ID NO: 24, and HCDR3 comprising or consisting of SEQ ID NO: 58; and / or the immunoglobulin light chain polypeptide comprises LCDR1 comprising or consisting of SEQ ID NO: 96, LCDR2 comprising or consisting of SEQ ID NO: 129, and LCDR3 comprising or consisting of SEQ ID NO: 152; (2) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 25, and an HCDR3 comprising or consisting of SEQ ID NO: 59; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 153; (3) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 3, an HCDR2 comprising or consisting of SEQ ID NO: 26, and an HCDR3 comprising or consisting of SEQ ID NO: 60; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 154; (4) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 4, an HCDR2 comprising or consisting of SEQ ID NO: 27, and an HCDR3 comprising or consisting of SEQ ID NO: 61; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 99, an LCDR2 comprising or consisting of SEQ ID NO: 130, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (5) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 5, an HCDR2 comprising or consisting of SEQ ID NO: 28, and an HCDR3 comprising or consisting of SEQ ID NO: 62; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 100, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 153; (6) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 6, an HCDR2 comprising or consisting of SEQ ID NO: 29, and an HCDR3 comprising or consisting of SEQ ID NO: 63; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 101, an LCDR2 comprising or consisting of SEQ ID NO: 131, and an LCDR3 comprising or consisting of SEQ ID NO: 156; (7) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 7, an HCDR2 comprising or consisting of SEQ ID NO: 30, and an HCDR3 comprising or consisting of SEQ ID NO: 64; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 102, an LCDR2 comprising or consisting of SEQ ID NO: 132, and an LCDR3 comprising or consisting of SEQ ID NO: 157; (8) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 31, and an HCDR3 comprising or consisting of SEQ ID NO: 65; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 103, an LCDR2 comprising or consisting of SEQ ID NO: 133, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (9) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 8, an HCDR2 comprising or consisting of SEQ ID NO: 32, and an HCDR3 comprising or consisting of SEQ ID NO: 66; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 104, an LCDR2 comprising or consisting of SEQ ID NO: 134, and an LCDR3 comprising or consisting of SEQ ID NO: 158; (10) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 9, an HCDR2 comprising or consisting of SEQ ID NO: 33, and an HCDR3 comprising or consisting of SEQ ID NO: 67; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 135, and an LCDR3 comprising or consisting of SEQ ID NO: 159; (11) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 7, an HCDR2 comprising or consisting of SEQ ID NO: 34, and an HCDR3 comprising or consisting of SEQ ID NO: 64; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 102, an LCDR2 comprising or consisting of SEQ ID NO: 132, and an LCDR3 comprising or consisting of SEQ ID NO: 160; (12) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 10, an HCDR2 comprising or consisting of SEQ ID NO: 35, and an HCDR3 comprising or consisting of SEQ ID NO: 68; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 105, an LCDR2 comprising or consisting of SEQ ID NO: 136, and an LCDR3 comprising or consisting of SEQ ID NO: 161; (13) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 25, and an HCDR3 comprising or consisting of SEQ ID NO: 69; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 106, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 162; (14) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 11, an HCDR2 comprising or consisting of SEQ ID NO: 36, and an HCDR3 comprising or consisting of SEQ ID NO: 70; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 107, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 163; (15) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 12, an HCDR2 comprising or consisting of SEQ ID NO: 37, and an HCDR3 comprising or consisting of SEQ ID NO: 71; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 108, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 164; (16) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 1, an HCDR2 comprising or consisting of SEQ ID NO: 38, and an HCDR3 comprising or consisting of SEQ ID NO: 72; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 109, an LCDR2 comprising or consisting of SEQ ID NO: 138, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (17) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 13, an HCDR2 comprising or consisting of SEQ ID NO: 39, and an HCDR3 comprising or consisting of SEQ ID NO: 73; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (18) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 40, and an HCDR3 comprising or consisting of SEQ ID NO: 74; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 110, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 166; (19) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 75; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 111, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (20) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 15, an HCDR2 comprising or consisting of SEQ ID NO: 42, and an HCDR3 comprising or consisting of SEQ ID NO: 74; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 139, and an LCDR3 comprising or consisting of SEQ ID NO: 152; (21) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 43, and an HCDR3 comprising or consisting of SEQ ID NO: 76; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 112, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (22) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 16, an HCDR2 comprising or consisting of SEQ ID NO: 44, and an HCDR3 comprising or consisting of SEQ ID NO: 77; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 113, an LCDR2 comprising or consisting of SEQ ID NO: 140, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (23) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 9, an HCDR2 comprising or consisting of SEQ ID NO: 45, and an HCDR3 comprising or consisting of SEQ ID NO: 78; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 114, an LCDR2 comprising or consisting of SEQ ID NO: 141, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (24) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 46, and an HCDR3 comprising or consisting of SEQ ID NO: 79; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (25) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 9, an HCDR2 comprising or consisting of SEQ ID NO: 25, and an HCDR3 comprising or consisting of SEQ ID NO: 80; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 115, an LCDR2 comprising or consisting of SEQ ID NO: 142, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (26) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 17, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 81; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 116, an LCDR2 comprising or consisting of SEQ ID NO: 143, and an LCDR3 comprising or consisting of SEQ ID NO: 167; (27) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 7, an HCDR2 comprising or consisting of SEQ ID NO: 47, and an HCDR3 comprising or consisting of SEQ ID NO: 82; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 117, an LCDR2 comprising or consisting of SEQ ID NO: 144, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (28) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 83; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 118, an LCDR2 comprising or consisting of SEQ ID NO: 131, and an LCDR3 comprising or consisting of SEQ ID NO: 168; (29) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 18, an HCDR2 comprising or consisting of SEQ ID NO: 48, and an HCDR3 comprising or consisting of SEQ ID NO: 84; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 119, an LCDR2 comprising or consisting of SEQ ID NO: 145, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (30) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 19, an HCDR2 comprising or consisting of SEQ ID NO: 49, and an HCDR3 comprising or consisting of SEQ ID NO: 85; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 120, an LCDR2 comprising or consisting of SEQ ID NO: 146, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (31) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 50, and an HCDR3 comprising or consisting of SEQ ID NO: 86; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 121, an LCDR2 comprising or consisting of SEQ ID NO: 147, and an LCDR3 comprising or consisting of SEQ ID NO: 169; (32) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 51, and an HCDR3 comprising or consisting of SEQ ID NO: 87; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 122, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 155; (33) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 20, an HCDR2 comprising or consisting of SEQ ID NO: 44, and an HCDR3 comprising or consisting of SEQ ID NO: 88; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 123, an LCDR2 comprising or consisting of SEQ ID NO: 148, and an LCDR3 comprising or consisting of SEQ ID NO: 170; (34) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 3, an HCDR2 comprising or consisting of SEQ ID NO: 52, and an HCDR3 comprising or consisting of SEQ ID NO: 60; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 98, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 171; (35) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 53, and an HCDR3 comprising or consisting of SEQ ID NO: 89; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 147, and an LCDR3 comprising or consisting of SEQ ID NO: 172; (36) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 21, an HCDR2 comprising or consisting of SEQ ID NO: 38, and an HCDR3 comprising or consisting of SEQ ID NO: 90; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 109, an LCDR2 comprising or consisting of SEQ ID NO: 150, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (37) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 22, an HCDR2 comprising or consisting of SEQ ID NO: 41, and an HCDR3 comprising or consisting of SEQ ID NO: 91; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 124, an LCDR2 comprising or consisting of SEQ ID NO: 151, and an LCDR3 comprising or consisting of SEQ ID NO: 173; (38) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 54, and an HCDR3 comprising or consisting of SEQ ID NO: 92; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 126, an LCDR2 comprising or consisting of SEQ ID NO: 129, and an LCDR3 comprising or consisting of SEQ ID NO: 165; (39) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 2, an HCDR2 comprising or consisting of SEQ ID NO: 55, and an HCDR3 comprising or consisting of SEQ ID NO: 93; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 97, an LCDR2 comprising or consisting of SEQ ID NO: 149, and an LCDR3 comprising or consisting of SEQ ID NO: 174; (40) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 23, an HCDR2 comprising or consisting of SEQ ID NO: 56, and an HCDR3 comprising or consisting of SEQ ID NO: 94; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 125, an LCDR2 comprising or consisting of SEQ ID NO: 142, and an LCDR3 comprising or consisting of SEQ ID NO: 175; (41) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 14, an HCDR2 comprising or consisting of SEQ ID NO: 43, and an HCDR3 comprising or consisting of SEQ ID NO: 76; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 127, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 176; (42) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 3, an HCDR2 comprising or consisting of SEQ ID NO: 57, and an HCDR3 comprising or consisting of SEQ ID NO: 95; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 128, an LCDR2 comprising or consisting of SEQ ID NO: 137, and an LCDR3 comprising or consisting of SEQ ID NO: 155; and / or (43) The immunoglobulin heavy chain polypeptide and the light chain polypeptide comprise any combination of the CDRs described in FIGS. 1A-D of PD-L1 type A binders 1-42.

[0107] In certain embodiments, the binder comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, the immunoglobulin heavy chain polypeptide comprising a first framework region, a second framework region, a third framework region, and / or a fourth framework region; and / or the immunoglobulin light chain polypeptide comprising a first framework region, a second framework region, a third framework region, and / or a fourth framework region; and / or the immunoglobulin heavy chain polypeptide and the light chain polypeptide each comprise any combination of the framework regions described in FIGS. 2A-D and FIGS. 3A-D.

[0108] PD-L1 binder - type B Disclosed herein is a PD-L1 binder (Type B) comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. In some embodiments, the PD-L1 binder (Type B) comprises any one of the immunoglobulin heavy chain variable regions of SEQ ID NOs: 430 to 450, or at least its CDRs, and any one of the immunoglobulin light chain variable regions of SEQ ID NOs: 451 to 471 or at least its CDRs. In other embodiments, the PD-L1 binder comprises an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 430 to 450, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 451 to 471. In still other embodiments, for the PD-L1 binder, the immunoglobulin heavy chain variable region polypeptide comprises a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 308 to 321, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 332 to 338, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 339 to 359; and / or the immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 360 to 374, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 131 and 375 to 386, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 387 to 398. Also provided are nucleic acids encoding the PD-L1 binder, or its individual heavy and light chains; vectors and cells comprising the nucleic acids; and compositions comprising the binder or nucleic acid.

[0109] In one embodiment, the PD-L1 binder (Type B) comprises any one immunoglobulin heavy chain variable region of SEQ ID NOs: 430 to 450, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, a sequence identical to SEQ ID NOs: 430 to 450, or at least its CDR, and / or any one immunoglobulin light chain variable region of SEQ ID NOs: 451 to 471, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, a sequence identical to SEQ ID NOs: 451 to 471 or at least its CDR.

[0110] As a further illustration, the PD-L1 binder (Type B) can include the following: (1) The immunoglobulin heavy chain variable region of SEQ ID NO: 429, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 450, or at least its CDR; (2) The immunoglobulin heavy chain variable region of SEQ ID NO: 430, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 451, or at least its CDR; (3) The immunoglobulin heavy chain variable region of SEQ ID NO: 431, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 452, or at least its CDR; (4) The immunoglobulin heavy chain variable region of SEQ ID NO: 432, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 453, or at least its CDR; (5) The immunoglobulin heavy chain variable region of SEQ ID NO: 433, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 454, or at least its CDR; (6) The immunoglobulin heavy chain variable region of SEQ ID NO: 434, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 455, or at least its CDR; (7) The immunoglobulin heavy chain variable region of SEQ ID NO: 435, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 456, or at least its CDR; (8) The immunoglobulin heavy chain variable region of SEQ ID NO: 436, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 457, or at least its CDR; (9) The immunoglobulin heavy chain variable region of SEQ ID NO: 437, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 458, or at least its CDR; (10) The immunoglobulin heavy chain variable region of SEQ ID NO: 438, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 459, or at least its CDR; (11) The immunoglobulin heavy chain variable region of SEQ ID NO: 439, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 460, or at least its CDR; (12) The immunoglobulin heavy chain variable region of SEQ ID NO: 440, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 461, or at least its CDR; (13) The immunoglobulin heavy chain variable region of SEQ ID NO: 441, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 462, or at least its CDR; (14) The immunoglobulin heavy chain variable region of SEQ ID NO: 442, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 463, or at least its CDR; (15) The immunoglobulin heavy chain variable region of SEQ ID NO: 443, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 464, or at least its CDR; (16) The immunoglobulin heavy chain variable region of SEQ ID NO: 444, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 465, or at least its CDR; (17) The immunoglobulin heavy chain variable region of SEQ ID NO: 445, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 466, or at least its CDR; (18) The immunoglobulin heavy chain variable region of SEQ ID NO: 446, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 467, or at least its CDR; (19) The immunoglobulin heavy chain variable region of SEQ ID NO: 447, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 468, or at least its CDR; (20) The immunoglobulin heavy chain variable region of SEQ ID NO: 448, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 469, or at least its CDR; and / or (21) The immunoglobulin heavy chain variable region of SEQ ID NO: 449, or at least its CDR, and / or the immunoglobulin light chain variable region of SEQ ID NO: 470, or at least its CDR; and / or (22) The immunoglobulin heavy chain variable region of FIGS. 8A - B and / or the immunoglobulin light chain variable region of FIGS. 8C - D, or at least its CDR.

[0111] The CDR of a given heavy or light chain Ig sequence can be determined according to any of various known Ig numbering schemes (e.g., Kabat, Chothia, Martin (extended Chothia), IGMT, AbM). In certain embodiments, the PD - L1 binder comprises one or more of the following CDRs: An HCDR1 comprising or consisting of any one of SEQ ID NOs: 308 to 321, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 308 to 321; An HCDR2 comprising or consisting of any one of SEQ ID NOs: 322 to 338, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 322 to 338; and An HCDR3 comprising or consisting of any one of SEQ ID NOs: 339 to 359, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 339 to 359; and / or the immunoglobulin light chain polypeptide is An LCDR1 comprising or consisting of any one of SEQ ID NOs: 360 to 374, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 360 to 374; An LCDR2 comprising or consisting of any one of SEQ ID NOs: 375 to 386, or a sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 375 to 386; An LCDR3 comprising or consisting of an array that is any one of SEQ ID NOs: 387 to 398, or at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NOs: 387 to 398.

[0112] In certain embodiments, the binder comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, wherein (1) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 308, an HCDR2 comprising or consisting of SEQ ID NO: 322, and an HCDR3 comprising or consisting of SEQ ID NO: 339; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 360, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 387; (2) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 323, and an HCDR3 comprising or consisting of SEQ ID NO: 340; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 361, an LCDR2 comprising or consisting of SEQ ID NO: 376, and an LCDR3 comprising or consisting of SEQ ID NO: 388; (3) the immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 310, an HCDR2 comprising or consisting of SEQ ID NO: 324, and an HCDR3 comprising or consisting of SEQ ID NO: 341; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 360, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 387; (4) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 311, an HCDR2 comprising or consisting of SEQ ID NO: 325, and an HCDR3 comprising or consisting of SEQ ID NO: 342; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 362, an LCDR2 comprising or consisting of SEQ ID NO: 377, and an LCDR3 comprising or consisting of SEQ ID NO: 389; (5) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 312, an HCDR2 comprising or consisting of SEQ ID NO: 326, and an HCDR3 comprising or consisting of SEQ ID NO: 343; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 360, an LCDR2 comprising or consisting of SEQ ID NO: 378, and an LCDR3 comprising or consisting of SEQ ID NO: 387; (6) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 313, an HCDR2 comprising or consisting of SEQ ID NO: 327, and an HCDR3 comprising or consisting of SEQ ID NO: 344; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 363, an LCDR2 comprising or consisting of SEQ ID NO: 379, and an LCDR3 comprising or consisting of SEQ ID NO: 390; (7) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 314, an HCDR2 comprising or consisting of SEQ ID NO: 327, and an HCDR3 comprising or consisting of SEQ ID NO: 345; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 364, an LCDR2 comprising or consisting of SEQ ID NO: 380, and an LCDR3 comprising or consisting of SEQ ID NO: 391; (8) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 312, an HCDR2 comprising or consisting of SEQ ID NO: 328, and an HCDR3 comprising or consisting of SEQ ID NO: 346; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 365, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 387; (9) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 314, an HCDR2 comprising or consisting of SEQ ID NO: 329, and an HCDR3 comprising or consisting of SEQ ID NO: 347; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 366, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 389; (10) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 330, and an HCDR3 comprising or consisting of SEQ ID NO: 348; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 360, an LCDR2 comprising or consisting of SEQ ID NO: 381, and an LCDR3 comprising or consisting of SEQ ID NO: 392; (11) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 327, and an HCDR3 comprising or consisting of SEQ ID NO: 349; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 367, an LCDR2 comprising or consisting of SEQ ID NO: 382, and an LCDR3 comprising or consisting of SEQ ID NO: 389; (12) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 309, an HCDR2 comprising or consisting of SEQ ID NO: 322, and an HCDR3 comprising or consisting of SEQ ID NO: 350; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 360, an LCDR2 comprising or consisting of SEQ ID NO: 383, and an LCDR3 comprising or consisting of SEQ ID NO: 387; (13) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 315, an HCDR2 comprising or consisting of SEQ ID NO: 323, and an HCDR3 comprising or consisting of SEQ ID NO: 351; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 368, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 393; (14) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 316, an HCDR2 comprising or consisting of SEQ ID NO: 331, and an HCDR3 comprising or consisting of SEQ ID NO: 352; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 365, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 389; (15) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 317, an HCDR2 comprising or consisting of SEQ ID NO: 332, and an HCDR3 comprising or consisting of SEQ ID NO: 353; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 369, an LCDR2 comprising or consisting of SEQ ID NO: 384, and an LCDR3 comprising or consisting of SEQ ID NO: 394; (16) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 318, an HCDR2 comprising or consisting of SEQ ID NO: 333, and an HCDR3 comprising or consisting of SEQ ID NO: 354; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 370, an LCDR2 comprising or consisting of SEQ ID NO: 379, and an LCDR3 comprising or consisting of SEQ ID NO: 395; (17) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 310, an HCDR2 comprising or consisting of SEQ ID NO: 334, and an HCDR3 comprising or consisting of SEQ ID NO: 355; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 371, an LCDR2 comprising or consisting of SEQ ID NO: 375, and an LCDR3 comprising or consisting of SEQ ID NO: 387; (18) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 310, an HCDR2 comprising or consisting of SEQ ID NO: 335, and an HCDR3 comprising or consisting of SEQ ID NO: 356; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 360, an LCDR2 comprising or consisting of SEQ ID NO: 385, and an LCDR3 comprising or consisting of SEQ ID NO: 396; (19) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 319, an HCDR2 comprising or consisting of SEQ ID NO: 336, and an HCDR3 comprising or consisting of SEQ ID NO: 357; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 372, an LCDR2 comprising or consisting of SEQ ID NO: 386, and an LCDR3 comprising or consisting of SEQ ID NO: 397; (20) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 320, an HCDR2 comprising or consisting of SEQ ID NO: 337, and an HCDR3 comprising or consisting of SEQ ID NO: 358; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 373, an LCDR2 comprising or consisting of SEQ ID NO: 379, and an LCDR3 comprising or consisting of SEQ ID NO: 398; (21) The immunoglobulin heavy chain polypeptide comprises an HCDR1 comprising or consisting of SEQ ID NO: 321, an HCDR2 comprising or consisting of SEQ ID NO: 338, and an HCDR3 comprising or consisting of SEQ ID NO: 359; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO: 374, an LCDR2 comprising or consisting of SEQ ID NO: 379, and an LCDR3 comprising or consisting of SEQ ID NO: 389; and / or (22) The immunoglobulin heavy chain polypeptide and the light chain polypeptide comprise any combination of the CDRs listed in FIGS. 5A - B (Type B).

[0113] In certain embodiments, the binder comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, the immunoglobulin heavy chain polypeptide comprising a first framework region, a second framework region, a third framework region, and / or a fourth framework region; and / or the immunoglobulin light chain polypeptide comprising a first framework region, a second framework region, a third framework region, and / or a fourth framework region; and / or the immunoglobulin heavy chain polypeptide and the light chain polypeptide each comprise any combination of the framework regions listed in FIGS. 6A - B and / or FIGS. 7A - B (Type B).

[0114] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen - binding domain that specifically recognizes and binds HER2.

[0115] In certain embodiments, the immunoconjugates of the invention comprise an anti-HER2 antibody. In one embodiment of the invention, the anti-HER2 antibody of the immunoconjugate of the invention is a humanized anti-HER2 antibody, such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in Table 3 of US 5821337, which is specifically incorporated herein by reference. These antibodies contain human framework regions having the complementarity determining regions of a mouse antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also known as trastuzumab and is commercially available under the trade name HERCEPTIN (Genentech, Inc.).

[0116] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1κ monoclonal antibody that is a humanized version of a mouse anti-HER2 antibody (4D5) that selectively binds with high affinity (Kd = 5 nM) to the extracellular domain of HER2 in cell-based assays (US 5677171; US 5821337; US 6054297; US 6165464; US 6339142; US 6407213; US 6639055; US 6719971; US 6800738; US 7074404; Coussens et al (1985) Science 230:1132-9, Slamon et al (1989) Science 244:707-12, Slamon et al (2001) New Engl. J. Med. 344:783-792).

[0117] In one embodiment of the present invention, the antibody construct or antigen-binding domain comprises the CDR region of trastuzumab. In one embodiment of the present invention, the anti-HER2 antibody further comprises the framework region of trastuzumab. In one embodiment of the present invention, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0118] In another embodiment of the present invention, the anti-HER2 antibody of the immunoconjugate of the present invention comprises a humanized anti-HER2 antibody, such as humanized 2C4, as described in US7862817. An exemplary humanized 2C4 antibody is pertuzumab (CAS Registry Number 380610-27-5), PERJETA™ (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI) and functions to inhibit the ability of HER2 to form active heterodimers or homodimers with other HER receptors (such as EGFR / HER1, HER2, HER3, and HER4). See, for example, Harari and Yarden, Oncogene 19:6102-14 (2000); Yarden and Sliwkowski, Nat Rev Mol Cell Biol 2:127-37 (2001); Sliwkowski, Nat Struct Biol 10:158-9 (2003); Cho et al., Nature 421:756-60 (2003); and Malik et al., Pro Am Soc Cancer Res 44:176-7 (2003). PERJETA™ is approved as a therapeutic for breast cancer.

[0119] In one embodiment of the present invention, the antibody construct or antigen-binding domain comprises the CDR region of pertuzumab. In one embodiment of the present invention, the anti-HER2 antibody further comprises the framework region of pertuzumab. In one embodiment of the present invention, the anti-HER2 antibody further comprises one or both variable regions of pertuzumab.

[0120] In an exemplary embodiment, the immunoconjugate of the present invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds CEA. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CD66e (Cluster of Differentiation 66e), is a member of the carcinoembryonic antigen (CEA) gene family.

[0121] In an exemplary embodiment, the immunoconjugate of the present invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds Caprin-1 (Ellis JA, Luzio JP (1995) J Biol Chem. 270(35):20717-23; Wang B, et al (2005) J Immunol. 175(7):4274-82; Solomon S, et al (2007) Mol Cell Biol. 27(6):2324-42). Caprin-1 is also known as GPIAP1, GPIP137, GRIP137, M11S1, RNG105, p137GPI, and cell cycle-related protein 1.

[0122] Cytoplasmic activation / proliferation-related protein-1 (caprin-1) is an RNA-binding protein involved in the regulation of cell cycle control-related genes. Caprin-1 selectively binds to c-Myc and cyclin D2 mRNAs, accelerates cell progression from the G1 phase to the S phase, enhances cell survival rate, promotes cell growth, indicating that caprin-1 may play an important role in tumor formation (Wang B, et al (2005) J Immunol. 175:4274-4282). Caprin-1 acts alone or in combination with other RNA-binding proteins such as RasGAP SH3 domain-binding protein 1 and fragile X mental retardation protein. In the tumor formation process, caprin-1 mainly functions by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, which contributes to radiation and chemotherapy resistance. Considering its role in various clinical malignancies, caprin-1 has the potential to be used as a biomarker and a target for the development of novel therapeutic agents (Yang, Z-S, et al (2019) Oncology Letters 18:15-21).

[0123] Antibodies targeting caprin-1 for treatment and detection have been described (WO2011 / 096519; WO2013 / 125654; WO2013 / 125636; WO2013 / 125640; WO2013 / 125630; WO2013 / 018889; WO2013 / 018891; WO2013 / 018883; WO2013 / 018892; WO2014 / 014082; WO2014 / 014086; WO2015 / 020212; WO2018 / 079740).

[0124] In an exemplary embodiment, the immunoconjugate of the present invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to CEA.

[0125] Elevated expression of carcinoembryonic antigen (CEA, CD66e, CEACAM5) is associated with various biological aspects of neoplasms, particularly tumor cell adhesion, metastasis, blockade of cellular immune mechanisms, and anti-apoptotic functions. CEA is also used as a blood marker for many cancers. Labetuzumab (CEA-CIDE™, Immunomedics, CAS registration number 219649-07-7), also known as MN-14 and hMN14, is a humanized IgG1 monoclonal antibody that has been studied for the treatment of colorectal cancer (Blumenthal, R. et al (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analog (labetuzumab govitecan, IMMU-130) targets carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and has been studied in patients with recurrent or refractory metastatic colorectal cancer (Sharkey, R. et al, (2018), Molecular Cancer Therapeutics 17(1):196-203; Cardillo, T. et al(2018) Molecular Cancer Therapeutics 17(1):150-160).

[0126] In embodiments of the invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMN-14 / labetuzumab of SEQ ID NO: 472 (US6676924). DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK SEQ ID NO: 472

[0127] In embodiments of the invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light chain CDR (complementary determining region) or light chain framework (LFR) sequences of hMN-14 / labetuzumab of SEQ ID NOs: 473-479 (US6676924).

Table 1

[0128] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of hMN-14 / rituximab of SEQ ID NO: 480 (US6676924). EVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPGKGLEWVAEIHPDSSTINYAPSLKDRFTISRDNSKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS SEQ ID NO: 480

[0129] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy chain CDR (complementary determining region) or heavy chain framework (HFR) sequences of hMN-14 / rituximab of SEQ ID NOS: 481-487 (US6676924).

Table 2

[0130] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hPR1A3 of SEQ ID NO: 488 (US8642742). DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIK SEQ ID NO: 488

[0131] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light chain CDR (complementary determining region) or light chain framework (LFR) sequences of hPR1A3 of SEQ ID NOS: 489-495 (US8642742).

Table 3

[0132] In embodiments of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy chain CDRs (complementary determining regions) or heavy chain framework (HFR) sequences of hPR1A3 of SEQ ID NOs: 496-502 (US8642742). [Table 4]

[0133] In embodiments of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMFE-23 of SEQ ID NO: 503 (US723288). ENVLTQSPSSMSASVGDRVNIACSASSSVSYMHWFQQKPGKSPKLWIYSTSNLASGVPSRFSGSGSGTDYSLTISSMQPEDAATYYCQQRSSYPLTFGGGTKLEIK SEQ ID NO: 503

[0134] In embodiments of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light chain CDRs (complementary determining regions) or light chain framework (LFR) sequences of hMFE-23 of SEQ ID NOs: 504-510 (US723288). [Table 5]

[0135] In embodiments of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of hMFE-23 of SEQ ID NO: 511 (US723288). QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS SEQ ID NO: 511

[0136] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy-chain CDRs (complementary determining regions) or heavy-chain framework (HFR) sequences of hMFE-23 of SEQ ID NOs: 512 to 518 (US723288). [Table 6]

[0137] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of SM3E of SEQ ID NO: 519 (US723288). ENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK SEQ ID NO: 519

[0138] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light-chain CDRs (complementary determining regions) or light-chain framework (LFR) sequences of SM3E of SEQ ID NOs: 520 to 526 (US723288). [Table 7]

[0139] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of SM3E of SEQ ID NO: 527 (US723288). QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS SEQ ID NO: 527

[0140] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy-chain CDRs (complementary determining regions) or heavy-chain framework (HFR) sequences of SM3E of SEQ ID NOs: 528 to 534 (US723288). [Table 8]

[0141] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light-chain CDRs (complementary determining regions) or light-chain framework (LFR) sequences of NP-4 / arcitumomab of SEQ ID NOs: 535 to 541. [Table 9]

[0142] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of NP-4 / arcitumomab of SEQ ID NO: 542. EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKALEWLGFIGNKANGYTTEYSASVKGRFTISRDKSQSILYLQMNTLRAEDSATYYCTRDRGLRFYFDYWGQGTTLTVSS SEQ ID NO: 542.

[0143] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy-chain CDRs (complementary determining regions) or heavy-chain framework (HFR) sequences of NP-4 of SEQ ID NOs: 543 to 549. [Table 10]

[0144] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of M5A / hT84.66 of SEQ ID NO: 550 (US7776330). DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIK SEQ ID NO: 550

[0145] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light chain CDRs (complementary determining regions) or light chain framework (LFR) sequences of M5A / hT84.66 of SEQ ID NOs: 551-557 (US7776330). [Table 11]

[0146] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of M5A / hT84.66 of SEQ ID NO: 558 (US7776330). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSS (SEQ ID NO: 558)

[0147] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy chain CDRs (complementary determining regions) or heavy chain framework (HFR) sequences of M5A / hT84.66 of SEQ ID NOs: 559-565. [Table 12]

[0148] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hAb2-3 of SEQ ID NO: 566 (US9617345). DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 566

[0149] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light chain CDRs (complementary determining regions) or light chain framework (LFR) sequences of hAb2-3 of SEQ ID NOs: 567 to 573 (US9617345). [Table 13]

[0150] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of SEQ ID NO: 574 (US9617345). EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS SEQ ID NO: 574

[0151] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy chain CDRs (complementary determining regions) or heavy chain framework (HFR) sequences of hAb2-3 of SEQ ID NOs: 575 to 581. [Table 14]

[0152] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of A240VL-B9VH / AMG-211 of SEQ ID NO: 582 (US9982063). QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL SEQ ID NO: 582

[0153] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the light chain CDR (complementary determining region) or light chain framework (LFR) sequences of A240VL-B9VH / AMG-211 of SEQ ID NOs: 583 to 589 (US9982063). [Table 15]

[0154] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of B9VH of SEQ ID NO: 590 (US9982063). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS SEQ ID NO: 590

[0155] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy chain CDR (complementary determining region) or heavy chain framework (HFR) sequences of SEQ ID NOs: 591 to 598 (US9982063). This embodiment includes two variants of CDR-H2, namely SEQ ID NO: 594 and SEQ ID NO: 595. [Table 16]

[0156] In an embodiment of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of E12VH of SEQ ID NO: 599 (US9982063). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFILNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS SEQ ID NO: 599

[0157] In embodiments of the present invention, the CEA-targeted antibody construct or antigen-binding domain comprises the heavy chain CDRs (complementary determining regions) or heavy chain framework (HFR) sequences of SEQ ID NOs: 600-606 (US9982063). [Table 17]

[0158] In some embodiments, the antibody construct further comprises an Fc domain. In certain embodiments, the antibody construct is an antibody. In certain embodiments, the antibody construct is a fusion protein. The antigen-binding domain can be a single-chain variable region fragment (scFv). A single-chain variable region fragment (scFv) is a shortened Fab fragment that includes the V domain of the antibody heavy chain linked to the variable (V) domain of the antibody light chain via a synthetic peptide and can be generated using conventional recombinant DNA technology techniques. Similarly, a disulfide-stabilized variable region fragment (dsFv) can be prepared by recombinant DNA technology. The antibody construct or antigen-binding domain may include one or more variable regions (e.g., two variable regions) of the antigen-binding domain of an anti-PD-L1 antibody, an anti-HER2 antibody, or an anti-CEA antibody, and each variable region includes CDR1, CDR2, and CDR3.

[0159] In some embodiments, the antibody in the immunoconjugate comprises a modified Fc region, and the modification regulates the binding of the Fc region to one or more Fc receptors.

[0160] In some embodiments, the Fc region is modified by including a TGFβ1 receptor or a fragment thereof that can bind to transforming growth factor beta 1 (TGFβ1). For example, the receptor can be TGFβ receptor II (TGFβRII). In some embodiments, the TGFβ receptor is a human TGFβ receptor. In some embodiments, the IgG has a C-terminal fusion to the TGFβRII extracellular domain (ECD) as described in US9676863, which is incorporated herein by reference. An "Fc linker" can be used to attach the IgG to the TGFβRII extracellular domain. The Fc linker can be a short and flexible peptide that allows for proper three-dimensional folding of the molecule while maintaining binding specificity to the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to the Fc of the antibody construct (with or without an Fc linker). In some embodiments, the C-terminus of the antibody construct heavy chain is fused to the TGFβ receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the antibody construct heavy chain is mutated to alanine.

[0161] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0162] In some embodiments, the antibody in the immunoconjugate provides site - specific conjugation of an adjuvant, label, or drug moiety to the antibody through cysteine substitution at a site where the engineered cysteine is available for conjugation, and is a cysteine - engineered antibody that does not disrupt immunoglobulin folding and assembly or modify antigen binding and effector functions. (Junutula, et al., 2008b Nature Biotech., 26(8):925 - 932; Dornan et al. (2009) Blood 114(13):2721 - 2729; US 7521541; US 7723485; US 2012 / 0121615; WO 2009 / 052249). A “cysteine - engineered antibody” or “cysteine - engineered antibody variant” is an antibody in which one or more residues of the antibody have been replaced with cysteine residues. Cysteine - engineered antibodies can be conjugated to a thienoazepine - adjuvant moiety as a thienoazepine - linker compound with uniform stoichiometry (e.g., for an antibody with a single engineered cysteine site, up to two thienoazepine moieties per antibody).

[0163] In some embodiments, the cysteine - engineered antibody used to prepare the immunoconjugates of Table 3 has a cysteine residue introduced at the 149 - lysine site of the light chain (LC K149C). In other embodiments, the cysteine - engineered antibody has a cysteine residue introduced at the 118 - alanine site (EU numbering) of the heavy chain (HC A118C). This site is numbered 121 in sequential numbering and 114 in Kabat numbering. In other embodiments, the cysteine - engineered antibody has a cysteine residue introduced into the light chain at G64C or R142C according to Kabat numbering, or into the heavy chain at D101C, V184C or T205C according to Kabat numbering.

[0164] Thienoazepine adjuvant compound The immunoconjugates of the present invention include a thienoazepine adjuvant moiety. The adjuvant moieties described herein are compounds that induce an immune response (i.e., immunostimulants). Generally, the adjuvant moieties described herein are TLR agonists. TLRs are type I transmembrane proteins involved in the initiation of the innate immune response in vertebrates. TLRs recognize various pathogen-associated molecular patterns from bacteria, viruses, and fungi and function as the first line of defense against invading pathogens. TLRs induce overlapping but distinct biological responses due to differences in their cellular expression and the signal transduction pathways they initiate. Once engaged (e.g., by a natural stimulus or a synthetic TLR agonist), TLRs initiate a signal transduction cascade that leads to the activation of nuclear factor-κB (NF-κB) via the recruitment of the adapter protein myeloid differentiation primary response gene 88 (MyD88) and interleukin-1 receptor-associated kinase (IRAK). The phosphorylation of IRAK leads to the recruitment of TNF receptor-associated factor 6 (TRAF6), and thus, the inhibitor of NF-κB, I-κB, is phosphorylated. As a result, NF-κB enters the cell nucleus and initiates the transcription of genes containing NF-κB binding sites, such as cytokines, as an example. Additional modes of regulation of TLR signaling include the induction of TNF receptor-associated factor 6 (TRAF6) dependent on the TIR domain-containing adapter inducing interferon-β (TRIF), and the activation of the MyD88-independent pathway via TRIF and TRAF3, which results in the phosphorylation of interferon response factor 3 (IRF3). Similarly, the MyD88-dependent pathway also activates several IRF family members, including IRF5 and IRF7, while the TRIF-dependent pathway also activates the NF-κB pathway.

[0165] Typically, the adjuvant moieties described herein are TLR7 and / or TLR8 agonists. Both TLR7 and TLR8 are expressed in monocytes and dendritic cells. In humans, TLR7 is also expressed in plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is mainly expressed in cells derived from the bone marrow, namely monocytes, granulocytes, and bone marrow dendritic cells. TLR7 and TLR8 can detect the presence of intracellular "foreign" single-stranded RNA as a means of responding to viral invasion. Treatment of TLR8-expressing cells with TLR8 agonists can lead to the production of high levels of IL-12, IFN-γ, IL-1, TNF-α, IL-6, and other inflammatory cytokines. Similarly, stimulation of TLR7-expressing cells such as pDCs with TLR7 agonists can lead to the production of high levels of IFN-α and other inflammatory cytokines. TLR7 / TLR8 involvement and the resulting cytokine production activate dendritic cells and other antigen-presenting cells, promoting diverse innate immune response mechanisms and acquired immune response mechanisms leading to tumor destruction.

[0166] Exemplary thienoazepine compounds (TAZs) of the invention are shown in Tables 1a - c. Each compound was synthesized, purified, characterized by mass spectrometry, and shown to have the indicated mass. Further experimental procedures are described in the Examples. Activity against HEK293 NFκB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. The thienoazepine compounds in Tables 1a - c exhibit surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders.

Table 18 - 1

Table 18 - 2

Table 18 - 3

Table 18 - 4

Table 18-5

Table 18-6

Table 18-7

Table 19-1

Table 19-2

Table 19-3

Table 19-4

Table 19-5

Table 19-6

Table 19-7

Table 19-8

Table 19-9

Table 19-10

Table 19-11

Table 19-12

Table 19-13

Table 19-14

Table 19-15

Table 19-16

Table 19-17

Table 19-18

Table 19-19

Table 19-20

Table 19-21

Table 19-22

Table 20-1

Table 20-2

Table 20-3

Table 20-4

Table 20-5

Table 20-6

Table 20-7

Table 20-8

Table 20-9

Table 20-10

Table 20-11

Table 20-12

Table 20-13

Table 20-14

Table 20-15

Table 20-16

Table 20-17

Table 20-18

Table 20-19

Table 20-20

Table 20-21

Table 20-22

Table 20-23

Table 20-24

Table 20-25

Table 20-26

Table 20-27

Table 20-28

Table 20-29

Table 20-30

Table 20-31

Table 20-32

Table 20-33

Table 20-34

Table 20-35

Table 20-36

Table 20-37

Table 20-38

[0167] Thienoazepine-linker compound The immunoconjugate of the present invention is prepared by conjugating an antibody with a thienoazepine-linker compound. The thienoazepine-linker compound includes a thienoazepine (TAZ) moiety covalently bonded to a linker unit. The linker unit includes functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy characteristics of the immunoconjugate. The linker unit includes reactive functional groups that react with, i.e., conjugate with, the reactive functional groups of the antibody. For example, nucleophilic groups such as the lysine side chain amino of the antibody react with the electrophilic reactive functional groups of the TAZ-linker compound to form an immunoconjugate. Also, for example, the cysteine thiol of the antibody reacts with the maleimide or bromoacetamide group of the TAZ-linker compound to form an immunoconjugate.

[0168] Suitable electrophilic reactive functional groups for the TAZ-linker compound include, but are not limited to, N-hydroxysuccinimidyl (NHS) ester and N-hydroxysulfosuccinimidyl (sulfo-NHS) ester (amine reactive); carbodiimide (amine and carboxyl reactive); hydroxymethylphosphine (amine reactive); maleimide (thiol reactive); haloacetamide such as N-iodoacetamide (thiol reactive); aryl azide (primary amine reactive); fluorinated aryl azide (reactive via carbon-hydrogen (C-H) insertion); pentafluorophenyl (PFP) ester (amine reactive); tetrafluorophenyl (TFP) ester (amine reactive); imido ester (amine reactive); isocyanate (hydroxyl reactive); vinyl sulfone (thiol, amine, and hydroxyl reactive); pyridyl disulfide (thiol reactive); and benzophenone derivative (reactive via C-H bond insertion). Further reagents include, but are not limited to, those described in Hermanson, Bioconjugate Techniques 2nd Edition, Academic Press, 2008.

[0169] The present invention provides solutions to limitations and problems with respect to the design, preparation, and use of immunoconjugates. Some linkers are unstable in the bloodstream, which can result in the release of unacceptable amounts of adjuvant / drug prior to internalization in target cells (Khot, A. et al (2015) Bioanalysis 7(13):1633-1648). Other linkers may provide stability in the bloodstream but can adversely affect the effectiveness of intracellular release. Linkers that provide desired intracellular release are typically poor in stability in the bloodstream. In other words, bloodstream stability and intracellular release are typically inversely related. Further, in standard conjugation processes, the amount of adjuvant / drug moiety loaded on the antibody, i.e., the drug load, the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are interrelated. For example, aggregate formation generally correlates positively with the number of equivalents of adjuvant / drug moiety and its derivatives conjugated to the antibody. At high drug loads, the aggregates formed must be removed for therapeutic use. As a result, drug load-mediated aggregate formation can reduce the yield of the immunoconjugate and make scale-up of the process difficult.

[0170] Exemplary embodiments include 5-aminothienoazepine-linker compounds of formula II:

Chemical formula

[0171] Exemplary embodiments of the thienoazepine-linker compounds of Formula II are those in which PEP is of the formula:

Chemical formula

[0172] Exemplary embodiments of the thienoazepine linker compound of Formula II include AA1 or AA2 forming a 5-membered ring with an adjacent nitrogen atom to form a proline amino acid.

[0173] Exemplary embodiments of the thienoazepine linker compound of Formula II are such that PEP has the formula:

Chemical formula

[0174] Exemplary embodiments of the thienoazepine linker compound of Formula II are such that Mcgluc has the formula:

Chemical formula

[0175] Exemplary embodiments of the thienoazepine-linker compound of Formula II include AA1 and AA2 being independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2.

[0176] Exemplary embodiments of the thienoazepine-linker compound of Formula II include AA1 being -CH(CH3)2 and AA2 being -CH2CH2CH2NHC(O)NH2.

[0177] Exemplary embodiments of the thienoazepine-linker compound of Formula II include AA1 and AA2 being independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H.

[0178] Exemplary embodiments of the thienoazepine-linker compounds of Formula II include those in which R 1 or R 3 of NR 5 (C2-C5 heteroaryl) is: [Chemical Formula] selected from the group consisting of.

[0179] Exemplary embodiments of the thienoazepine-linker compounds of Formula II include those in which X 1 is a bond and R 1 is H.

[0180] Exemplary embodiments of the thienoazepine-linker compounds of Formula II include those in which X 2 is a bond and R 2 is C1-C8 alkyl.

[0181] Exemplary embodiments of the thienoazepine-linker compounds of Formula II include those in which X 2 and X 3 are each a bond and R 2 and R 3 are independently selected from C1-C8 alkyl, -O-(C1-C 12 alkyl), -(C1-C 12 ) alkyldiyl)-OR 5 , -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 alkyl)-N(R 5 )CO2R 5 selected from the group consisting of.

[0182] Exemplary embodiments of the thienoazepine linker compounds of Formula II include those in which R 2 and R 3 are each independently selected from -CH2CH2CH3, -OCH2CH3, -CH2CH2CF3, and -CH2CH2CH2OH.

[0183] Exemplary embodiments of the thienoazepine-linker compounds of formula II are those in which R 2 is C1-C8 alkyl and R 3 is -(C1-C8 alkyldiyl)-N(R 5 )CO2R 4 .

[0184] Exemplary embodiments of the thienoazepine linker compounds of formula II are those in which R 2 is -CH2CH2CH3 and R 3 is -CH2CH2CH2NHCO2(t-Bu).

[0185] Exemplary embodiments of the thienoazepine-linker compounds of formula II are those in which R 2 and R 3 are each -CH2CH2CH3.

[0186] Exemplary embodiments of the thienoazepine-linker compounds of formula II are those in which X 3 -R 3 is selected from the group consisting of:

Chemical formula

[0187] Exemplary embodiments of the thienoazepine linker compounds of formula II are those in which one of R 2 and R 3 is: -(C1-C 12 alkyldiyl)-N(R 5 )- * ; -(C1-C 12 alkyldiyl)-O-(C1-C 12 alkyldiyl)-N(R 5 )- * ; -(C1-C 12 alkyldiyl)-N(R 5 )C(=NR 5 )-N(R 5 )- * ; -(C1-C12 (Alkyldiyl)-(C6-C 20 (Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )- * ; -(C1-C 12 Alkyldiyl)-(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-C(=NR 5 )N(R 5 )- * ; -(C2-C6 Alkynyldiyl)-N(R 5 )- * ; and -(C2-C6 Alkynyldiyl)-N(R 5 )C(=NR 5 )N(R 5 )- * ; selected from the group consisting of X 2 and X 3 are bonds, and the asterisk * indicates the binding site of L.

[0188] Exemplary embodiments of the thienoazepine linker compounds of Formula II are those in which L is: Q-C(=O)-(PEG)-; Q-C(=O)-(PEG)-C(=O)-; Q-C(=O)-(PEG)-O-; Q-C(=O)-(PEG)-N(R 5 )-; and Q-C(=O)-(PEG)-N(R 5 )C(=O)- selected from the group consisting of.

[0189] Exemplary embodiments of the thienoazepine linker compounds of Formula II are Formulas IIa-IIc:

Chemical formula

[0190] Exemplary embodiments of the thienoazepine linker compounds of Formula II are of Formulas IId-IIh:

Chemical formula

[0191] Exemplary embodiments of the thienoazepine linker compounds of Formula II are such that Q is:

Chemical formula

[0192] Exemplary embodiments of the thienoazepine linker compounds of Formula II include that Q is phenoxy-substituted with one or more Fs.

[0193] Exemplary embodiments of the thienoazepine linker compounds of Formula II include that Q is 2,3,5,6-tetrafluorophenoxy.

[0194] Exemplary embodiments of the thienoazepine-linker (TAZ-L) compounds of Formula II are selected from Tables 2a-c. Each compound was synthesized, purified, characterized by mass spectrometry, and shown to have the indicated mass. Further experimental procedures are described in the examples. The thienoazepine-linker compounds in Tables 2a-c exhibit surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. The thienoazepine linker compounds in Tables 2a-c are used by complexing with an antibody by the method of Example 201 to form the immunoconjugates in Tables 3a-c.

Table 21-1

Table 21-2

Table 21-3

Table 22-1

Table 22-2

Table 22-3

Table 22-4

Table 22-5

Table 22-6

Table 22-7

Table 22-8

Table 22-9

Table 22-10

Table 22-11

Table 22-12

Table 22-13

Table 22-14

Table 22-15

Table 23-1

Table 23-2

Table 23-3

Table 23-4

Table 23-5

Table 23-6

Table 23-7

Table 23-8

Table 23-9

Table 23-10

Table 23-11

Table 23-12

Table 23-13

Table 23-14

Table 23-15

Table 23-16

Table 23-17

Table 23-18

Table 23-19

Table 23-20

Table 23-21

Table 23-22

Table 23-23

Table 23-24

Table 23-25

Table 23-26

Table 23-27

Table 23-28

Table 23-29

Table 23-30

Table 23-31

Table 23-32

Table 23-33

Table 23-34

Table 23-35

Table 23-36

Table 23-37

Table 23-38

Table 23-39

Table 23-40

Table 23-41

Table 23-42

Table 23-43

Table 23-44

Table 23-45

[0195] Immunoconjugate Exemplary embodiments of the immunoconjugate covalently link to one or more 5-aminothienoazepine (TAZ) moieties via a linker and have the formula I: Ab-[L-TAZ] p I or a pharmaceutically acceptable salt thereof, comprising an antibody (wherein, Ab is an antibody; p is an integer from 1 to 8; TAZ is the formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0196] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to PD-L1.

[0197] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab, or biosimilars or bio-betters thereof.

[0198] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to HER2.

[0199] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is selected from the group consisting of trastuzumab and pertuzumab, or biosimilars or bio-betters thereof.

[0200] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to CEA.

[0201] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is labetuzumab, or a biosimilar or bio-better thereof.

[0202] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to Caprin-1.

[0203] Exemplary embodiments of the immunoconjugate of Formula I include those in which the PEP has the formula:

Chemical formula

[0204] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 or AA2 forms a 5-membered ring proline amino acid together with an adjacent nitrogen atom.

[0205] Exemplary embodiments of the immunoconjugate of Formula I include those in which the PEP has the formula: [Chemical formula] including having.

[0206] Exemplary embodiments of the immunoconjugate of Formula I include those in which the MCgluc has the formula: [Chemical formula] including having.

[0207] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2.

[0208] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0209] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H.

[0210] Exemplary embodiments of the immunoconjugate of Formula I include those in which R 1 and R 4 either one of which has the formula: [Chemical formula] selected from.

[0211] Exemplary embodiments of the immunoconjugate of Formula I include those in which R 1 and R 4One of which is -C(=O)NR 5 -(C1-C 20 heteroaryldiyl)-(C2-C 20 heterocyclicdiyl)-C(=O)NR 5 -(C1-C 12 ) alkyldiyl)-NR 5 -L, including the fact that it is -L.

[0212] Exemplary embodiments of the immunoconjugate of formula I include C1-C 20 heteroaryldiyl being pyridinediyl and C2-C 20 heterocyclicdiyl being piperidinediyl.

[0213] Exemplary embodiments of the immunoconjugate of formula I include R 1 , R 2 , R 3 , and R 4 One of them being -(C1-C8 alkyldiyl)-NR 5 (C2-C5 heteroaryl).

[0214] Exemplary embodiments of the immunoconjugate of formula I include R 1 , R 2 , R 3 , and R 4 NR of 5 (C2-C5 heteroaryl) being:

Chemical formula

[0215] Exemplary embodiments of the immunoconjugate of formula I include X 1 being a bond and R 1 being H.

[0216] Exemplary embodiments of the immunoconjugate of formula I include X 2 being a bond and R 2 being C1-C8 alkyl.

[0217] Exemplary embodiments of the immunoconjugates of formula I are X 2 and X 3 are each a bond, R 2 and R 3 are each independently selected from C1-C8 alkyl, -O-(C1-C 12 alkyl), -(C1-C 12 alkyl diyl)-OR 5 , -(C1-C8 alkyl diyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 alkyl)-N(R 5 )CO2R 5 and includes being selected independently.

[0218] Exemplary embodiments of the immunoconjugates of formula I are R 2 and R 3 each independently selected from -CH2CH2CH3, -OCH2CH3, -CH2CH2CF3, and -CH2CH2CH2OH and includes being selected.

[0219] Exemplary embodiments of the immunoconjugates of formula I are R 2 is C1-C8 alkyl, R 3 is -(C1-C8 alkyl diyl)-N(R 5 )CO2R 4 and includes being.

[0220] Exemplary embodiments of the immunoconjugates of formula I are R 2 is -CH2CH2CH3, R 3 is -CH2CH2CH2NHCO2(t-Bu) and includes being.

[0221] Exemplary embodiments of the immunoconjugates of formula I are R 2 and R 3 each is -CH2CH2CH3 and includes being.

[0222] Exemplary embodiments of the immunoconjugates of formula I are X 3 -R 3 is

Chem.

[0223] Exemplary embodiments of the immunoconjugate of Formula I are those in which one of R 2 and R 3 is -(C1-C 12 alkyl diyl)-N(R 5 )- * ; -(C1-C 12 alkyl diyl)-O-(C1-C 12 alkyl diyl)-N(R 5 )- * ; -(C1-C 12 alkyl diyl)-N(R 5 )C(=NR 5 )-N(R 5 )- * ; -(C1-C 12 alkyl diyl)-(C6-C 20 aryl diyl)-(C1-C 12 alkyl diyl)-N(R 5 )- * ; -(C1-C 12 alkyl diyl)-(C6-C 20 aryl diyl)-(C1-C 12 alkyl diyl)-N(R 5 )-C(=NR 5 )N(R 5 )- * ; -(C2-C6 alkynyl diyl)-N(R 5 )- * ; and -(C2-C6 alkynyl diyl)-N(R 5 )C(=NR 5 )N(R 5 )- * , including being selected from; X 2 and X 3 are bonds, where the asterisk *shows the binding site of L.

[0224] Exemplary embodiments of the immunoconjugate of Formula I are those in which L is: -C(=O)-(PEG)-; -C(=O)-(PEG)-C(=O)-; -C(=O)-(PEG)-O-; -C(=O)-(PEG)-N(R 5 )-; and -C(=O)-(PEG)-N(R 5 )C(=O)- selected from the group consisting of.

[0225] Exemplary embodiments of the immunoconjugate of Formula I are Formulas Ia - Ic:

Chemical formula

[0226] Exemplary embodiments of the immunoconjugate of Formula I are Formulas Id - Ih:

Chemical formula

[0227] The present invention includes all reasonable combinations and permutations of features of the embodiments of Formula I.

[0228] In certain embodiments, the immunoconjugate compounds of the present invention include those having immunostimulatory activity. The antibody - drug conjugates of the present invention selectively deliver an effective dose of the thienoazepine drug to tumor tissue, thereby achieving a higher selectivity (i.e., a lower effective dose) while increasing the therapeutic index (the "therapeutic concentration range") compared to unconjugated thienoazepine.

[0229] The drug load is the number of TAZ moieties per antibody in the immunoconjugate of Formula I, represented by p. The drug (TAZ) load can range from 1 to 8 drug moieties (D) per antibody. The immunoconjugate of Formula I comprises a mixture or aggregate of antibodies conjugated to a range of from 1 to about 8 drug moieties. In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of reactive or available amino acid side chain residues such as lysine and cysteine. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. In such embodiments, p can be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges thereof, for example, 1-8 or 2-5. In such embodiments, p and n are equal (i.e., p = n = 1, 2, 3, 4, 5, 6, 7, or 8, or ranges therebetween). Exemplary immunoconjugates of Formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine residues are already present in the antibody without the use of engineering to form intra-chain disulfide bonds. In that case, the existing free cysteine residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation of the antibody to generate one or more free cysteine residues.

[0230] For some anti-immunoconjugates, p can be limited by the number of binding sites on the antibody. For example, as in certain exemplary embodiments described herein, when the binding is a cysteine thiol, the antibody can have only one or a limited number of cysteine thiol groups, or only one or a limited number of sufficiently reactive thiol groups to which the drug can be bound. In other embodiments, one or more lysine amino groups in the antibody are available and can be reactive towards conjugation with the TAZ-linker compound of Formula II. In certain embodiments, a higher drug load, e.g., p greater than 5, can cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the average drug load of the immunoconjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0231] The loading (drug / antibody ratio) of the immunoconjugate can be controlled in different ways and, for example, by (i) limiting the molar excess of the TAZ-linker intermediate compound compared to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reductive denaturing conditions for optimized antibody reactivity.

[0232] It should be understood that when two or more nucleophilic groups of an antibody react with a drug, the resulting product is a mixture of immunoconjugate compounds in which one or more drug moieties conjugated to the antibody are distributed. The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay that is specific for the antibody and specific for the drug. Individual immunoconjugate molecules may be identified in the mixture by mass spectrometry and separated by HPLC, for example, by hydrophobic interaction chromatography (see, e.g., McDonagh et al (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, K.J., et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous immunoconjugates having a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.

[0233] Exemplary embodiments of the immunoconjugates of Formula I are selected from the immunoconjugates in Tables 3a - c. Evaluation of the immunoconjugate activity in vitro was carried out according to the method of Example 203. [Table 24] [Table 25 - 1] [Table 25 - 2] [Table 25 - 3] [Table 26 - 1] [Table 26 - 2] [Table 26 - 3] [Table 26 - 4] [Table 26 - 5] [Table 26 - 6] [Table 26 - 7] [Table 26 - 8] [Table 26 - 9] [Table 26 - 10]

[0234] Composition of the immunoconjugate The present invention provides a composition, such as a pharmaceutically or pharmacologically acceptable composition or formulation, comprising a plurality of immunoconjugates described herein and optionally a carrier therefor, such as a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates may have the same or different compositions, i.e., the composition may comprise immunoconjugates having the same number of adjuvants linked to the same position on the antibody construct, and / or immunoconjugates having the same number of TAZ adjuvants linked to different positions on the antibody construct, immunoconjugates having different numbers of adjuvants linked to the same position on the antibody construct, or immunoconjugates having different numbers of adjuvants linked to different positions on the antibody construct.

[0235] In an exemplary embodiment, a composition comprising an immunoconjugate compound comprises a mixture of immunoconjugate compounds, and the average drug (TAZ) load per antibody in the mixture of immunoconjugate compounds is from about 2 to about 5.

[0236] The composition of the immunoconjugates of the present invention can have an average adjuvant to antibody construct ratio (DAR) of from about 0.4 to about 10. One of ordinary skill in the art will recognize that the number of thienoazepine adjuvants conjugated to the antibody construct may vary among the immunoconjugates in a composition comprising a plurality of immunoconjugates of the present invention. Thus, the adjuvant to antibody construct (e.g., antibody) ratio can be measured on average, and this may be referred to as the drug to antibody ratio (DAR). The adjuvant to antibody construct (e.g., antibody) ratio can be evaluated by any suitable means, many of which are known in the art.

[0237] The average number of adduct moieties per antibody (DAR) in preparing an immunoconjugate from a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the immunoconjugate in the composition in units of p can also be determined. In some cases, the separation, purification, and characterization of immunoconjugates of the same species with a particular value of p from immunoconjugates with other drug loads may be achieved by means such as reverse-phase HPLC or electrophoresis.

[0238] In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the present invention can be formulated for parenteral administration such as intravenous administration or administration into a body cavity or the lumen of an organ. Alternatively, the immunoconjugate can be injected intratumorally. Injectable compositions generally comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be used include water and isotonic solutions of one or more salts such as sodium chloride, for example, Ringer's solution. In addition, sterile, fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose, any bland, fixed oil containing synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid can be used in the preparation of injectables as well. These compositions are preferably sterile and generally free of undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, toxicity adjusting agents, and the like that are required to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0239] The composition can contain an immunoconjugate at any suitable concentration. The concentration of the immunoconjugate in the composition can vary widely and is selected mainly based on factors such as fluid volume, viscosity, body weight, etc., according to the specific mode of administration selected and the needs of the patient. In certain embodiments, the concentration of the immunoconjugate in an injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0240] Method for treating cancer with an immunoconjugate The present invention provides a method for treating cancer. This method involves administering a therapeutically effective amount of the immunoconjugate described herein (e.g., as a composition described herein) to a subject in need thereof, such as a subject having cancer and in need of treatment for cancer. This method involves administering a therapeutically effective amount of an immunoconjugate (IC) selected from Tables 3a and 3b.

[0241] It is contemplated that the immunoconjugates of the present invention can be used to treat various hyperproliferative diseases or disorders, such as those characterized by overexpression of tumor antigens. Exemplary hyperproliferative disorders include benign or malignant solid tumors, as well as hematological diseases such as leukemia and lymphoid tumors.

[0242] In another aspect, an immunoconjugate for use as a medicament is provided. In certain embodiments, the present invention provides an immunoconjugate for use in a method of treating an individual, the method comprising administering an effective amount of the immunoconjugate to the individual. In such an embodiment, the method further comprises, for example, administering to the individual an effective amount of at least one additional therapeutic agent as described herein.

[0243] In a further aspect, the present invention provides the use of an immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer, and the method comprises administering to an individual having cancer an effective amount of the medicament. In such an embodiment, the method further comprises, for example, administering to the individual an effective amount of at least one additional therapeutic agent as described herein.

[0244] A carcinoma is a malignant tumor derived from epithelial tissue. Epithelial cells cover the outer surface of the body, line internal cavities, and form the lining of glandular tissue. Examples of carcinomas include adenocarcinoma (cancer that occurs in glandular (secretory) cells such as breast cancer, pancreatic cancer, lung cancer, prostate cancer, gastric cancer, gastroesophageal junction cancer, and colon cancer), adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian cancer; carcinoma in situ; ductal carcinoma; breast cancer; basal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; colon cancer; nasopharyngeal carcinoma; multilocular cystic renal cell carcinoma; oat cell carcinoma; large cell lung cancer; small cell lung cancer; non-small cell lung cancer; and the like, but are not limited thereto. Carcinomas may be found in the prostate, pancreas, colon, brain (usually as secondary metastases), lung, breast, and skin. In some embodiments, a method for treating non-small cell lung cancer comprises administering an immunoconjugate comprising an antibody construct (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or bio-betters thereof) that can bind to PD-L1. In some embodiments, a method for treating breast cancer comprises administering an immunoconjugate comprising an antibody construct (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or bio-betters thereof) that can bind to PD-L1. In some embodiments, a method for treating triple-negative breast cancer comprises administering an immunoconjugate comprising an antibody construct (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or bio-betters thereof) that can bind to PD-L1.

[0245] Soft tissue tumors are a very diverse group of rare tumors that originate from connective tissues. Examples of soft tissue tumors include alveolar soft part sarcoma; angiomatoid fibrous histiocytoma; chondromyxoid fibroma; osteosarcoma of soft tissue origin; extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplastic small round cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing sarcoma; desmoid (fibromatosis); infantile fibrosarcoma; gastrointestinal stromal tumor; giant cell tumor of bone; tenosynovial giant cell tumor; inflammatory myofibroblastic tumor; uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindle cell lipoma or pleomorphic lipoma; atypical lipoma; chondroid lipoma; well-differentiated liposarcoma; myxoid / round cell liposarcoma; pleomorphic liposarcoma; myxoid malignant fibrous histiocytoma; high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignant peripheral nerve sheath tumor; mesothelioma; neuroblastoma; osteochondroma; osteosarcoma; undifferentiated neuroectodermal tumor; alveolar rhabdomyosarcoma; fetal rhabdomyosarcoma; benign or malignant schwannoma; synovial sarcoma; Evans tumor; nodular fasciitis; desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis (PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovial sarcoma; malignant peripheral nerve sheath tumor; neurofibroma; pleomorphic adenoma of soft tissue; and neoplasms derived from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and Schwann cells, but are not limited to these.

[0246] A sarcoma is a rare type of cancer that develops in cells of mesenchymal origin, such as bone or soft tissues of the body that contain cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissues. Different types of sarcomas are based on where the cancer occurs. For example, osteosarcoma occurs in bone, liposarcoma in fat, and rhabdomyosarcoma in muscle. Examples of sarcomas include, but are not limited to, Askin tumor; botryoid sarcoma; chondrosarcoma; Ewing sarcoma; malignant angioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcoma (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodes; dermatofibrosarcoma protuberans (DFSP); desmoid tumor; fibromatosis; fibrous histiocytoma; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); perivascular cell tumor; hemangiosarcoma (more commonly referred to as "angiosarcoma"); Kaposi sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0247] A teratoma is a type of germ cell tumor that may contain several different types of tissue, such as hair, muscle, and bone (e.g., it can include tissue derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm). Teratomas occur most frequently in the ovaries in women, the testicles in men, and the coccyx in children.

[0248] Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma may begin as a mole (cutaneous melanoma), but it can also begin in other pigmented tissues, such as the eye or intestine.

[0249] Merkel cell carcinoma is a rare type of skin cancer that typically presents as a skin-colored or bluish-red nodule on the face, head, or neck. Merkel cell carcinoma is also referred to as a neuroendocrine carcinoma of the skin. In some embodiments, a method for treating Merkel cell carcinoma comprises administering an immunoconjugate containing an antibody construct (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or bio-betters thereof) that can bind to PD-L1. In some embodiments, the Merkel cell carcinoma is metastatic at the time of administration.

[0250] Leukemia is a cancer that occurs in hematopoietic tissues such as the bone marrow and produces large numbers of abnormal blood cells that enter the bloodstream. For example, leukemia can occur in bone marrow-derived cells that normally mature in the bloodstream. Leukemia is named according to the speed of onset and progression of the disease (e.g., acute and chronic), and the type of white blood cells affected (e.g., myeloid and lymphoid). Myeloid leukemia is also called myelogenous leukemia or myeloblastic leukemia. Lymphocytic leukemia is also called lymphoblastic leukemia or lymphocytic leukemia. Lymphocytic leukemia cells can aggregate in lymph nodes and cause swelling. Examples of leukemia include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0251] Lymphoma is a cancer that begins in cells of the immune system. For example, lymphoma can occur in bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphoma. One category of lymphoma is Hodgkin lymphoma (HL), which is characterized by the presence of a type of cell called Reed-Sternberg cells. Currently, there are six recognized types of HL. Examples of Hodgkin lymphoma include nodular sclerosis classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-depleted CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0252] Other categories of lymphoma are non-Hodgkin lymphoma (NHL), which includes a large and diverse group of cancers of immune system cells. Non-Hodgkin lymphoma can be further divided into cancers with a slow (slow-growing) course and cancers with an aggressive (fast-growing) course. Currently, there are 61 recognized types of NHL. Examples of non-Hodgkin lymphoma include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, immunoblastic lymphoma of blood, blastic NK cell lymphoma, Burkitt lymphoma, Burkitt-like lymphoma (small non-cleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T cell lymphoma, diffuse large B cell lymphoma, enteropathy-type T cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T cell lymphoma, T cell leukemia, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T cell lymphoma, pediatric lymphoma, peripheral T cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T cell lymphoma, and Waldenström macroglobulinemia.

[0253] Brain cancer includes any cancer of the brain tissue. Examples of brain cancer include, but are not limited to, gliomas (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningiomas, pituitary adenomas, and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastoma).

[0254] The immunoconjugates of the present invention can be used in a treatment method either alone or in combination with other agents. For example, the immunoconjugate can be co-administered with at least one additional therapeutic agent such as a chemotherapeutic agent. Such combination therapies include combined administration (two or more therapeutic agents are included in the same or separate formulations) and separate administration, and in the case of separate administration, the administration of the immunoconjugate can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The immunoconjugate can also be used in combination with radiation therapy.

[0255] The immunoconjugates of the present invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intratracheal, and intranasal, and, if desired for local therapy, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The dosage can be by any suitable route, e.g., by injection such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including single or multiple administrations, bolus dosing, and pulse infusions at various times, but are not limited thereto.

[0256] Atezolizumab, durvalumab, avelumab, their biosimilars, and their bio-betters are known to be useful for the treatment of cancer, particularly breast cancer, particularly triple-negative (negative for estrogen receptor, progesterone receptor, and overexpression of HER2 protein tests) breast cancer, bladder cancer, and Merkel cell carcinoma. Using the immunoconjugates described herein, the same types of cancer as in the case of atezolizumab, durvalumab, avelumab, their biosimilars, and their bio-betters can be treated, particularly breast cancer, particularly triple-negative (negative for estrogen receptor, progesterone receptor, and overexpression of HER2 protein tests) breast cancer, bladder cancer, and Merkel cell carcinoma.

[0257] The immunoconjugate is administered, in a therapeutically effective amount, to a subject in need thereof using any suitable dosing regimen, such as the dosing regimens utilized for atezolizumab, durvalumab, abevizumab, biosimilars thereof, and biobetters thereof. For example, the method can include administering an immunoconjugate to provide a dose of about 100 ng / kg to about 50 mg / kg to the subject. The dose of the immunoconjugate can be in the range of about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of the immunoconjugate can be outside of these ranges depending on the particular conjugate, as well as the type and severity of the cancer being treated. The frequency of administration can range from single administration per week to multiple administrations per week, or be more frequent. In some embodiments, the immunoconjugate is administered about once a month to about five times a week. In some embodiments, the immunoconjugate is administered once a week.

[0258] In another aspect, the present invention provides a method for preventing cancer. The method includes administering to a subject a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above). In certain embodiments, the subject is susceptible to a particular cancer to be prevented. For example, the method can include administering an immunoconjugate to provide a dose to the subject in the range of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can be in the range of about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of the immunoconjugate can also be outside these ranges, depending on the particular conjugate, as well as the type and severity of the cancer being treated. The frequency of administration can range from single administration per week to multiple administrations per week, or can be more frequent. In some embodiments, the immunoconjugate is administered about once a month to about five times a week. In some embodiments, the immunoconjugate is administered once a week.

[0259] Some embodiments of the present invention provide a method for treating cancer as described above, wherein the cancer is breast cancer. Breast cancer can originate from various regions of the breast and is characterized by multiple types of breast cancers. For example, the immunoconjugates of the present invention can be used to treat non-invasive ductal carcinoma; invasive ductal carcinoma (e.g., tubular carcinoma of the breast; medullary carcinoma; mucinous carcinoma; papillary carcinoma; or cribriform carcinoma); non-invasive lobular carcinoma; invasive lobular carcinoma; inflammatory breast cancer; and other forms of breast cancer such as triple-negative (negative for estrogen receptor, progesterone receptor, and overexpression of HER2 protein) breast cancer. In some embodiments, the method for treating breast cancer comprises administering an immunoconjugate containing an antibody construct (e.g., trastuzumab, pertuzumab, their biosimilars, or biobetters) capable of binding to HER2 and an antibody construct (e.g., atezolizumab, durvalumab, avelumab, their biosimilars, or biobetters) capable of binding to PD-L1. In some embodiments, the method for treating colon cancer, lung cancer, kidney cancer, pancreatic cancer, gastric cancer, and esophageal cancer comprises administering an immunoconjugate containing an antibody construct (e.g., ravulizumab, their biosimilars, or biobetters) capable of binding to CEA or tumors overexpressing CEA.

[0260] In some embodiments, the cancer is sensitive to an inflammation-inducing response induced by TLR7 and / or TLR8.

Example

[0261] Preparation of thienoazepine compounds (TAZ) and intermediates Example 1 Synthesis of 5-amino-2-bromo-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-1

Chem.

[0262] Example 2 Synthesis of 5-amino-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-2

Chemical formula

[0263] Example 3 Synthesis of tert-butyl (2-(1-(5-(5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepine-2-carboxamido)pyridin-2-yl)piperidine-4-carboxamido)ethyl)carbamate, TAZ-3

Chemical formula

[0264] Preparation of 5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepine-2-carboxylic acid, TAZ-22 To a solution of methyl 5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepine-2-carboxylate, TAZ-20 (450 mg, 1.29 mmol, 1 equiv) in MeOH (10 mL) and H2O (10 mL) was added LiOH.H2O (270 mg, 6.44 mmol, 5 equiv), and then the mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with 30 mL of H2O and extracted with EtOAc (10 mL × 2). The pH of the aqueous phase was adjusted to about 4 with aq (aqueous) HCl (1 M), and then extracted with EtOAc (10 mL × 3). The organic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated to give TAZ-22 (0.2 g, 596.27 μmol, yield 46.30%) as a pale yellow solid. 11H NMR (DMSO-d6, 400 MHz) δ 7.49 (s, 1H), 6.99 (s, 1H), 3.32 - 3.28 (m, 4H), 3.16 (s, 2H), 1.61 - 1.46 (m, 4H), 0.82 (br s, 6H).

[0265] tert-Butyl (2-(1-(5-(5-Amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepine-2-carboxamido)pyridin-2-yl)piperidine-4-carboxamido)ethyl)carbamate, Preparation of TAZ-3 To a solution of 5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepine-2-carboxylic acid (150 mg, 447 μmol, 1 equiv) in DMF (2 mL), 7-aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, PYAOP (256 mg, 491.9 μmol, 1.1 equiv), Et3N (45.0 mg, 447.2 μmol, 62.25 μL, 1 equiv) and tert-butyl N-[2-[[1-(5-amino-2-pyridyl)piperidine-4-carbonyl]amino]ethyl]carbamate (195. mg, 536.6 μmol, 1.2 equiv) were added and stirred at 25 °C for 12 h. The mixture was filtered and purified by preparative HPLC (column: Welch Xtimate C18 150 * 25 mm * 5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 30% - 60%, 10.5 min) to give TAZ-3 (62 mg, 91.06 μmol, yield 20.36%) as a gray solid. 11H NMR (MeOD-d4, 400 MHz) δ 8.35 (d, J = 2.0 Hz, 1H), 7.89 - 7.82 (m, 1H), 7.60 (s, 1H), 7.51 (s, 1H), 6.93 (s, 1H), 6.91 - 6.84 (m, 1H), 4.28 (d, J = 12.8 Hz, 2H), 3.44 - 3.35 (m, 4H), 3.27 - 3.21 (m, 2H), 3.18 - 3.12 (m, 2H), 2.97 (s, 2H), 2.88 (t, J = 11.6 Hz, 2H), 2.48 - 2.32 (m, 1H), 1.90 - 1.80 (m, 2H), 1.79 - 1.56 (m, 6H), 1.43 (s, 9H), 0.90 (s, 6H). LC / MS [M+H] 681.3 (calculated); LC / MS [M+H] 681.4 (measured).

[0266] Example 4 Synthesis of 5-Amino-2-[3-[3-(Hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-4 [Chemical Structure] To a solution of 5-Amino-2-bromo-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-1 (71.0 mg, 192 μmol, 1.1 equiv) in dioxane (1 mL) and H2O (0.5 mL), [1-[3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methanol (62.0 mg, 174 μmol, 1 equiv), K2CO3 (48.0 mg, 348 μmol, 2 equiv) and [1,1'-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride, Pd(dppf)Cl2 (6.0 mg, 8.7 μmol, 0.05 equiv) were added under N2 at 25 °C, then the mixture was stirred at 110 °C for 2 h. The mixture was filtered and concentrated. The residue was purified by preparative HPLC (column: Welch Xtimate C18 150 * 25 mm *5 μm; Mobile phase: [Water (10 mM NH4HCO3) - ACN]; B%: 30% - 60%, 10.5 min), and it was purified to obtain TAZ - 4 (22 mg, 42.58 μmol, yield 24.45%) as a pale yellow solid. 1 1H NMR (MeOD - d4, 400 MHz) δ8.04 - 7.97 (m, 2H), 7.79 - 7.75 (m, 1H), 7.73 - 7.67 (m, 1H), 7.31 (s, 1H), 6.92 (s, 1H), 3.85 (t, J = 8.2 Hz, 2H), 3.62 - 3.56 (m, 2H), 3.45 - 3.37 (m, 6H), 2.99 (s, 2H), 2.63 - 2.52 (m, 1H), 1.71 - 1.59 (m, 4H), 0.99 - 0.83 (m, 6H). LC / MS [M + H] 517.2 (calculated value); LC / MS [M + H] 517.2 (measured value)

[0267] Example 5 Synthesis of [4 - [[(2S) - 2 - [[(2S) - 2 - (9H - Fluoren - 9 - ylmethoxycarbonylamino) - 3 - methyl - butanoyl]amino] - 5 - ureido - pentanoyl]amino]phenyl]methyl N - [2 - [[1 - [5 - [[5 - amino - 7 - (dipropylcarbamoyl) - 6H - thieno[3,2 - b]azepin - 2 - carbonyl]amino] - 2 - pyridyl]piperidine - 4 - carbonyl]amino]ethyl]carbamate, TAZ - 5

Chemical Structure

[0268] Preparation of TAZ-5 To a solution of 5a (50 mg, 61.8 μmol, 1 equiv, 2TFA) in DMF (1 mL) was added DIEA (32.0 mg, 247.2 μmol, 43.0 μL, 4 equiv) and [[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (52.0 mg, 68.0 μmol, 1.1 equiv), and then the mixture was stirred at 25 °C for 1 h. The mixture was filtered and subjected to preparative HPLC (column: Nano-micro Kromasil C18 100 *30 mm 8 um; Mobile phase: [Water (0.1% TFA) - ACN]; Purified by B%: 25% - 45%, 10 min), and TAZ-5 (36 mg, 27.2 μmol, yield 44.03%, TFA) was obtained as a pale yellow solid. 1 H NMR (MeOD-d4, 400 MHz) δ8.58 (s, 1H), 8.07 (br d, J = 10.4 Hz, 1H), 7.93 (br s, 1H), 7.86 - 7.72 (m, 3H), 7.63 (br t, J = 7.2 Hz, 2H), 7.57 (br d, J = 8.2 Hz, 2H), 7.41 - 7.34 (m, 2H), 7.33 - 7.25 (m, 4H), 7.13 (s, 1H), 5.03 (br s, 2H), 4.41 - 4.29 (m, 3H), 4.23 - 4.15 (m, 1H), 4.13 - 4.03 (m, 2H), 3.98 - 3.91 (m, 1H), 3.51 - 3.34 (m, 5H), 3.25 - 3.07 (m, 9H), 2.52 - 2.31 (m, 1H), 2.08 (br d, J = 7.6 Hz, 1H), 1.99 - 1.36 (m, 12H), 1.04 - 0.82 (m, 12H). LC / MS [M+H] 1208.6 (calculated value); LC / MS [M+H] 1208.5 (measured value).

[0269] Example 6 Synthesis of tert-butyl N-[5-[5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepin-2-yl]pent-4-ynyl]carbamate, TAZ-6

Chemical Structure

[0270] Example 7 Synthesis of 5-amino-2-methyl-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-7

Chem.

[0271] Example 8 Synthesis of tert-butyl N-[3-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]propyl]carbamate, TAZ-8

Chem.

[0272] Example 9 Synthesis of tert-butyl N-[3-[(5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]propyl]carbamate, TAZ-9

Chemical Structure

[0273] Example 10 Synthesis of tert-butyl N-[4-[(5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]but-2-ynyl]carbamate, TAZ-10

Chemical formula

[0274] Example 11 Synthesis of 5-amino-N-(3-aminopropyl)-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-11

Chemical Structure

[0275] Example 12 Synthesis of 5-amino-2-[1-[3-(hydroxymethyl)azetidin-1-yl]sulfonylpyrazol-4-yl]-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-12

Chem.

[0276] Preparation of methyl 1-(4-bromopyrazol-1-yl)sulfonylazetidine-3-carboxylate, 12c To a mixture of 4-bromo-1H-pyrazole (1.58 g, 10.77 mmol, 1.0 eq) in DCM (40 mL), DABCO (1.57 g, 14.0 mmol, 1.54 mL, 1.3 eq) and 12b (2.3 g, 10.8 mmol, 1.0 eq) were added at 25 °C all at once, and the mixture was stirred for 2 h. The mixture was diluted with water and extracted with EtOAc (50 mL×3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 3 / 1) to give 12c (3 g, 9.25 mmol, yield 85.97%) as a yellow oil. 1 H NMR (CDCl3, 400 MHz) δ8.03 (s, 1H), 7.77 (s, 1H), 4.32 - 4.27 (m, 4H), 3.73 (s, 3H), 3.41 - 3.34 (m, 1H).

[0277] Preparation of [1-(4-bromopyrazol-1-yl)sulfonylazetidin-3-yl]methanol, 12d To a solution of 12c (3.3 g, 10.2 mmol, 1 equiv) in DCM (50 mL), DIBAL-H (1 M, 40.7 mL, 4 equiv) was slowly added at 0 °C under N2, and then the mixture was stirred at this temperature for 2 h. The mixture was quenched with water (1.5 mL), dried over Na2SO4, filtered, and concentrated to give 12d (1.19 g, crude) as a yellow oil. 1 1H NMR (CDCl3, 400 MHz) δ 8.04 (s, 1H), 7.77 (s, 1H), 4.18 - 4.14 (t, J = 8.4 Hz, 2H), 3.96 (dd, J = 5.6, 8.4 Hz, 2H), 3.66 (d, J = 5.6 Hz, 2H).

[0278] Preparation of [1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazol-1-yl]sulfonylazetidin-3-yl]methanol, 12e To a mixture of 12d (0.1 g, 338 μmol, 1.0 equiv) in dioxane (2 mL), Pin2B2 (129 mg, 507 μmol, 1.5 equiv), potassium acetate, KOAc (66.3 mg, 675 μmol, 2.0 equiv) and Pd(dppf)Cl2 (12.4 mg, 16.9 μmol, 0.05 equiv) were added at once at 25 °C under N2, and the mixture was stirred at 100 °C for 2 h. Then the mixture was diluted with water and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give 12e (0.1 g, crude) as a black oil.

[0279] Preparation of TAZ-12 A mixture of 5-amino-2-bromo-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-1 (54 mg, 146 μmol, 1.0 equiv), and 12e (50 mg, 146 μmol, 1.0 equiv) in dioxane (2 mL) and H2O (0.2 mL) was treated with K2CO3 (60.4 mg, 437 μmol, 3.0 equiv) and Pd(dppf)Cl2 (5.3 mg, 7.28 μmol, 0.05 equiv) all at once at 25 °C under N2. The mixture was stirred at 90 °C for 2 h. Then the reaction was diluted with water and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was further purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 * 30 mm * 3 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 30%-50%, 10.5 min) to give 5-amino-2-[1-[3-(hydroxymethyl)azetidin-1-yl]sulfonylpyrazol-4-yl]-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide (9 mg, 17.8 μmol, 12.19% yield) as a white solid. 1 1H NMR (MeOD, 400 MHz) δ 8.56 (s, 1H), 8.28 (s, 1H), 7.42 (s, 1H), 7.38 - 7.35 (m, 2H), 6.85 (s, 1H), 4.13 (t, J = 8.8 Hz, 2H), 3.89 (dd, J = 6.0, 8.4 Hz, 2H), 3.49 (d, J = 6.0 Hz, 2H), 3.43 - 3.37 (m, 4H), 2.97 (s, 2H), 2.72 - 2.67 (m, 1H), 1.69 - 1.61 (m, 4H), 0.93 - 0.87 (m, 6H). LC / MS [M+H] 507.2 (calcd); LC / MS [M+H] 507.2 (found).

[0280] Example 13 Synthesis of tert-butyl N-[5-[5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepin-2-yl]pentyl]carbamate, TAZ-13

Chem.

[0281] Example 14 Synthesis of 5-amino-2-(5-aminopentyl)-N,N-dipropyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-14

Chem.

[0282] Example 15 Synthesis of 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, TAZ-15

Chemical Structure

[0283] Preparation of methyl 5-bromo-3-(tert-butoxycarbonylamino)thiophene-2-carboxylate, 15c To a solution of 15b (8.9 g, 34.6 mmol, 1 equiv) in THF (50 mL) was added LDA (2 M, 60.0 mL, 3.5 equiv) at -78 °C. The mixture was stirred at -78 °C for 1 h, then 1,2-dibromo-1,1,2,2-tetrafluoro-ethane (53.92 g, 207.54 mmol, 6 equiv) was added and the mixture was stirred at this temperature for 1 h. While stirring vigorously, the mixture was poured into cold ammonium chloride solution (100 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 10 g SepaFlash® silica flash column, 0 - 20% ethyl acetate / petroleum ether gradient, eluent at 45 mL / min) to give 15c (4 g, 11.90 mmol, 34.40% yield) as an off-white solid. 1 H NMR (CDCl3, 400 MHz) δ9.33 (s, 1H), 7.97 (s, 1H), 3.86 (s, 3H), 1.54 (s, 9H)

[0284] Preparation of tert-butyl N-[5-bromo-2-(hydroxymethyl)-3-thienyl]carbamate, 15d To a solution of 15c (4 g, 11.9 mmol, 1 equiv) in DCM (60 mL) was added DIBAL-H (1 M, 59.0 mL, 5 equiv) at 0 °C under N2 and the mixture was stirred at 25 °C for 2 h. The reaction mixture was quenched by adding 1 mL of H2O at 0 °C, then 15% NaOH (0.5 mL) and H2O (1 mL) were added at 0 °C. The mixture was stirred at 25 °C for 30 min, filtered, and the cake was washed with EtOAc (50 mL). The filtrate was concentrated to give 15d (3 g, 9.7 mmol, 81.82% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ7.21 (s, 1H), 6.68 (s, 1H), 4.60 (s, 2H), 1.51 (s, 9H).

[0285] Preparation of tert-Butyl N-(5-Bromo-2-formyl-3-thienyl)carbamate, 15e To a solution of 15d (3 g, 9.73 mmol, 1 equiv) in DCM (30 mL) was added MnO2 (8.5 g, 97.34 mmol, 10 equiv) at 25 °C. The mixture was stirred at 50 °C for 12 h. The mixture was filtered and concentrated to give 15e (1.5 g, 4.90 mmol, 50.33% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 9.82 (s, 1H), 9.51 (s, 1H), 8.03 (s, 1H), 1.53 (s, 9H).

[0286] Preparation of Ethyl (E)-3-[5-Bromo-3-(tert-butoxycarbonylamino)-2-thienyl]-2-(cyanomethyl)prop-2-enoate, 15f To a solution of 15e (1.5 g, 4.90 mmol, 1 equiv) in toluene (15 mL) was added ethyl 3-cyano-2-(triphenyl-phosphanylidene)propanoate (2.5 g, 6.4 mmol, 1.3 equiv) at 25 °C, and then the mixture was stirred at 75 °C for 2 h. The mixture was concentrated and the residue was purified by flash silica gel chromatography (ISCO (registered trademark); 2 g SepaFlash (registered trademark) silica flash column, 0 - 80% ethyl acetate / petroleum ether gradient, eluent at 45 mL / min) to give 15f (1.65 g, 3.97 mmol, 81.10% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 7.81 (s, 1H), 7.73 (s, 1H), 6.72 (s, 1H), 4.35 (q, J = 7.2 Hz, 2H), 3.68 (s, 2H), 1.54 (s, 9H), 1.39 (t, J = 7.2 Hz, 3H).

[0287] Preparation of Ethyl 5-Amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylate, 15g To a solution of 15f (1.3 g, 3.13 mmol, 1 equiv) in EtOAc (10 mL) was added HCl / EtOAc (4 M, 13.00 mL, 16.6 equiv) at 25 °C. The mixture was stirred at 25 °C for 2 h and then concentrated to give 15g (1 g, crude) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ10.13 ( s, 1H), 9.29 ( s, 1H), 7.91 (s, 1H), 7.37 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H), 3.52 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H).

[0288] Preparation of 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, TAZ-15 To a solution of 15g (1 g, 3.17 mmol, 1 equiv) in EtOH (10 mL) and H2O (1 mL) was added LiOH.H2O (665 mg, 15.8 mmol, 5 equiv), and then the mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure to remove EtOH. The residue was diluted with H2O (30 mL), then the pH of the mixture was adjusted to 4 with aqueous HCl (1 M), and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine (10 mL), dried over Na2SO4, filtered, and concentrated to give TAZ-15 (0.85 g, 2.96 mmol, 93.30% yield) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ7.65 (s, 1H), 7.34 (br s, 2H), 6.97 (s, 1H), 2.97 (s, 2H).

[0289] Example 16 Synthesis of tert-butyl N-[4-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]but-2-ynyl]carbamate, TAZ-16 [Chemical formula] Preparation of 5-amino-6H-thieno[3,2-b]azepine-7-carboxylic acid, 16a A solution of 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, TAZ-15 (1 g, 3.48 mmol, 1 equiv) in MeOH (20 mL) was added with Pd / C (10%, 0.2 g) and aqueous ammonium hydroxide, NH3.H2O (4.88 g, 34.8 mmol, 5.37 mL, purity 25%, 10 equiv) under N2. The suspension was degassed under vacuum and purged several times with H2, then stirred at 25 °C for 12 h under H2 (50 psi). The reaction mixture was filtered through Celite® (Johns Manville), the pH of the filtrate was adjusted to about 6 with 2N HCl at 0 °C, and then concentrated under reduced pressure to remove MeOH. The solid was filtered and dried under reduced pressure to obtain 16a (0.54 g, 2.59 mmol, yield 74.46%) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ7.71 (s, 1H), 7.61 (d, J = 5.2 Hz, 1H), 6.98 (br s, 2H), 6.83 (d, J = 5.2 Hz, 1H), 2.91 (s, 2H).

[0290] Preparation of TAZ-16 To a solution of 16a (0.33 g, 1.58 mmol, 1 equiv) in DMF (4 mL) were added HATU (662.82 mg, 1.74 mmol, 1.1 equiv) and DIPEA (1.02 g, 7.92 mmol, 1.38 mL, 5 equiv) at 0 °C. After 10 min, tert-butyl N-[4-(propylamino)but-2-ynyl]carbamate (394.51 mg, 1.74 mmol, 1.1 equiv) was added at 0 °C, and then the resulting mixture was stirred at 25 °C for 30 min. The reaction mixture was quenched by adding H2O (30 mL) at 0 °C, and then extracted with EtOAc (15 mL × 3). The combined organic layers were washed with brine (5 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex Luna C18 100 * 30 mm *5 um; Mobile phase: [Water (0.1% TFA) - ACN]; Purified by B%: 15% - 45% for 10 minutes, and TAZ - 16 (0.185 g, 444.14 μmol, yield 28.03%) was obtained as a pale yellow solid. 1 1H NMR (MeOD, 400 MHz) δ7.74 (d, J = 5.6 Hz, 1H), 7.29 (s, 1H), 7.13 (d, J = 5.6 Hz, 1H), 4.30 (s, 2H), 3.84 (s, 2H), 3.54 - 3.52 (m, 2H), 3.38 (s, 2H), 1.76 - 1.67 (m, 2H), 1.43 (s, 9H), 0.94 (t, J = 7.6 Hz, 3H). LC / MS [M + H] 417.2 (calculated value); LC / MS [M + H] 417.2 (measured value).

[0291] Example 17 Synthesis of 5 - amino - N - (4 - aminobut - 2 - ynyl) - N - propyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 17

Chemical Structure

[0292] Example 18 Synthesis of 5 - Amino - 2 - phenyl - N,N - dipropyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 18 [Chemical formula] To a mixture of phenylboronic acid (34.5 mg, 283 μmol, 1.5 equiv), K2CO3 (52.0 mg, 378 μmol, 2.0 equiv) and 5 - amino - 2 - bromo - N,N - dipropyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 1 (70.0 mg, 190 μmol, 1.0 equiv) in dioxane (2 mL) and H2O (0.2 mL), Pd(dppf)Cl2 (7.0 mg, 9.45 μmol, 0.05 equiv) was added at 25 °C under N2, and then the mixture was stirred at 100 °C for 1 hour. The mixture was filtered and purified by preparative HPLC (column: Nano - micro Kromasil C18 100 * 30 mm 8 μm; mobile phase: [water (0.1% TFA) - ACN]; B%: 25% - 50%, 10 min), and TAZ - 18 (54 mg, 147 μmol, yield 77.7%) was obtained as a white solid. 11H NMR (MeOD, 400 MHz) δ 7.69 (d, J = 7.2 Hz, 2H), 7.49 - 7.37 (m, 4H), 7.11 (s, 1H), 3.54 - 3.38 (m, 6H), 1.68 (sxt, J = 7.4 Hz, 4H), 0.99 - 0.92 (m, 6H). LC / MS [M+H] 368.2 (calculated value); LC / MS [M+H] 368.1 (measured value).

[0293] Example 19 Synthesis of 5 - Amino - N,N - dipropyl - 2 - (1 - ((2 - (trimethylsilyl)ethoxy)methyl)-1H - pyrazol - 4 - yl)-6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 19 [Chemical Structure] To a mixture of 5 - Amino - 2 - bromo - N,N - dipropyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 1 (100 mg, 270 μmol, 1.0 equiv), K2CO3 (75.0 mg, 540 μmol, 2.0 equiv), and trimethyl - [2 - [[4 - (4,4,5,5 - tetramethyl - 1,3,2 - dioxaborolan - 2 - yl)pyrazol - 1 - yl]methoxy]ethyl]silane (96 mg, 297 μmol, 1.1 equiv) in dioxane (3 mL) and H2O (0.2 mL), Pd(dppf)Cl2 (9.88 mg, 13.50 μmol, 0.05 equiv) was added at 25 °C under N2, and then the mixture was stirred at 95 °C for 1 h. The mixture was filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini - NX C18 75 * 30 mm * 3 μm; mobile phase: [water (10 mM NH4HCO3) - ACN]; B%: 40% - 60%, 10.5 min) to give TAZ - 19 (80 mg, 164 μmol, 60.7% yield) as a pale yellow solid. 11H NMR (MeOD, 400 MHz) δ 8.09 (s, 1H), 7.80 (s, 1H), 6.97 (s, 1H), 6.85 (s, 1H), 5.45 (s, 2H), 3.61 (t, J = 8.0 Hz, 2H), 3.46 - 3.36 (m, 4H), 2.96 (s, 2H), 1.72 - 1.57 (m, 4H), 0.90 (t, J = 8.0 Hz, 8H), 0.00 (s, 9H). LC / MS [M+H] 488.2 (calculated value); LC / MS [M+H] 488.2 (measured value).

[0294] Example 20 Synthesis of Methyl 5 - Amino - 7 - (dipropylcarbamoyl) - 6H - thieno[3,2 - b]azepine - 2 - carboxylate, TAZ - 20 [Chemical formula] To a solution of 5 - Amino - 2 - bromo - N,N - dipropyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 1 (1.2 g, 3.24 mmol, 1.0 equiv) and Et3N (984 mg, 9.72 mmol, 1.35 mL, 3 equiv) in MeOH (20 mL) was added Pd(dppf)Cl2 (118.56 mg, 162.03 μmol, 0.05 equiv) under N2. The suspension was degassed under vacuum and purged several times with CO. The mixture was stirred at 80 °C for 12 h under CO (50 psi). The mixture was filtered and concentrated in vacuo to give TAZ - 20 (1.1 g, 3.15 mmol, yield 97.14%) as a white solid. 1 1H NMR (MeOD, 400 MHz) δ 7.72 (s, 1H), 7.14 (s, 1H), 3.92 (s, 3H), 3.58 - 3.37 (m, 6H), 1.69 - 1.62 (m, 4H), 0.99 - 0.90 (m, 6H). LC / MS [M+H] 350.2 (calculated value); LC / MS [M+H] 350.2 (measured value).

[0295] Example 21 Synthesis of 5-Amino-N,N-dipropyl-2-(1H-pyrazol-4-yl)-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-21

Chem.

[0296] Example 22 Synthesis of 5-Amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepine-2-carboxylic acid, TAZ-22

Chem.

[0297] Example 23 Synthesis of 5-amino-N2-phenyl-N7,N7-dipropyl-6H-thieno[3,2-b]azepine-2,7-dicarboxamide, TAZ-23

Chem.

[0298] Example 24 Synthesis of 5 - Amino - N2 - ethyl - N7,N7 - dipropyl - 6H - thieno[3,2 - b]azepine - 2,7 - dicarboxamide, TAZ - 24

Chemical Structure

[0299] Example 25 Synthesis of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[5-[5-Amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepin-2-yl]pentyl-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, TAZ-25

Chemical formula

[0300] Preparation of TAZ - 25 To a solution of 25a (0.39 g, 406 μmol, 1.0 equiv) in H2O (20 mL) was added TFA (927 mg, 8.13 mmol, 602 μL, 20 equiv) at 25 °C. The mixture was stirred at 85 °C for 1 h and then concentrated at 50 °C under reduced pressure. The residue was purified by preparative HPLC (column: Nano - micro Kromasil C18 100 * 30 mm 8 um; mobile phase: [water (0.1% TFA) - ACN]; purified by B%: 10% - 30%, 15 min, and TAZ - 25 (0.18 g, 199.30 μmol, yield 49.02%) was obtained as a pale yellow oil. 1 1H NMR (MeOD, 400 MHz) δ 7.02 (s, 1H), 6.90 (s, 1H), 3.84 - 3.82 (m, 2H), 3.75 - 3.59 (m, 41H), 3.45 - 3.42 (m, 4H), 3.35 (s, 2H), 2.96 - 2.87 (m, 5H), 2.54 (t, J = 6.4 Hz, 2H), 1.84 - 1.76 (m, 4H), 1.71 - 1.60 (m, 4H), 1.54 - 1.44 (m, 2H), 0.94 - 0.89 (m, 6H). LC / MS [M + H] 903.5 (calculated); LC / MS [M + H] 903.5 (measured).

[0301] Example 26 Synthesis of 5 - amino - 2 - benzyl - N,N - dipropyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 26

Chemical Structure

[0302] Example 27 Synthesis of 5-amino-N7,N7-dipropyl-N2-pyrimidin-5-yl-6H-thieno[3,2-b]azepine-2,7-dicarboxamide, TAZ-27

Chemical formula

[0303] Example 28 Synthesis of 2-amino-N,N-dipropyl-3H-benzo[4,5]thieno[3,2-b]azepine-4-carboxamide, TAZ-28 [Chemical formula] Preparation of 3-[bis(tert-butoxycarbonyl)amino]benzothiophene-2-carboxylate methyl, 28b To a mixture of pyridine, Pyr (30 mL), and methyl 3-aminobenzothiophene-2-carboxylate, 28a (3 g, 14.5 mmol, 1.0 equiv), DMAP (177 mg, 1.45 mmol, 0.1 equiv) was added. Then, a solution of Boc2O (6.32 g, 29.0 mmol, 6.65 mL, 2.0 equiv) in pyridine (10 mL) was slowly added to the mixture at 0 °C, and then the mixture was stirred at 25 °C for 16 h. The mixture was concentrated in vacuo. The residue was dissolved in EtOAc (20 ml) and washed successively with aqueous saturated NaHCO3 and brine. The mixture was dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 5 / 1) to give methyl 3-[bis(tert-butoxycarbonyl)amino]benzothiophene-2-carboxylate (5.6 g, 13.7 mmol, 94.94% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 7.83 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.52 - 7.42 (m, 2H), 3.94 (s, 3H), 1.35 (s, 18H).

[0304] Preparation of tert-butyl N-[2-(hydroxymethyl)benzothiophen-3-yl]carbamate, 28c To a mixture of 28b (3.8 g, 9.33 mmol, 1.0 equiv) in DCM (40 mL), DIBAL-H (1 M, 37.3 mL, 4.0 equiv) was slowly added at 0 °C under N2, and the mixture was stirred at the same temperature for 2 h. The reaction was quenched with water (2 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 3 / 1) to give 28c (2.3 g, 8.23 mmol, 88.29% yield) as a yellow solid. 11H NMR (CDCl3, 400 MHz) δ 7.83 - 7.78 (m, 1H), 7.63 (dd, J = 2.0, 6.8 Hz, 1H), 7.43 - 7.35 (m, 2H), 4.75 (d, J = 6.4 Hz, 2H), 1.55 (s, 9H).

[0305] tert-Butyl N-(2-formylbenzothiophen-3-yl)carbamate, preparation of 28d To a solution of 28c (1.8 g, 6.44 mmol, 1.0 eq) in DCM (30 mL) was added MnO2 (4.48 g, 51.6 mmol, 8.0 eq) at once at 25 °C, and then stirred for 12 h. The reaction mixture was filtered and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 - 5 / 1) to give 28d (1.2 g, 4.33 mmol, yield 67.15%) as a yellow solid. 1 1H NMR (CDCl3, 400 MHz) δ 10.07 (s, 1H), 8.22 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.55 - 7.50 (m, 1H), 7.47 - 7.43 (m, 1H), 1.57 (s, 9H).

[0306] Ethyl (E)-3-[3-(tert-butoxycarbonylamino)benzothiophen-2-yl]-2-(cyanomethyl)prop-2-enoate, preparation of 28e To a solution of 28d (0.6 g, 2.16 mmol, 1.0 eq) and ethyl 3-cyano-2-(triphenyl-phosphanylidene)propanoate (1.09 g, 2.81 mmol, 1.3 eq) in toluene (15 mL) at 25 °C was stirred at 80 °C for 12 h. Then the mixture was concentrated. The residue was diluted with water and extracted with EtOAc (30 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 1 / 0, 5 / 1) to give 28e (0.6 g, 1.55 mmol, yield 71.88%) as a yellow solid.1 1H NMR (DMSO, 400 MHz) δ 8.08 (d, J = 7.2 Hz, 1H), 8.00 (s, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.56 - 7.47 (m, 2H), 4.28 (q, J = 7.2 Hz, 2H), 3.90 (s, 2H), 1.47 (s, 9H), 1.29 (t, J = 7.2 Hz, 3H).

[0307] Preparation of ethyl 2 - amino - 3H - benzothieno[3,2 - b]azepine - 4 - carboxylate, 28f To a solution of 28e (0.2 g, 518 μmol, 1.0 equiv) in EtOAc (2 mL), HCl / EtOAc (10 mL) was added at 25 °C all at once, and it was stirred at 50 °C for 12 h. The mixture was concentrated to give 28f (0.25 g, crude) as a green solid.

[0308] Preparation of 2 - amino - 3H - benzothieno[3,2 - b]azepine - 4 - carboxylic acid, 28g To a mixture of 28f (0.25 g, 774 μmol, 1.0 equiv) in MeOH (6 mL), a solution of LiOH·H2O (162 mg, 3.87 mmol, 5.0 equiv) in H2O (1 mL) was added at 25 °C. The mixture was stirred at 50 °C for 12 h. The mixture was quenched with aqueous HCl (1 M) until the pH reached 5, and then concentrated to remove MeOH. The desired solid was precipitated from the mixture and then filtered to give 28g (0.15 g, crude) as a yellow solid.

[0309] Preparation of 2 - amino - N,N - dipropyl - 3H - benzo[4,5]thieno[3,2 - b]azepine - 4 - carboxamide, TAZ - 28 To a mixture of 28 gm (0.05 g, 194 μmol, 1.0 equiv) in DMF (2 mL), HATU (88.3 mg, 232 μmol, 1.2 equiv) and DIEA (125 mg, 968 μmol, 169 μL, 5.0 equiv) were added and the mixture was stirred at 25 °C for 1 h. N-Propylpropan-1-amine (25.5 mg, 252 μmol, 34.7 μL, 1.3 equiv) was added to the mixture and stirred for 1 h. The reaction mixture was filtered and purified by preparative HPLC (column: Nano-micro Kromasil C18 100 * 30 mm 8um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 10 min) to give TAZ-28 (19 mg, 55.64 μmol, yield 28.74%) as a white solid. 1 1H NMR (MeOD, 400 MHz) δ8.00-7.92 (m, 2H), 7.58-7.53 (m, 2H), 7.18 (s, 1H), 3.48 (s, 6H), 1.74-1.65(m, 4H), 0.94 (s, 6H). LC / MS [M+H] 342.2 (calculated); LC / MS [M+H] 342.2 (measured).

[0310] Example 29 Synthesis of tert-butyl (4-((5-amino-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide)methyl)benzyl)carbamate, TAZ-29

Chemical Structure

[0311] Preparation of tert-butyl (4-((5-amino-2-bromo-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide)methyl)benzyl)carbamate, 29c 5-Amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, TAZ-15 (0.330 g, 1.15 mmol, 1 equiv) and 29b (0.32 g, 1.15 mmol, 1 equiv) were suspended in 5 mL of DMF. DIPEA (1.2 mL, 6.9 mmol, 6 equiv) was added, followed by 7-aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, PyAOP (0.90 g, 1.72 mmol, 1.5 equiv). The reaction was monitored by LCMS. When the starting material was consumed, the reaction mixture was added to 100 mL of water, filtered, and the precipitate was purified by flash chromatography (MeOH / DCM containing 1% TEA) to give 29c (0.35 g, 0.64 mmol, 56%). LC / MS [M+H] 547.14 / 549.14 (calcd); LC / MS [M+H] 547.40 / 549.35 (found).

[0312] Preparation of TAZ-29 Intermediate 29c (0.35 g, 0.64 mmol, 1 equiv) was dissolved in 5 ml of THF. Triethylamine (0.89 ml, 6.4 mmol, 10 equiv) and dichlorobis(1,1'-bis(diphenylphosphino)ferrocene)palladium(II), Pd(dppf)Cl2 (0.023 g, 0.032 mmol, 0.05 equiv) were added, followed by sodium borohydride (0.12 g, 3.2 mmol, 5 equiv). After 2 h, another portion of sodium borohydride (0.073 g, 1.9 mmol, 3 equiv) was added and the reaction mixture was stirred for 30 min. The reaction mixture was concentrated and purified by HPLC to give TAZ-29 (0.129 g, 0.28 mmol, 43%). LC / MS [M+H] 469.23 (calcd); LC / MS [M+H] 469.42 (found).

[0313] Example 30 Synthesis of 5-amino-N-(4-(aminomethyl)benzyl)-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-30

Chem.

[0314] Example 52 Synthesis of 5-amino-N-[[4-(aminomethyl)-2-(trifluoromethyl)phenyl]methyl]-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-52

Chem.

[0315] Preparation of tert-butyl N-[[4-(aminomethyl)-3-(trifluoromethyl)phenyl]methyl]carbamate, 52c To a solution of 52b (0.5 g, 1.67 mmol, 1.0 equiv) in MeOH (10 mL) was added NH₃·H₂O (17 mg, 166 μmol, purity 33%, 0.1 equiv) and Raney-Ni (1.43 g, 1.67 mmol, purity 10%, 1.0 equiv) at 25 °C under N₂. The suspension was degassed under vacuum and purged several times with H₂, then stirred at 25 °C under H₂ (50 psi) for 10 h. It was then filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 10 / 1, 1 / 1) to give 52c (200 mg, 657.23 μmol, yield 39.47%) as a yellow oil. 1 H NMR (MeOD, 400 MHz) δ 6.99 - 6.92 (m, 2H), 6.89 - 6.86 (m, 1H), 3.62 (s, 2H), 2.66 (s, 2H), 0.80 (s, 9H)

[0316] Preparation of tert-butyl N-[[4-(propylaminomethyl)-3-(trifluoromethyl)phenyl]methyl]carbamate, 52d To a mixture of 52c (190 mg, 624 μmol, 1 equiv) in MeOH (2 mL) and THF (2 mL) was added propanal (47 mg, 812 μmol, 1.3 equiv). After 30 min, NaBH₃CN (117 mg, 1.87 mmol, 3.0 equiv) and AcOH (3 mg, 62 μmol, 3 μL, 0.1 equiv) were added at 25 °C and then stirred for 2 h. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75 * 30 mm * 3 μm; mobile phase: [water (10 mM NH₄HCO₃)-ACN]; B%: 30% - 60%, 12 min) to give 52d (100 mg, 277 μmol, yield 44.39%, purity 96%) as a white solid. 11H NMR (MeOD, 400 MHz) δ 7.65 - 7.57 (m, 2H), 7.53 - 7.50 (m, 1H), 4.27 (s, 2H), 3.91 (s, 2H), 2.58 (t, J = 7.6 Hz, 2H), 1.63 - 1.59 (m, 2H), 1.45 (s, 9H), 0.93 (t, J = 7.2 Hz, 3H)

[0317] tert-Butyl N-[[4-[[(5-Amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]methyl]-3-(trifluoromethyl)phenyl]methyl]carbamate, Preparation of 52e To a solution of 5-amino-6H-thieno[3,2-b]azepine-7-carboxylic acid, 16a (24.0 mg, 115 μmol, 1.0 equiv) in DMF (1 mL), DIEA (74 mg, 577 μmol, 100 μL, 5 equiv) and PyAOP (66 mg, 127 μmol, 1.1 equiv) were added at once at 25 °C, stirred for 30 min, then 52d (60 mg, 173.22 μmol, 1.5 equiv) was added, and then stirred for another 2 h. Then, the reaction solution was concentrated and purified by preparative HPLC (column: Phenomenex Synergi C18 150 * 25 * 10 μm; mobile phase: [water (0.1% TFA) - ACN]; B%: 25% - 50%, 10 min), and 52e (15 mg, 27.95 μmol, yield 24.21%) was obtained as a white solid. 1 1H NMR (MeOD, 400 MHz) δ 7.75 - 7.68 (m, 1H), 7.65 (s, 1H), 7.57 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.30 - 7.10 (m, 2H), 4.92 (s, 2H), 4.29 (s, 2H), 3.48 (t, J = 7.2 Hz, 2H), 3.32 (s, 2H), 1.66 - 1.64 (m, 2H), 1.45 (s, 9H), 0.94 - 0.86 (m, 3H). LC / MS [M + H] 537.2 (calculated); LC / MS [M + H] 537.1 (measured)

[0318] Preparation of TAZ-52 To a solution of 52e (10 mg, 18.6 μmol, 1.0 equiv) in EtOAc (2 mL) was added HCl / EtOAc (4 M, 140 μL, 30 equiv) at once at 25 °C. The mixture was stirred at 25 °C for 3 h. Subsequently, the mixture was concentrated to afford TAZ-52 (8 mg, 18.3 μmol, yield 98.35%) as a yellow solid. 1 H NMR (MeOD, 400 MHz) δ 7.80 (s, 1H), 7.70 - 7.63 (m, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.20 - 7.17 (m, 1H), 7.03 (d, J = 4.0 Hz, 1H), 4.85 (s, 2H), 4.13 (s, 2H), 3.45 - 3.37 (m, 2H), 3.31 (s, 2H), 1.62 - 1.50 (m, 2H), 0.87 - 0.68 (m, 3H). LC / MS [M + H] 437.2 (calcd); LC / MS [M + H] 437.1 (found).

[0319] Example 54 Synthesis of 5 - amino - N - [[4 - (aminomethyl)-2-(trifluoromethyl)phenyl]methyl]-N - propyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 54 [Chemical formula] Preparation of 4 - cyano - N - propyl - 2 - (trifluoromethyl)benzamide, 54b To a solution of 4 - bromo - 3 - (trifluoromethyl)benzonitrile, 54a (4.00 g, 16.0 mmol, 1.0 equiv) in DMF (20 mL) were added propan - 1 - amine (2.84 g, 48.0 mmol, 3.95 mL, 3.0 equiv), Et3N (4.86 g, 48.0 mmol, 6.68 mL, 3.0 equiv) and Pd(dppf)Cl2 (585 mg, 800 μmol, 0.05 equiv) under N2. The suspension was degassed under vacuum and purged several times with CO, and the mixture was stirred at 80 °C for 15 h under CO (50 psi). Water (50 mL) was added to the mixture, and the aqueous phase was extracted with ethyl acetate (30 mL* It was extracted in (3), and the combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 1 / 1) to obtain 54b (4.00 g, 15.6 mmol, yield 97.5%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ8.13 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 6.49 (br s, 1H), 3.37 (q, J = 7.2 Hz, 2H), 1.66 - 1.54 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H)

[0320] Preparation of tert-butyl N-[[4-(propylcarbamoyl)-3-(trifluoromethyl)phenyl]methyl]carbamate, 54c To a solution of 54b (2.30 g, 8.98 mmol, 1.0 equiv) in MeOH (30 mL) was added Raney Ni (0.5 g, 1.0 equiv) and Boc2O (9.80 g, 44.8 mmol, 10.3 mL, 5.0 equiv) at 25 °C under N2. The suspension was degassed under vacuum and purged several times with H2. The mixture was stirred at 25 °C under H2 (50 psi) for 5 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 1 / 1) to obtain 54c (2.50 g, 6.94 mmol, yield 77.2%) as a white solid. 11H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 6.26 (s, 1H), 5.02 (s, 1H), 4.53 (d, J = 6.0 Hz, 2H), 3.44 (q, J = 6.8 Hz, 2H), 1.70 - 1.62 (m, 2H), 1.46 (s, 9H), 1.00 (t, J = 7.6 Hz, 3H).

[0321] tert-Butyl N-[[4-(propylaminomethyl)-3-(trifluoromethyl)phenyl]methyl]carbamate, Preparation of 54d To a mixture of 54c (1.03 g, 2.86 mmol, 1.0 equiv) and RhH(CO)(PPh3)3 (263 mg, 286 μmol, 0.1 equiv) in THF (30 mL), diphenylsilane (3.16 g, 17.1 mmol, 3.16 mL, 6.0 equiv) was added all at once under N2 at 20 °C, and then the mixture was stirred at 20 °C for 8 h. The reaction mixture was concentrated in vacuo, and the residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 0 / 1 to ethyl acetate / methanol = 5 / 1) to give 54d (0.4 g, 1.15 mmol, yield 40.40%) as a yellow oil. 1 1H NMR (400 MHz, MeOD) δ 7.89 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 4.48 (s, 2H), 4.29 (s, 2H), 3.08 - 3.01 (m, 2H), 1.82 - 1.71 (m, 2H), 1.48 (s, 9H), 1.05 (t, J = 7.6 Hz, 3H)

[0322] tert-Butyl N-[[4-[[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]methyl]-3-(trifluoromethyl)phenyl]methyl]carbamate, Preparation of 54e A solution of 5-amino-6H-thieno[3,2-b]azepine-7-carboxylic acid, 16a (12.0 mg, 57.7 μmol, 1.0 equiv) in DMF (1 mL) was added with HATU (19.7 mg, 52.0 μmol, 0.9 equiv) and Et3N (17.53 mg, 173 μmol, 24.1 μL, 3.0 equiv) at 20 °C under N2. The mixture was stirred at 20 °C for 10 min, then 54d (20 mg, 57.7 μmol, 1.0 equiv) was added, and then stirred at 20 °C for 2 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150 * 25 * 10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-55%, 10 min) to obtain 54e (5.00 mg, 7.47 μmol, yield 12.9%, purity 97.1%, TFA) as a white solid. 1 1H NMR (400 MHz, MeOD) δ 7.74 (d, J = 5.6 Hz, 1H), 7.60 - 7.52 (m, 3H), 7.22 (s, 1H), 7.12 (br d, J = 5.6 Hz, 1H), 4.82 (br s, 2H), 4.45 (s, 2H), 3.51 (br t, J = 7.2 Hz, 2H), 3.42 - 3.35 (m, 2H), 1.75 - 1.64 (m, 2H), 1.49 (s, 9H), 0.96 - 0.90 (m, 3H). LC / MS [M+H] 537.2 (calculated); LC / MS [M+H] 537.1 (measured).

[0323] Preparation of TAZ-54 To a solution of 54e (50.0 mg, 93.2 μmol, 1.0 equiv) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 2.33 mL, 100 equiv) at 20 °C, and then stirred at 20 °C for 2 h. The reaction mixture was concentrated in vacuo and lyophilized to obtain TAZ-54 (30.0 mg, 62.8 μmol, yield 67.4%, purity 99.0%, HCl) as a yellow solid. 11H NMR (400 MHz, MeOD) δ 7.67 (br s, 1H), 7.65 - 7.61 (m, 3H), 7.10 (s, 1H), 7.04 (d, J = 5.2 Hz, 1H), 4.75 (s, 2H), 4.22 (s, 2H), 3.45 - 3.38 (m, 2H), 3.29 (br s, 2H), 1.63 - 1.52 (m, 2H), 0.79 (br t, J = 7.2 Hz, 3H). LC / MS [M+H] 437.2 (calculated); LC / MS [M+H] 437.1 (measured).

[0324] Example 65 Synthesis of 5 - Amino - 2 - [5 - (dimethylamino)pentyl] - N - [[4 - (methylaminomethyl)phenyl]methyl] - N - propyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 65 [Chemical Structure] Preparation of tert - butyl N - [[4 - [[(5 - amino - 2 - bromo - 6H - thieno[3,2 - b]azepine - 7 - carbonyl)-propyl - amino]methyl]phenyl]methyl] - N - methylcarbamate, 65b To a solution of 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, 65a (1.0 g, 3.48 mmol, 1.0 equiv) in DMF (20 mL), HATU (1.46 g, 3.83 mmol, 1.1 equiv), DIEA (1.35 g, 10.45 mmol, 1.82 mL, 3.0 equiv) and tert-butyl N-methyl-N-[[4-(propylaminomethyl)phenyl]methyl]carbamate (1.07 g, 3.66 mmol, 1.05 equiv) were added, and then the mixture was stirred at 25 °C for 1 h. The reaction mixture was quenched by adding H2O (100 mL), and then extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. Finally, the residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 1 - 0 / 1) to give 65b (1.80 g, 3.21 mmol, yield 92.04%) as a yellow oil. 1 H NMR (CDCl3, 400 MHz) δ7.25 - 7.15 (m, 4H), 6.92 (s, 1H), 6.77 (s, 1H), 4.72 (s, 2H), 4.42 (s, 2H), 3.40 (t, J = 7.2 Hz, 2H), 2.86 (s, 2H), 2.81 (s, 3H), 1.69 - 1.58 (m, 2H), 1.49 (s, 9H), 0.89 (t, J = 7.2 Hz, 3H).

[0325] Preparation of tert-butyl N-[[4-[[[5-amino-2-(4-cyanobut-1-ynyl)-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]methyl]phenyl]methyl]-N-methyl-carbamate, 65c To a mixture of 65b (2.25 g, 4.01 mmol, 1.0 equiv) and pent-4-ynenitrile (951 mg, 12.0 mmol, 3.0 equiv) in TEA (13.2 mL) and DMF (44 mL) were added Pd(PPh3)2Cl2 (140.6 mg, 200.34 μmol, 0.050 equiv), CuI (152.6 mg, 801.38 μmol, 0.20 equiv) and PPh3 (210 mg, 801 μmol, 0.20 equiv) at 15 °C, and then the mixture was stirred at 140 °C for 3 h under a N2 atmosphere. The reaction mixture was quenched by adding H2O (220 mL) at 25 °C, and then extracted with DCM / isopropanol = 3 / 1 (200 mL × 3). The combined organic layers were washed with H2O (40 mL × 4), dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 1 to 0 / 1 to ethyl acetate / MeOH = 50 / 1 to 5 / 1) to give 65c (1.62 g, 2.89 mmol, 72% yield) as a yellow oil. 1 H NMR (CDCl3, 400 MHz) δ 7.21-7.20 (m, 4H), 7.00 (s, 1H), 6.81 (s, 1H), 4.73 (s, 2H), 4.42 (s, 2H), 3.40 (t, J = 6.4 Hz, 2H), 2.83-2.81 (m, 7H), 2.68-2.65 (m, 2H), 1.64-1.62 (m, 2H), 1.49 ( s, 9H), 0.89 (t, J = 7.2 Hz, 3H)

[0326] Preparation of tert-butyl N-[[4-[[[5-amino-2-(4-cyanobutyl)-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]methyl]phenyl]methyl]-N-methyl-carbamate, 65d To a solution of 65c (1.47 g, 2.63 mmol, 1.0 eq) in MeOH (30 mL), Pd(OH)2 / C (369 mg, 262 μmol, purity 10%, 0.10 eq) was added under a N2 atmosphere. The suspension was degassed and purged with H2 three times. The mixture was stirred at 25 °C for 12 h under H2 (50 Psi). The reaction mixture was filtered and the filtrate was concentrated to give 65d (1.15 g, 2.04 mmol, yield 77.67%) as a yellow oil. 1 H NMR (CDCl3, 400 MHz) δ7.21 - 7.19 (m, 4H), 6.86 (s, 1H), 6.67 (s, 1H), 4.74 (s, 2H), 4.42 (s, 2H), 3.40 (t, J = 6.4 Hz, 2H), 2.86 - 2.82 (m, 5H), 2.37 (t, J = 6.8 Hz, 2H), 1.88 - 1.83 (m, 2H), 1.78 - 1.74 (m, 2H), 1.64 - 1.61 (m, 2H), 1.49 (m, 9H), 0.89 (t, J = 7.6 Hz, 3H).

[0327] Preparation of tert-butyl N-[[4-[[[5-amino-2-(5-aminopentyl)-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]methyl]phenyl]methyl]-N-methyl-carbamate, 65e To a solution of 65d (1.15 g, 2.04 mmol, 1.0 eq) in MeOH (23 mL), NH3·H2O (2.86 g, 20.4 mmol, 3.14 mL, purity 25%, 10 eq) and Ni (100 mg) were added under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred at 25 °C for 3 h under H2 (50 psi). The reaction mixture was filtered and the filtrate was concentrated to give 65e (1.03 g, 1.81 mmol, yield 88.93%) as a yellow oil.

[0328] Preparation of tert-butyl N-[[4-[[[5-amino-2-[5-(dimethylamino)pentyl]-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]methyl]phenyl]methyl]-N-methyl-carbamate, 65f To a solution of 65e (300 mg, 528 μmol, 1.0 equiv) in MeOH (8 mL) were added AcOH (3.1 mg, 52.8 μmol, 0.10 equiv), HCHO (171.5 mg, 2.11 mmol, 157.3 μL, purity 37%, 4.0 equiv), and NaBH3CN (99.6 mg, 1.59 mmol, 3.0 equiv), and the mixture was then stirred at 25 °C for 1 h. The mixture was quenched by adding H2O (10 mL) and concentrated to remove MeOH. The aqueous phase was extracted with DCM / isopropanol = 3 / 1 (10 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated to give 65f (314 mg, crude) as a yellow oil.

[0329] Preparation of TAZ-65 To a solution of 65f (50 mg, 83.9 μmol, 1.0 equiv) in H2O (0.5 mL) was added HCl (12 M, 140 μL, 20.0 equiv), and the mixture was stirred at 80 °C for 1 h. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150 * 25 * 10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-35%, 10 min) to give TAZ-65 (10 mg, 13.74 μmol, yield 16.37%, purity 99.44%, 2TFA) as a colorless oil. 1 1H NMR (MeOD, 400 MHz,) δ 7.50 - 7.40 (m, 4H), 7.09 (s, 1H), 6.89 (s, 1H), 4.78 (s, 2H), 4.19 (s, 2H), 3.45 - 3.35 (m, 4H), 3.32 (s, 2H), 3.14 - 3.10 (m, 2H), 2.88 (s, 6H), 2.72 (s, 3H), 1.80 - 1.74 (m, 4H), 1.66 - 1.64 (m, 2H), 1.49 - 1.47 (m, 2H), 0.89 - 0.86 (m, 3H). LC / MS [M + H] 496.3 (calculated); LC / MS [M + H] 496.3 (measured).

[0330] Example 109 Synthesis of 5-Amino-2-(5-aminopentyl)-N-(3-(3,3-dimethylbutanamido)propyl)-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-109 [Chemical Structure] 5-Amino-2-(5-(bis(tert-butoxycarbonyl)amino)pentyl)-6H-thieno[3,2-b]azepine-7-carboxylic acid, 109a (115 mg, 0.23 mmol, 1 equiv) and 3,3-dimethyl-N-(3-(propylamino)propyl)butanamide (50 mg, 0.23, 1 equiv) were taken up in 8:3 ACN:DCM (2.75 ml). DIPEA (0.121 ml, 0.7 mmol, 3 equiv) was added, followed by 7-aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, PyAOP (0.122 m, 0.23 mmol, 1 equiv). The solution was stirred at ambient temperature. After confirmation of completion by LCMS, the reaction mixture was concentrated and purified by HPLC to obtain [amide], which was subsequently dissolved in minimal TFA and left for 15 minutes. The solution was concentrated and triturated with diethyl ether to obtain TAZ-109 (61.4 mg, 0.102 mmol, 44%) as the trifluoroacetate salt. LC / MS [M+H] 490.32 (calculated); LC / MS [M+H] 490.39 (measured).

[0331] Example 133 Synthesis of tert-Butyl (3-(5-amino-2-(5-aminopentyl)-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide)propyl)carbamate, TAZ-133 [Chemical Structure] Preparation of 5-(5-Amino-7-(ethoxycarbonyl)-6H-thieno[3,2-b]azepin-2-yl)pentan-1-aminium trifluoroacetate, 133b Ethyl 5-amino-2-(5-(bis(tert-butoxycarbonyl)amino)pentyl)-6H-thieno[3,2-b]azepine-7-carboxylate, 133a (0.188 g, 0.36 mmol, 1 equiv) was dissolved in minimal TFA. After complete deprotection, the solution was concentrated and the product was precipitated from diethyl ether to afford 133b (0.082 g, 0.188 mmol, 52%) as a yellow powder. LC / MS [M+H] 322.16 (calcd); LC / MS [M+H] 322.25 (found).

[0332] Preparation of 5-amino-2-(5-(((benzyloxy)carbonyl)amino)pentyl)-6H-thieno[3,2-b]azepine-7-carboxylic acid, 133c Intermediate 133b (0.296 g, 0.68 mmol, 1 equiv) was suspended in 2 ml of DMF. Collidine (0.27 ml, 2 mmol, 3 equiv) was added, followed by benzyl chloroformate, Cbz-Cl, CAS No. 501-53-1 (0.1 ml, 0.68 mmol, 1 equiv). The reaction was monitored by LCMS. After consumption of the amine starting material, the reaction was concentrated and redissolved in 7 ml of 3:1:3 THF:MeOH:water. Lithium hydroxide (0.16 g, 6.8 mmol, 10 equiv) was added and the reaction was stirred at room temperature. Upon completion, the reaction mixture was concentrated and purified by reverse phase HPLC to afford 133c (0.246 g, 0.58 mmol, 85%). LC / MS [M+H] 428.16 (calcd); LC / MS [M+H] 428.32 (found).

[0333] Preparation of Benzyl (5-(5-amino-7-((3-((tert-butoxycarbonyl)amino)propyl)(propyl)carbamoyl)-6H-thieno[3,2-b]azepin-2-yl)pentyl)carbamate, 133d Intermediate 133c (0.29 g, 0.68 mol, 1 equivalent) and tert-butyl (3-(propylamino)propyl) carbamate (0.225 g, 1.0 mmol, 1.53 equivalents) were dissolved in 1 ml of DMF. Diisopropylethylamine, DIPEA (0.59 ml, 1.0 mmol, 1.53 equivalents) was added, followed by PyAOP (0.541 g, 1.0 mmol, 1.53 equivalents). The reaction mixture was stirred at room temperature and then concentrated, and purified by reverse-phase flash chromatography to obtain 133d (0.201 g, 0.32 mmol, 47%). LC / MS [M+H] 626.34 (calculated); LC / MS [M+H] 626.51 (measured).

[0334] Preparation of TAZ-133 Intermediate 133d (0.2 g, 0.32 mmol, 1 equivalent) was dissolved in 2 ml of MeOH. Triethylamine (0.1 ml) and formic acid (0.049 ml, 1.29 mmol, 4 equivalents) were added, followed by 10% w / w Pd / C (0.04 g). The stirred reaction mixture was heated to 60 °C. After 1 hour, 20% w / w Pd(OH)2 (0.02 g) was added. After completion, the reaction mixture was filtered, concentrated, and purified by HPLC to obtain TAZ-133 (0.139 g, 0.28 mmol, 88%). LC / MS [M+H] 492.30 (calculated); LC / MS [M+H] 492.45 (measured).

[0335] Example 176 Synthesis of 5-amino-N-ethoxy-2-[2-(4-piperidyl)ethyl]-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-176 [Chemical Structure] Preparation of 5-amino-2-[2-(1-tert-butoxycarbonyl-4-piperidyl)ethynyl]-6H-thieno[3,2-b]azepine-7-carboxylate ethyl, 176a To a mixture of ethyl 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylate, 15 g (2 g, 6.35 mmol, 1.0 eq) in CH3CN (60 mL) was added 4-ethynylpiperidine-1-carboxylic acid butyl (1.73 g, 8.25 mmol, 1.3 eq), Cs2CO3 (6.20 g, 19.0 mmol, 3.0 eq), CuI (242 mg, 1.27 mmol, 0.2 eq) and Pd(PPh3)2Cl2 (445 mg, 635 μmol, 0.1 eq) all at once under N2 at 25 °C, and the mixture was stirred at 100 °C for 2 h. The mixture was concentrated to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 10 / 1) to give 176a (3.5 g, crude) as a yellow solid.

[0336] Preparation of 5-amino-2-[2-(1-tert-butoxycarbonyl-4-piperidyl)ethynyl]-6H-thieno[3,2-b]azepine-7-carboxylic acid, 176b To a mixture of 176a (3.5 g, 7.89 mmol, 1.0 eq) in EtOH (50 mL) and H2O (8 mL) was added LiOH.H2O (1.32 g, 31.6 mmol, 4.0 eq) all at once at 25 °C. The mixture was stirred at 30 °C for 2 h. The mixture was concentrated and the residue was diluted with water (30 mL). Then the mixture was filtered. The filter cake was triturated with CH3CN at 25 °C for 0.5 h and then filtered to give 176b (2.3 g, 5.54 mmol, yield 70.2%) as a yellow solid.

[0337] Preparation of tert-butyl 4-[2-[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2-yl]ethynyl]piperidine-1-carboxylate, 176c To a mixture of 176b (1 g, 2.41 mmol, 1.0 equiv) in DCM (10 mL) and DMA (10 mL), N-ethoxypropan-1-amine (353 mg, 2.53 mmol, 1.05 equiv, HCl) and EDCI (1.85 g, 9.63 mmol, 4.0 equiv) were added at once at 25 °C, and the mixture was stirred at 25 °C for 1 h. The mixture was concentrated to remove DCM, the residue was diluted with water (50 mL), the pH of the mixture was adjusted to about 8 with saturated NaHCO3, and then extracted with EtOAc (30 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, ethyl acetate / MeOH = 1 / 0, 10 / 1) to give 176c (0.63 g, 1.26 mmol, 52.3% yield) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ7.27 (s, 1H), 6.88 (s, 1H), 3.89 (q, J = 7.2 Hz, 2H), 3.74 - 3.70 (m, 2H), 3.69 (t, J = 6.8 Hz, 2H), 3.25 - 3.19 (m, 3H), 2.99 (s, 2H), 1.89 - 1.85 (m, 2H), 1.79 - 1.67 (m, 2H), 1.65 - 1.59 (m, 2H), 1.47 (s, 9H), 1.15 (t, J = 7.2 Hz, 3H), 0.95 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 501.2 (calculated); LC / MS [M+H] 501.1 (measured).

[0338] Preparation of tert-butyl 4-[2-[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2-yl]ethyl]piperidine-1-carboxylate, 176d To a solution of 176c (0.45 g, 899 μmol, 1.0 equiv) in MeOH (15 mL), Pd(OH)2 / C (0.2 g, purity 10%) was added under N2. The suspension was degassed under vacuum and purged several times with H2. The mixture was stirred at 25 °C for 12 h under H2 (50 psi). The mixture was filtered and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to give 176d (0.3 g, 594 μmol, yield 66.13%) as a pale yellow solid. 1 1H NMR (MeOD, 400 MHz) δ 7.32 (s, 1H), 6.65 (s, 1H), 4.06 (d, J = 13.2 Hz, 2H), 3.89 (q, J = 7.2 Hz, 2H), 3.69 (t, J = 7.2 Hz, 2H), 2.98 (s, 2H), 2.85 (t, J = 7.6 Hz, 2H), 2.73 (s, 2H), 1.77 - 1.73 (m, 4H), 1.69 - 1.60 (m, 2H), 1.58 - 1.50 (m, 1H), 1.45 (s, 9H), 1.16 (t, J = 7.2 Hz, 3H), 1.13 - 1.05 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H). LC / MS [M + H] 501.2 (calcd); LC / MS [M + H] 505.3 (found).

[0339] Preparation of TAZ - 176 To a mixture of 176d (0.3 g, 594 μmol, 1.0 equiv) in EtOAc (5 mL), HCl / EtOAc (4 M, 10 mL) was added at once at 25 °C and the mixture was stirred at 25 °C for 0.5 min. The mixture was concentrated to give TAZ - 176 (0.3 g, crude, HCl) as a yellow solid. 11H NMR (MeOD, 400 MHz) δ 7.45 (s, 1H), 6.93 (s, 1H), 3.93 (q, J = 7.2 Hz, 2H), 3.72 (t, J = 7.2 Hz, 2H), 3.45 - 3.37 (m, 4H), 3.05 - 2.91 (m, 4H), 2.02 (d, J = 13.6 Hz, 2H), 1.80 - 1.65 (m, 5H), 1.52 - 1.35 (m, 2H), 1.18 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 405.2 (calculated value); LC / MS [M+H] 405.1 (measured value).

[0340] Example 183 Synthesis of 5 - amino - 2 - (azetidin - 3 - ylmethyl) - N - ethoxy - N - propyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 183

Chemical Structure

[0341] Preparation of 5-amino-2-[(1-tert-butoxycarbonylazetidin-3-yl)methyl]-6H-thieno[3,2-b]azepine-7-carboxylic acid, 183b To a mixture of 183a (2 g, 4.93 mmol, 1 eq) in THF (10 mL) and H2O (10 mL) was added LiOH.H2O (621 mg, 14.8 mmol, 3 eq), and the mixture was then stirred at 15 °C for 3 h. The mixture was concentrated to remove THF, and then the pH of the aqueous phase was adjusted to about 7 with HCl (4 M). The desired solid was precipitated from the mixture and filtered to give 183b (1.5 g, 3.97 mmol, 80.6% yield) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 7.65 (s, 1H), 6.94 (s, 2H), 6.66 (s, 1H), 4.05 - 3.93 (m, 2H), 3.70 - 3.55 (m, 2H), 3.07 (d, J = 7.6 Hz, 2H), 2.88 (s, 2H), 2.80 - 2.65 (m, 1H), 1.43 (s, 9H).

[0342] tert-Butyl 3-[[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2-yl]methyl]azetidine-1-carboxylate, Preparation of 183c A mixture of 183b (0.2 g, 530 μmol, 1 equiv) and N-ethoxypropan-1-amine (96.2 mg, 689 μmol, 1.3 equiv, HCl) in DMA (3 mL) and DCM (3 mL) was added with EDCI (406 mg, 2.12 mmol, 4 equiv), and then stirred at 15 °C for 2 h. The mixture was concentrated to obtain a residue. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150 * 25 * 10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 25% - 55%, 8 min) to give 183c (130 mg, 273 μmol, yield 51.6%, purity 97.2%) as a pale yellow solid. 1 1H NMR (400 MHz, MeOD) δ 7.47 (s, 1H), 6.93 (s, 1H), 4.07 (br t, J = 8.4 Hz, 2H), 3.95 (q, J = 7.2 Hz, 2H), 3.82 - 3.63 (m, 4H), 3.44 (s, 2H), 3.18 (d, J = 7.6 Hz, 2H), 3.02 - 2.81 (m, 1H), 1.76 (sxt, J = 7.2 Hz, 2H), 1.45 (s, 9H), 1.20 (t, J = 7.2 Hz, 3H), 0.99 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 463.2 (calculated); LC / MS [M+H] 463.1 (measured).

[0343] Preparation of TAZ-183 To a mixture of 183c (0.11 g, 238 μmol, 1 equiv) in DCM (10 mL) was added TFA (1.54 g, 13.5 mmol, 1 mL, 56.8 equiv). The mixture was stirred at 15 °C for 1 h. The pH of the mixture was adjusted to about 7 with a saturated aqueous solution of NaHCO3, and then extracted with DCM / i-PrOH (3:1, 10 mL * 3). The organic layer was dried over Na2SO4 and concentrated to give TAZ-183 (60 mg, 151 μmol, yield 63.3%, purity 90.95%) as a pale yellow solid. 1 1H NMR (400 MHz, MeOD) δ 7.20 (s, 1H), 6.63 (s, 1H), 4.04 (br t, J = 9.6 Hz, 2H), 3.83 - 3.75 (m, 4H), 3.59 (t, J = 7.2 Hz, 2H), 3.36 - 3.30 (m, 1H), 3.05 (d, J = 7.6 Hz, 2H), 2.88 (s, 2H), 1.62 (sxt, J = 7.2 Hz, 2H), 1.08 - 1.00 (m, 3H), 0.85 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 363.2 (calculated); LC / MS [M+H] 363.1 (measured).

[0344] Example 185 Cyclobutyl N-[3-[[5-amino-2-(4-piperidylmethyl)-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]propyl]carbamate, Synthesis of TAZ-185 [Chemical Structure] Preparation of ethyl 5-amino-2-[(1-tert-butoxycarbonyl-4-piperidyl)methyl]-6H-thieno[3,2-b]azepine-7-carboxylate, 185a A mixture of tert-butyl 4-methylenepiperidine-1-carboxylate (4.51 g, 22.8 mmol, 2.0 equiv) and 9-BBN (1 M, 57.1 mL, 5.0 equiv) was heated to 70 °C and stirred at 70 °C. After 2 h, ethyl 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylate, 15 g (3.60 g, 11.4 mmol, 1.0 equiv), Xantphos (1.59 g, 2.74 mmol, 0.24 equiv), Pd2(dba)3 (836 mg, 913 μmol, 0.08 equiv), K2CO3 (4.74 g, 34.2 mmol, 3.0 equiv), H2O (5 mL) and dioxane (50 mL) were cooled to 20 °C, and then the mixture was stirred at 100 °C for 4 h under N2. Water (200 mL) was added, and the aqueous phase was extracted with ethyl acetate (50 mL * 4), and the combined organic phases were washed with brine (100 mL * 1), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 10 / 1, 0 / 1) to give 185a (1.8 g, 4.15 mmol, 36.35% yield) as a brown oil.

[0345] Preparation of 5-amino-2-[(1-tert-butoxycarbonyl-4-piperidyl)methyl]-6H-thieno[3,2-b]azepine-7-carboxylic acid, 185b To a solution of L-185a (1.80 g, 4.15 mmol, 1.0 equiv) in EtOH (3 mL) and H2O (5 mL), LiOH·H2O (696 mg, 16.6 mmol, 4.0 equiv) was added in one portion at 20 °C under N2. The mixture was then stirred at 20 °C for 4 h. The reaction mixture was quenched with HCl (4 M) until pH = 6, and then EtOH was removed in vacuo. The precipitate was filtered and dried to give 185b (1.20 g, 2.96 mmol, 71.2% yield) as a yellow solid. 11H NMR (400 MHz, DMSO-d6) δ 7.61 (s, 1H), 6.58 (s, 1H), 3.86 (d, 2.0 Hz, 2H), 2.92 (s, 2H), 2.65 (d, J = 6.4 Hz, 2H), 1.65 - 1.60 (m, 3H), 1.35 (s, 9H), 1.07 - 0.96 (m, 2H)

[0346] tert-Butyl 4-[[5-amino-7-[3-(cyclobutoxycarbonylamino)propyl]propylcarbamoyl]-6H-thieno[3,2-b]azepin-2-yl]methyl]piperidine-1-carboxylate, Preparation of 185c To a mixture of L-185b (200 mg, 493 μmol, 1.0 equiv) and cyclobutyl N-[3-(propylamino)propyl]carbamate (148 mg, 591 μmol, 1.2 equiv, HCl) in DMF (2 mL), HATU (187 mg, 493 μmol, 1.0 equiv) and DIEA (191 mg, 1.48 mmol, 257 μL, 3.0 equiv) were added at once under N2 at 20 °C, and the mixture was stirred at 20 °C for 1 h. Water (10 mL) was added, and the aqueous phase was extracted with ethyl acetate (10 mL * 3), and the combined organic phases were washed with brine (15 mL * 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100 - 200 mesh silica gel, petroleum ether / ethyl acetate = 5 / 1, 0 / 1 to ethyl acetate / methanol = 10 / 1) to give 185c (180 mg, 299 μmol, 60.6% yield) as a brown solid. 11H NMR (400 MHz, MeOD) δ 6.88 (s, 1H), 6.68 (s, 1H), 4.88 - 4.80 (m, 1H), 4.08 (d, J = 12.4 Hz, 2H), 3.49 (t, J = 7.2 Hz, 2H), 3.41 (t, J = 7.6 Hz, 2H), 3.11 (s, 2H), 2.75 (d, J = 6.8 Hz, 3H), 2.36 - 2.23 (m, 2H), 1.86 - 1.59 (m, 10H), 1.47 (s, 9H), 1.22 - 1.09 (m, 2H), 0.90 (t, J = 4.0, 3H)

[0347] Preparation of TAZ - 185 To a solution of 185c (180 mg, 299 μmol, 1.0 equiv) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 3.74 mL, 50 equiv) at once under N2 at 20 °C, and then the mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated in vacuo to give TAZ - 185 (140 mg, 260 μmol, yield 86.98%, HCl) as a yellow oil.

[0348] Example 198 Synthesis of 5 - Amino - N - ethoxy - 2 - (4 - piperidylmethyl) - N - propyl - 6H - thieno[3,2 - b]azepine - 7 - carboxamide, TAZ - 198

Chemical Structure

[0349] Preparation of TAZ-198 To a solution of 198a (150 mg, 305 μmol, 1.0 equiv) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 3.82 mL, 50 equiv) under N2 at 20 °C, and the mixture was stirred at 20 °C for 1 h. The reaction mixture was concentrated in vacuo to give TAZ-198 (100 mg, 234 μmol, yield 76.6%, HCl) as a yellow oil.

[0350] Example 238 Synthesis of Cyclobutyl N-[2-[[5-amino-2-(azetidin-3-ylmethyl)-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]oxyethyl]carbamate, TAZ-238

Chemical formula

[0351] Preparation of TAZ - 238 To a mixture of 238a (0.31 g, 538 μmol, 1.0 equiv) in DCM (6 mL), TFA (1.23 g, 10.8 mmol, 797 μL, 20.0 equiv) was added at once at 25 °C, and the mixture was stirred at 25 °C for 2 h. The mixture was concentrated to obtain a residue, which was diluted with H2O (15 mL), and the mixture was extracted with MTBE (10 mL * 2) to remove the excess TFA. The aqueous phase was lyophilized to obtain TAZ - 238 (0.38 g, 521 μmol, yield 96.7%, purity 96.4%, TFA salt) as a yellow solid. 1 1H NMR (MeOD, 400 MHz) δ 7.47 (s, 1H), 6.96 (s, 1H), 4.81 - 4.74 (m, 1H), 4.22 - 4.13 (m, 2H), 3.96 - 3.86 (m, 4H), 3.71 (t, J = 7.2 Hz, 2H), 3.41 (s, 2H), 3.29 - 3.22 (m, 5H), 2.25 (d, J = 7.2 Hz, 2H), 2.03 - 1.89 (m, 2H), 1.80 - 1.55 (m, 4H), 0.96 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 476.2 (calculated); LC / MS [M+H] 476.1 (measured).

[0352] Example 253 Synthesis of 5-Amino-2-(azetidin-3-ylmethyl)-N-isopropoxy-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-253 [Chemical Structure] Preparation of N-Isopropoxypropan-1-amine To a solution of O-Isopropylhydroxylamine (2 g, 17.9 mmol, 1 equiv., HCl) in THF (15 mL) was added a solution of NaHCO3 (3.01 g, 35.8 mmol, 1.39 mL, 2 equiv.) in H2O (5 mL) and tert-Butoxycarbonyl tert-butyl carbonate (5.87 g, 26.9 mmol, 6.18 mL, 1.5 equiv.), and then the mixture was stirred at 20 °C for 2 h under a N2 atmosphere. H2O (50 mL) was added to the mixture, and then the mixture was extracted with EtOAc (80 mL × 3). The combined organic phases were washed with brine (50 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with (petroleum ether:ethyl acetate = 1:0, 1:1) to give tert-Butyl N-isopropoxycarbamate (2.81 g, 16.0 mmol, 89.46% yield) as a pale yellow oil. 1 1H NMR (MeOD, 400 MHz) δ 7.00 (br s, 1H), 4.10 - 1.00 (m, 1H), 1.49 (s, 9H), 1.22 (d, J = 6.4 Hz, 6H).

[0353] To a solution of tert-butyl N-isopropoxycarbamate (2.80 g, 15.9 mmol, 1 equiv) in DMF (10 mL) was added NaH (959 mg, 23.9 mmol, purity 60%, 1.5 equiv) at 0 °C under N2, and the mixture was stirred for 0.5 h. Then 1-iodopropane (5.43 g, 32.0 mmol, 3.12 mL, 2 equiv) was added. The mixture was stirred at 25 °C for 2 h under a N2 atmosphere. The reaction mixture was quenched at 0 °C by adding saturated NH4Cl solution (50 mL), then extracted with EtOAc (80 mL × 3). The combined organic phases were washed with brine (30 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate = 0:1 - 1:1. tert-Butyl N-isopropoxy-N-propyl-carbamate (3.8 g, crude) was obtained as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ4.12 - 4.02 (m, 1H), 3.39 (t, J = 7.2 Hz, 2H), 1.70 - 1.61 (m, 2H), 1.49 (s, 9H), 1.21 (d, J = 6.0 Hz, 6H), 0.90 (t, J = 7.2 Hz, 3H).

[0354] To a solution of tert-butyl N-isopropoxy-N-propyl-carbamate (3.2 g, 14.7 mmol, 1 equiv) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 55.2 mL, 15 equiv), and then the mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. N-Isopropoxypropan-1-amine (1.61 g, 10.48 mmol, yield 71.16%, HCl) was obtained as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ4.76 - 4.67 (m, 1H), 3.23 - 3.16 (m, 2H), 2.00 - 1.89 (m, 2H), 1.41 (d, J = 6.2 Hz, 6H), 1.03 (t, J = 7.6 Hz, 3H).

[0355] tert-Butyl 3-[[5-amino-7-[isopropoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2-yl]methyl]azetidine-1-carboxylate, Preparation of 253a To 5-amino-2-[(1-tert-butoxycarbonylazetidin-3-yl)methyl]-6H-thieno[3,2-b]azepine-7-carboxylic acid, 183b (400 mg, 1.06 mmol, 1 equiv), and N-isopropoxypropan-1-amine (244 mg, 1.59 mmol, 1.50 equiv, HCl) in DMA (2 mL) and DCM (2 mL) was added EDCI (610 mg, 3.18 mmol, 3 equiv), and the mixture was then stirred at 20 °C for 1 h. H2O (20 mL) was added to the reaction mixture, and then the pH of the mixture was adjusted to approximately 8 with aqueous NaHCO3. The mixture was extracted with EtOAc (30 mL × 3), and the combined organic phases were washed with brine (10 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with (ethyl acetate:methanol = 1:0.5:1) to give 253a (410 mg, 860 μmol, 81.17% yield) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ 7.34 (s, 1H), 6.73 (s, 1H), 4.20 - 4.15 (m, 1H), 4.09 - 4.00 (m, 2H), 3.74 - 3.63 (m, 4H), 3.06 (d, J = 8.0 Hz, 2H), 2.95 (s, 2H), 2.90 - 2.84 (m, 1H), 1.76 - 1.67 (m, 2H), 1.43 (s, 9H), 1.17 (d, J = 6.2 Hz, 6H), 0.94 (t, J = 7.6 Hz, 3H).

[0356] Preparation of TAZ-253 To a solution of 253a (410 mg, 860 μmol, 1 equiv) in CH3CN (2 mL) and H2O (2 mL), TFA (785 mg, 6.88 mmol, 510 μL, 8 equiv) was added, and the mixture was stirred at 80 °C for 2 h under a N2 atmosphere. The reaction mixture was concentrated under reduced pressure to remove CH3CN, and the aqueous phase was extracted with MTBE (15 mL × 3) to remove excess TFA. The aqueous phase was lyophilized to afford TAZ-253 (320 mg, 850 μmol, 98.80% yield) as a pale yellow solid. 1 1H NMR (MeOD, 400 MHz) δ 7.43 (s, 1H), 6.95 (s, 1H), 4.27 - 4.20 (m, 1H), 4.20 - 4.14 (m, 2H), 3.97 - 3.87 (m, 2H), 3.73 (br t, J = 7.2 Hz, 2H), 3.40 (s, 2H), 3.30 - 3.25 (m, 3H), 1.81 - 1.69 (m, 2H), 1.18 (d, J = 6.2 Hz, 6H), 0.96 (t, J = 7.4 Hz, 3H). LC / MS [M + H] 377.2 (calcd); LC / MS [M + H] 377.1 (found).

[0357] Example 260 Synthesis of Isopropyl N-[2-[[5-Amino-2-(azetidin-3-ylmethyl)-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl-amino]oxyethyl]carbamate, TAZ-260 [Chemical formula] Preparation of tert-Butyl 3-[[5-Amino-7-[2-(isopropoxycarbonylamino)ethoxy-propyl-carbamoyl]-6H-thieno[3,2-b]azepin-2-yl]methyl]azetidine-1-carboxylate, 260a Isopropyl N-[2-(propylaminooxy)ethyl]carbamate (158 mg, 654 μmol, 1.3 eq, HCl) and 5-amino-2-[(1-tert-butoxycarbonylazetidin-3-yl)methyl]-6H-thieno[3,2-b]azepine-7-carboxylic acid, a mixture of 261a (0.19 g, 503 μmol, 1.0 eq) in DCM (4 mL) and DMA (0.5 mL), was added to EDCI (289 mg, 1.51 mmol, 3.0 eq) at 25 °C all at once, and then stirred at 25 °C for 0.5 h. The mixture was concentrated to remove DCM. Then, the mixture was diluted with water (20 mL), the pH of the aqueous phase was adjusted to about 8 with saturated NaHCO3, and then the mixture was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, ethyl acetate / MeOH = 1 / 0, 10 / 1) to obtain 260a (0.18 g, 319 μmol, yield 63.44%) as a yellow oil. 1 H NMR (MeOH, 400 MHz) δ 7.30 (s, 1H), 6.67 (s, 1H), 4.83 - 4.75 (m, 1H), 4.07 - 4.03 (m, 2H), 3.89 (t, J = 5.4 Hz, 2H), 3.68 (t, J = 7.0 Hz, 4H), 3.30 - 3.26 (m, 2H), 3.08 (d, J = 7.6 Hz, 2H), 3.00 (s, 2H), 2.90 - 2.85 (m, 1H), 1.76 - 1.67 (m, 2H), 1.43 (s, 9H), 1.18 (d, J = 6.0 Hz, 6H), 0.94 (t, J = 7.2 Hz, 3H).

[0358] Preparation of TAZ-260 To a mixture of 260a (0.18 g, 319 μmol, 1.0 equiv) in CH3CN (2 mL) and H2O (2 mL), TFA (291 mg, 2.55 mmol, 189 μL, 8.0 equiv) was added at 25 °C, and the mixture was stirred at 80 °C for 0.5 h. The mixture was concentrated to remove CH3CN. Then, the mixture was extracted with MTBE (10 mL × 3) to remove excess TFA. The aqueous phase was lyophilized to give TAZ-260 (0.25 g, 310.30 μmol, yield 97.18%, TFA salt) as a yellow solid. 1 H NMR (MeOH, 400 MHz) δ7.47 (s, 1H), 6.96 (s, 1H), 4.81-4.76 (m, 1H), 4.23-4.09 (m, 2H), 3.98-3.86 (m, 4H), 3.71 (t, J = 6.8 Hz, 2H), 3.41 (s, 2H), 3.29-3.18 (m, 5H), 1.83-1.64 (m, 2H), 1.25-1.12 (m, 6H), 0.96 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 464.2 (calcd); LC / MS [M+H] 464.1 (found).

[0359] Example 261 Synthesis of 5-amino-2-(azetidin-3-ylmethyl)-N-[2-(ethylcarbamoylamino)ethoxy]-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TAZ-261

Chem.

[0360] Preparation of TAZ-261 To a solution of 261b (270 mg, 492 μmol, 1 equiv) in CH3CN (3.00 mL) and H2O (3.00 mL), TFA (449 mg, 3.94 mmol, 291 μL, 8 equiv) was added, and the mixture was stirred at 80 °C for 1 h. The mixture was concentrated, diluted with water (20 mL), extracted with MTBE (20 mL × 2) to remove excess TFA, and the aqueous phase was lyophilized to give TAZ-261 (300 mg, 443.38 μmol, yield 90.10%, 2TFA) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ7.45 (s, 1H), 6.96 (s, 1H), 4.21 - 4.13 (m, 2H), 3.97 - 3.86 (m, 4H), 3.71 (t, J = 7.2 Hz, 2H), 3.42 (s, 2H), 3.30 - 3.20 (m, 3H), 3.06 (q, J = 7.2 Hz, 2H), 1.80 - 1.68 (m, 2H), 1.05 (t, J = 7.2 Hz, 3H), 0.96 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 449.2 (calcd); LC / MS [M+H] 449.1 (found).

[0361] Example L-1 Synthesis of 2,3,5,6-tetrafluorophenyl (E)-40-(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-35-((3-cyanophenyl)imino)-4,7,10,13,16,19,22,25,28,31-decaoxa-34,36,40-triazatetratetracontanoate, TAZ-L-1

Chem.

[0362] (E)-40-(5-Amino-6H-thieno[3,2-b]azepine-7-carbonyl)-35-((3-cyanophenyl)imino)-4,7,10,13,16,19,22,25,28,31-decaoxa-34,36,40-triazatetratetracontanoic acid, Preparation of L-1b L-1a (0.102 g, 0.100 mmol, 1 equivalent.) was dissolved in 100 μl of TFA. After 15 minutes, the product was triturated with diethyl ether and then concentrated under vacuum to give L-1b (94.4 mg, 0.98 mmol, 98%). LC / MS [M+H] 962.49 (calculated); LC / MS [M+H] 962.85 (measured).

[0363] Preparation of TAZ-L-1 L-1b (0.094 g, 0.098 mmol, 1 equivalent) and 2,3,5,6-tetrafluorophenol, TFP (0.033 g, 0.20 mmol, 2 equivalents) were dissolved in DMF. Collidine (0.064 ml, 0.49 mmol, 5 equivalents) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC-HCl (0.038 g, 0.20 mmol, 2 equivalents). The reaction mixture was stirred at room temperature until completion, and then purified by HPLC to obtain TAZ-L-1 (0.057 g, 0.051 mmol, 52%). LC / MS [M+H] 1110.48 (calculated); LC / MS [M+H] 1110.87 (measured).

[0364] Example L-2 Synthesis of 2,3,5,6-tetrafluorophenyl (E)-41-(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-35-((3-cyanophenyl))imino)-4,7,10,13,16,19,22,25,28,31-decaoxa-34,36,41-triazatetratetracont-38-enoate, TAZ-L-2 [Chemical Formula] tert-Butyl (E)-41-(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-35-((3-cyanophenyl)imino)-4,7,10,13,16,19,22,25,28,31-decaoxa-34,36,41-triazatetratetracont-38-enoate, L-2a TAZ-17 (0.05 g, 0.16 mmol, 1 equivalent) and tert-butyl 1-((3-cyanophenyl)imino)-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azapentatriacont-1-en-35-oate, PEG10-diimide (0.112 g, 0.16 mmol, 1 equivalent) were dissolved in DMF. Triethylamine (0.066 ml, 0.47 mmol, 3 equivalents) was added and the reaction mixture was stirred at ambient temperature. When the amine starting material was consumed, the reaction mixture was concentrated and purified by HPLC to afford L-2a (0.120 g, 0.12 mmol, 74%). LC / MS [M+H] 1028.54 (calculated); LC / MS [M+H] 1028.92 (measured).

[0365] (E)-41-(5-Amino-6H-thieno[3,2-b]azepine-7-carbonyl)-35-((3-cyanophenyl)imino)-4,7,10,13,16,19,22,25,28,31-decaoxa-34,36,41-triazatetratetracont-38-enoic acid, Preparation of L-2b L-2a (0.120 g, 0.12 mmol, 1 equivalent) was dissolved in 100 μl of TFA. After 15 minutes, the product was concentrated and purified by HPLC to afford L-2b (84.9 mg, 0.087 mmol, 75%). LC / MS [M+H] 972.47 (calculated); LC / MS [M+H] 972.83 (measured).

[0366] Preparation of TAZ-L-2 L-2b (0.085 g, 0.087 mmol, 1 equiv) and TFP (0.029 g, 0.17 mmol, 2 equiv) were dissolved in DMF. Collidine (0.058 ml, 0.44 mmol, 5 equiv) was added, followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC-HCl (0.033 g, 0.17 mmol, 2 equiv). The reaction was stirred at room temperature until completion and then purified by HPLC to obtain TAZ-L-2 (0.057 g, 0.055 mmol, 62%). LC / MS [M+H] 1120.47 (calculated); LC / MS [M+H] 1120.85 (measured).

[0367] Example L-3 Synthesis of 2,3,5,6-tetrafluorophenyl 39-(5-amino-7-(dipropylcarbamoyl)-6H-thieno[3,2-b]azepin-2-yl)-34-methyl-4,7,10,13,16,19,22,25,28,31-decaoxa-34-azanonatriacontanoate, TAZ-L-3

Chemical formula

Claims

1. An immunoconjugate comprising an antibody covalently attached to one or more 5-aminothienoazepine moieties by a linker and having the formula I: Ab−[L-TAZ] ｐ I or a pharmaceutically acceptable salt thereof wherein Ab is said antibody; p is an integer from 1 to 8; TAZ is a 5-aminothienoazepine moiety having the formula: 【Chemical 1】 and R １ 、R ２ 、R ３ 、and R ４ are independently selected from the group consisting of H, C １ -C １２ alkyl, C ２ -C ６ alkenyl, C ２ -C ６ alkynyl, C ３ -C １２ carbocyclic, C ６ -C ２０ aryl, C ２ -C ９ heterocyclic, and C １ -C ２０ heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclic, aryl, heterocyclic, and heteroaryl are -(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C １ -C １２ alkyldiyl)-OR ５ -(C ３ -C １２ carbocyclyl); -(C ３ -C １２ carbocyclic)- ＊ ; -(C ３ -C １２ -carbocyclyl)-(C １ -C １２ -alkyldiyl)-NR ５ - ＊ ; -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyl diyl)-N(R ５ ) ２ ; -(C ３ -C １２ carbocyclyl)-NR ５ -C(=NR ５ )NR ５ - ＊ ; -(C ６ -C ２０ aryl); -(C ６ -C ２０ aryl)- ＊ ; -(C ６ -C ２０ aryl diyl)-N(R ５ )- ＊ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-(C ２ -C ２０ heterocyclicdiyl)- ＊ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-NR ５ -C(=NR ５ａ )N(R ５ )- ＊ ; -(C ２ -C ２０ heterocyclyl); -(C ２ -C ２０ heterocyclyl)- ＊ ; -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyl diyl)-NR ５ - ＊ ; -(C ２ -C ９ -heterocyclyl)-(C １ -C １２ -alkyldiyl)-N(R ５ ) ２ ; -(C ２ -C ９ -heterocyclyl)-C(=O)-(C １ -C １２ -alkyl diyl)-N(R ５ )- ＊ ; -(C ２ -C ９ -heterocyclyl)-NR ５ -C(=NR ５ａ )NR ５ - ＊ ; -(C ２ -C ９ heterocyclyl)-NR ５ -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C ２ -C ９ -heterocyclyl)-(C ６ -C ２０ -aryldiyl)- ＊ ; -(C １ -C ２０ heteroaryl); -(C １ -C ２０ heteroaryl)- ＊ ; -(C １ -C ２０ -heteroaryl)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C ２０ -heteroaryl)-(C １ -C １２ -alkyldiyl)-N(R ５ ) ２ ; -(C １ -C ２０ -heteroaryl)-NR ５ -C(=NR ５ａ ))N(R ５ )- ＊ ; -(C １ -C ２０ -heteroaryl)-N(R ５ )(C=O)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -C(=O)- ＊ ; -C(=O)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -C(=O)-(C ２ -C ２０ -heterocyclic diyl)- ＊ ; -C(=O)N(R ５ ) ２ ; -C(=O)N(R ５ )- ＊ ; -C(=O)N(R ５ )-(C １ -C １２ -alkyldiyl)-N(R ５ )(=O)R ５ ; -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)N(R ５ ) ２ ; -C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-N(R ５ )CO ２ R ５ ; -C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-N(R ５ )(C=NR ５ａ )(N(R ５ ) ２ ; -C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-NR ５ C(=NR ５ａ )R ５ ; -C(=O)NR ５ -(C １ -C ８ -alkyldiyl)-NR ５ (C ２ -C ５ -heteroaryl); -C(=O)NR ５ -(C １ -C ２０ -heteroaryldiyl)-N(R ５ )- ＊ ; -C(=O)NR ５ -(C １ -C ２０ -heteroaryldiyl)- ＊ ; -C(=O)NR ５ -(C １ -C ２０ -heteroaryldiyl)-(C １ -C １２ -alkyldiyl)N(R ５ ) ２ ; -C(=O)NR ５ -(C １ -C ２０ -heteroaryldiyl)-(C ２ -C ２０ -heterocyclyldiyl)-C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-NR ５ - ＊ ; -N(R ５ ) ２ ; -N(R ５ )- ＊ ; -N(R ５ ),C(=O)R ５ ; -N(R ５ )(=O)- ＊ ; -N(R ５ )(C=O)N(R ５ ); ２ ; -N(R ５ )(C=O)N(R ５ )- ＊ ; -N(R ５ )CO ２ R ５ ; -NR ５ C(=NR ５ａ )N(R ５ ) ２ ; -NR ５ C(=NR ５ａ )N(R ５ )- ＊ ; -NR ５ C(=NR ５ａ )R ５ ; -N(R ５ )(C=O)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -N(R ５ )-(C ２ -C ５ heteroaryl); -N(R ５ )-S(=O) ２ -(C １ -C １２ alkyl); -O-(C １ -C １２ alkyl); -O-(C １ -C １２ -alkyl-diyl)-N(R ５ ) ２ ; -O-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -S(=O) ２ -(C ２ -C ２０ -heterocyclic diyl)- ＊ ; -S(=O) ２ -(C ２ -C ２０ -heterocyclic diyl)-(C １ -C １２ -alkyl diyl)-N(R ５ ) ２ ; -S(=O) ２ -(C ２ -C ２０ -heterocyclic diyl)-(C １ -C １２ -alkyl diyl)-NR ５ - ＊ ; and -S(=O) ２ -(C ２ -C ２０ -heterocyclyldiyl)-(C １ -C １２ -alkyldiyl)-OH; and is independently optionally substituted with one or more groups selected from; or R ２ and R ３ together form a 5- or 6-membered heterocyclyl ring; X １ , X ２ , X ３ , and X ４ is independently selected from the group consisting of a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N(R ５ ); R ５ is selected from the group consisting of H, C ６ -C ２０ -aryl, C ３ -C １２ -carbocyclic, C ６ -C ２０ -aryldiyl, C １ -C １２ -alkyl, and C １ -C １２ -alkyldiyl, or two R ５ groups together form a 5- or 6-membered heterocyclic ring; R ５ａ is selected from the group consisting of C ６ -C ２０ -aryl and C １ -C ２０ -heteroaryl; Here, the asterisk ＊ indicates the binding site of L, where R １ , R ２ , R ３ and R ４ one of which is bound to L; L is -C(=O)-(PEG)-; -C(=O)-(PEG)-C(=O)-; -C(=O)-(PEG)-O-; -C(=O)-(PEG)-C(=O)-(PEP)-; -C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ alkanediyl); -C(=O)-(PEG)-C(=O)N(R ５ )(-C １ -C １２ -alkyldiyl)-N(R ５ )(=O)-C(=O)-(C ２ -C ５ -monoheterocyclyldiyl)-; -C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ -alkyldiyl)-(Mcgluc)-; -C(=O)-(PEG)-C(=O)-(Mcgluc)-; -C(=O)-(PEG)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ alkyldiyl); -C(=O)-(PEG)-C(=O)-(PEP)-N(R ５ )(C １ -C １２ -alkyl diyl)-N(R ５ )(C=O)-(C ２ -C ５ -monoheterocyclyl diyl)-; -C(=O)-(PEG)-N(R ５ )-; -C(=O)-(PEG)-N(R ５ )C(=O)-; -C(=O)-(PEG)-N(R ５ )-(PEG)-C(=O)-(PEP)-; -C(=O)-(PEG)-N ＋ (R ５ ) ２ -(PEG)-C(=O)-(PEP)-; -C(=O)-(PEG)-C(=O)-N(R ５ )CH(AA １ )C(=O)-(PEG)-C(=O)-(PEP)-; -C(=O)-(PEG)-C(=O)-N(R ５ )(CH(AA １ ))C(=O)-N(R ５ )-(C １ -C １２ alkyldiyl)-; -C(=O)-(PEG)-SS-(C １ -C １２ -alkyl diyl)-OC(=O)-; -(C(=O)-(PEG)-SS-(C １ -C １２ -alkyl diyl)-C(=O)-; -C(=O)-(C １ -C １２ -alkyldiyl)-C(=O)-(PEP)-; -C(=O)-(C １ -C １２ -alkyldiyl)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ -alkyldiyl)-; -C(=O)-(C １ -C １２ -alkyldiyl)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ -alkyldiyl)-N(R ５ )-C(=O); -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-; -C(=O)-CH ２ CH ２ OCH ２ CH ２ -(C １ -C ２０ heteroaryldiyl)-CH ２ O-(PEG)-C(=O)-(Mcgluc)-; -C(=O)-CH ２ CH ２ OCH ２ CH ２ -(C １ -C ２０ heteroaryldiyl)-CH ２ O-(PEG)-C(=O)-(Mcgluc)-N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )(C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-; and -(Succinimidyl)-(CH ２ ) ｍ -C(=O)-(PEP)-N(R ５ )-(C １ -C １２ Alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ Monocyclic heterocyclyldiyl)-selected from the group consisting of; Here, PEG has the formula: -(CH ２ CH ２ O) ｎ -(CH ２ ) ｍ -; m is an integer from 1 to 5, and n is an integer from 2 to 50; PEP is a formula: [Chemical Formula 2] having, wherein AA １ and AA ２ is independently selected from amino acid side chains, or AA １ or AA ２ and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the bonding point; R ６ is selected from the group consisting of C ６ -C ２０ -aryl diyl and C １ -C ２０ -heteroaryl diyl, -CH ２ O-C(=O)- and optionally: 【Chemical Formula 3】 substituted by: and Mcgluc is a group: 【Chemical Formula 4】 selected from, where q is from 1 to 8 and AA is an amino acid side chain; Here, alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl, and heteroaryldiyl are F, Cl, Br, I, -CN, -CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH(CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH ３ ) ２ , -CO ２ H, -COCH ３ , -CO ２ CH ３ , -CO ２ C(CH ３ ) ３ , -COCH(OH)CH ３ , -CONH ２ , -CONHCH ３ , -CON(CH ３ ) ２ , -C(CH ３ ) ２ CONH ２ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , -NHCOCH ３ , -N(CH ３ )COCH ３ , -NHSO ２ CH ３ , -N(CH ３ )C(CH ３ ) ２ CONH ２ , -N(CH ３ )CH ２ CH ２ SO ２ CH ３ , -NHC(=NH)H, -NHC(=NH)CH ３ , -NHC(=NH)NH ２ , -NHC(=O)NH ２ , -NO ２ , =O, -OH, -OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ , -OCH ２ CH ２ , -N(CH ３ ) ２ , -O(CH ２ CH ２ O) ｎ -(CH ２ ) ｍ CO ２ H, -O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N(CH ３ ), ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ one or more groups independently selected from H and optionally substituted).

2. The immunoconjugate according to claim 1, wherein the antibody is an antibody construct having an antigen-binding domain that binds to a target selected from PD-L1, HER2, and CEA.

3. The immunoconjugate according to claim 2, wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and ravulizumab, or a biosimilar or bio-better thereof.

4. PEP has the formula: 【Chemical Formula 5】 and wherein AA １ and AA ２ are independently selected from the side chains of naturally occurring amino acids, or AA １ or AA ２ forms a 5-membered ring proline amino acid, the immunoconjugate according to any one of claims 1 to 3.

5. PEP has the formula: 【Chemical Formula 6】 The immunoconjugate according to any one of claims 1 to 3.

6. Mcgluc has the formula: 【Chemical Formula 7】 The immunoconjugate according to any one of claims 1 to 3.

7. AA １ and AA ２ are independently selected from H, -CH ３ , -CH(CH ３ ) ２ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NH C(NH)NH ２ , -CHCH(CH ３ )CH ３ , -CH ２ SO ３ H, and -CH ２ CH ２ CH ２ NH C(O)NH2, the immunoconjugate according to any one of claims 1 to 3.

8. AA １ is -CH(CH ３ ) ２ and AA ２ is -CH ２ CH ２ CH ２ NH C(O)NH ２ The immunoconjugate according to claim 7, wherein

9. AA １ and AA ２ are independently selected from GlcNAc aspartic acid, -CH ２ SO ３ H, and -CH ２ OPO ３ H, and the immunoconjugate according to any one of claims 1 to 3.

10. X １ is a bond, and R １ is H, the immunoconjugate according to any one of claims 1 to 3.

11. X ２ is a bond, and R ２ is C １ -C ８ is alkyl, the immunoconjugate according to any one of claims 1 to 3.

12. X ２ and X ３ are each a bond, R ２ and R ３ is C １ -C ８ -alkyl, -O-(C １ -C １２ -alkyl), -(C １ -C １２ -alkyldiyl)-OR ５ , -(C １ -C ８ -alkyldiyl)-N(R ５ ),CO ２ R ５ , and -O-(C １ -C １２ -alkyl)-N(R ５ ),CO ２ R ５ independently selected from, the immunoconjugate according to any one of claims 1 to 3.

13. R ２ and R ３ are each independently selected from -CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -CH ２ CH ２ CF ３ , and -CH ２ CH ２ CH ２ OH, the immunoconjugate according to claim 12.

14. R ２ is C １ -C ８ alkyl, and R ３ is -(C １ -C ８ alkyldiyl)-N(R ５ )CO ２ R ４ The immunoconjugate according to claim 12, wherein

15. R ２ is -CH ２ CH ２ CH ３ and R ３ is -CH ２ CH ２ CH ２ NHCO ２ (t-Bu), the immunoconjugate according to claim 14.

16. R ２ and R ３ are each —CH ２ CH ２ CH ３ The immunoconjugate according to claim 12, wherein the immunoconjugate is

17. X ３ -R ３ The immunoconjugate according to any one of claims 1 to 3, wherein X-R is selected from the group consisting of: 【Chemical Formula 8】

18. R ２ and R ３ one of which is -(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-O-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ -alkyldiyl)-N(R ５ )(=NR ５ )-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyl diyl)-(C ６ -C ２０ aryl diyl)-(C １ -C １２ alkyl diyl)-N(R ５ )-C(=NR ５ )N(R ５ )- ＊ ; -(C ２ -C ６ alkynyl diyl)-N(R ５ )- ＊ ; and -(C ２ -C ６ alkynyl diyl)-N(R ５ )(=NR ５ )(R ５ )- ＊ ; selected from X ２ and X ３ are linked, where the asterisk ＊ indicates the binding site of L, the immunoconjugate according to any one of claims 1 to 3.

19. The immunoconjugate according to any one of claims 1 to 3, wherein L is selected from the group consisting of: -C(=O)-(PEG)-; -C(=O)-(PEG)-C(=O)-; -C(=O)-(PEG)-O-; -C(=O)-(PEG)-N(R ５ )-; and -C(=O)-(PEG)-N(R ５ )C(=O)-。

20. The immunoconjugate according to any one of claims 1 to 3, selected from the formulas Ia to Ih: 【Chemical Formula 9】 【Chem.】

21.

22. R ２ and R ３ are C １ -C ８ alkyl, -O-(C １ -C １２ alkyl), -(C １ -C １２ alkanediyl)-OR ５ , -(C １ -C ８ alkanediyl)-N(R ５ ),CO ２ R ５ , and -O-(C １ -C １２ alkyl)-N(R ５ ),CO ２ R ５ The immunoconjugate according to claim 20, which is independently selected from

23. R ２ and R ３ are each independently selected from -CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -CH ２ CH ２ CF ３ , and -CH ２ CH ２ CH ２ OH, the immunoconjugate according to claim 21 5-aminothienoazepine-linker compound of formula II L is 【Chemical 11】 (wherein, R １ , R ２ , R ３ , and R ４ one of which is connected to L; R １ 、 R ２ 、 R ３ 、 and R ４ are independently selected from the group consisting of H, C １ -C １２ alkyl, C ２ -C ６ alkenyl, C ２ -C ６ alkynyl, C ３ -C １２ carbocyclic, C ６ -C ２０ aryl, C ２ -C ９ heterocyclic, and C １ -C ２０ heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclic, aryl, heterocyclic, and heteroaryl are -(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C １ -C １２ alkyldiyl)-OR ５ -(C ３ -C １２ carbocyclic); -(C ３ -C １２ carbocyclic)- ＊ ; -(C ３ -C １２ Carbocyclyl)-(C １ -C １２ Alkyldiyl)-NR ５ - ＊ ; -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C ３ -C １２ carbocyclyl)-NR ５ -C(=NR ５ )NR ５ - ＊ ; -(C ６ -C ２０ aryl); -(C ６ -C ２０ aryl)- ＊ ; -(C ６ -C ２０ aryldiyl)-N(R ５ )- ＊ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-(C ２ -C ２０ heterocyclicdiyl)- ＊ ; -(C ６ -C ２０ arylene diyl)-(C １ -C １２ alkylene diyl)-N(R ５ ) ２ ; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-NR ５ -C(=NR ５ａ ))N(R ５ )- ＊ ; -(C ２ -C ２０ heterocyclyl); -(C ２ -C ２０ heterocyclyl)- ＊ ; -(C ２ -C ９ -heterocyclyl)-(C １ -C １２ -alkyl diyl)-NR ５ - ＊ ; -(C ２ -C ９ -heterocyclyl)-(C １ -C １２ -alkyldiyl)-N(R ５ ) ２ ; -(C ２ -C ９ -heterocyclyl)-C(=O)-(C １ -C １２ -alkyl diyl)-N(R ５ )- ＊ ; -(C ２ -C ９ -heterocyclyl)-NR ５ -C(=NR ５ａ )NR ５ - ＊ ; -(C ２ -C ９ heterocyclyl)-NR ５ -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C ２ -C ９ heterocyclyl)-(C ６ -C ２０ aryldiyl)- ＊ ; -(C １ -C ２０ heteroaryl); -(C １ -C ２０ heteroaryl)- ＊ ; -(C １ -C ２０ -heteroaryl)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C ２０ -heteroaryl)-(C １ -C １２ -alkyldiyl)-N(R ５ ) ２ ; -(C １ -C ２０ -heteroaryl)-NR ５ -C(=NR ５ａ ))N(R ５ )- ＊ ; -(C １ -C ２０ -heteroaryl)-N(R ５ )(C=O)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -C(=O)- ＊ ; -C(=O)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -C(=O)-(C ２ -C ２０ -heterocyclic diyl)- ＊ ; -C(=O)N(R ５ ) ２ ; -C(=O)N(R ５ )- ＊ ; -C(=O)N(R ５ )-(C １ -C １２ -alkyldiyl)-N(R ５ )C(=O)R ５ ; -C(=O)N(R ５ )-(C １ -C １２ -alkyldiyl)-N(R ５ )(=O)N(R ５ ) ２ ; -C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-N(R ５ )CO ２ R ５ ; -C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-N(R ５ )(C=NR ５ａ )(N(R ５ ) ２ ; -C(=O)NR ５ -(C １ -C １２ -alkyldiyl)-NR ５ C(=NR ５ａ )R ５ ; -C(=O)NR ５ -(C １ -C ８ -alkyldiyl)-NR ５ (C ２ -C ５ -heteroaryl); -C(=O)NR ５ -(C １ -C ２０ -heteroaryldiyl)-N(R ５ )- ＊ ; -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)- ＊ ; -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-(C １ -C １２ alkyldiyl)N(R ５ ) ２ ; -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-(C ２ -C ２０ heterocyclyldiyl)-C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５ - ＊ ; -N(R ５ ) ２ ; -N(R ５ )- ＊ ; -N(R ５ )(C=O)R ５ ; -N(R ５ )(=O)- ＊ ; -N(R ５ )(=O)N(R ５ ); ２ ; -N(R ５ )(=O)N(R ５ )- ＊ ; -N(R ５ )CO ２ R ５ ; -NR ５ C(=NR ５ａ )N(R ５ ) ２ ; -NR ５ C(=NR ５ａ )N(R ５ )- ＊ ; -NR ５ C(=NR ５ａ )R ５ ; -N(R ５ )(=O)-(C １ -C １２ -alkyldiyl)-N(R ５ )- ＊ ; -N(R ５ ),-(C ２ -C ５ heteroaryl); -N(R ５ )-S(=O) ２ -(C １ -C １２ alkyl); -O-(C １ -C １２ -alkyl); -O-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -O-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -S(=O) ２ -(C ２ -C ２０ -heterocyclyldiyl)- ＊ ; -S(=O) ２ -(C ２ -C ２０ -heterocyclic diyl)-(C １ -C １２ -alkyl diyl)-N(R ５ ) ２ ; -S(=O) ２ -(C ２ -C ２０ -heterocyclyldiyl)-(C １ -C １２ -alkyldiyl)-NR ５ - ＊ ; and -S(=O) ２ -(C ２ -C ２０ -heterocyclic diyl)-(C １ -C １２ -alkyl diyl)-OH; and is independently optionally substituted with one or more groups selected from; or R ２ and R ３ together form a 5- or 6-membered heterocyclic ring; X １ , X ２ , X ３ , and X ４ is independently selected from the group consisting of a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N(R ５ ); R ５ is H, C ６ -C ２０ -aryl, C ３ -C １２ -carbocyclic, C ６ -C ２０ -aryldiyl, C １ -C １２ -alkyl, and C １ -C １２ -alkyldiyl, or is selected from the group consisting of; or two R ５ groups together form a 5- or 6-membered heterocyclic ring; R ５ａ is selected from the group consisting of C ６ -C ２０ -aryl and C １ -C ２０ -heteroaryl; Here, the asterisk ＊ indicates the binding site of L, where R １ , R ２ , R ３ and R ４ of which one is bound to L; Q-C(=O)-(PEG)-; Q-C(=O)-(PEG)-C(=O)-; Q-C(=O)-(PEG)-O-; Q-C(=O)-(PEG)-C(=O)-(PEP)-; ​ Q-C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ alkanediyl)-; Q-C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ monocyclic heterodiyl)-; Q-C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-(Mcgluc)-; Q-C(=O)-(PEG)-C(=O)-(Mcgluc)-; Q-C(=O)-(PEG)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ alkyl diyl); Q-C(=O)-(PEG)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ -alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ -monoheterocyclyldiyl)-; Q-C(=O)-(PEG)-N(R ５ )-; Q-C(=O)-(PEG)-N(R ５ )C(=O)-; Q-C(=O)-(PEG)-N(R ５ )-(PEG)-C(=O)-(PEP)-; Q-C(=O)-(PEG)-N ＋ (R ５ ) ２ -(PEG)-C(=O)-(PEP)-; Q-C(=O)-(PEG)-C(=O)-N(R ５ ),CH(AA １ ),C(=O)-(PEG)-C(=O)-(PEP)-; Q-C(=O)-(PEG)-C(=O)-N(R ５ )(CH(AA １ )(C(=O)-N(R ５ )-(C １ -C １２ alkyldiyl)-; Q-C(=O)-(PEG)-SS-(C １ -C １２ -alkyl diyl)-OC(=O)-; Q-C(=O)-(PEG)-SS-(C １ -C １２ -alkyl diyl)-C(=O)-; Q-C(=O)-(C １ -C １２ -alkyldiyl)-C(=O)-(PEP)-; Q-C(=O)-(C １ -C １２ alkanediyl)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ alkanediyl)-; Q-C(=O)-(C １ -C １２ -alkyl diyl)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ -alkyl diyl)-N(R ５ )-C(=O); Q-C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-(PEP)-N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-; Q-C(=O)-CH ２ CH ２ OCH ２ CH ２ -(C １ -C ２０ heteroaryldiyl)-CH ２ O-(PEG)-C(=O)-(Mcgluc)-; Q-C(=O)-CH ２ CH ２ OCH ２ CH ２ -(C １ -C ２０ heteroaryldiyl)-CH ２ O-(PEG)-C(=O)-(Mcgluc)-N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )(C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-; and Q-(CH ２ ) ｍ -C(=O)-(PEP)-N(R ５ )-(C １ -C １２ -alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ -monoheterocyclyldiyl)- selected from the group consisting of; Here, PEG has the formula: -(CH ２ CH ２ O) ｎ -(CH ２ ) ｍ - and m is an integer from 1 to 5, and n is an integer from 2 to 50; PEP has the formula: 【Chemical 12】 has, wherein AA １ and AA ２ is independently selected from amino acid side chains, or AA １ or AA ２ and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the bonding point; R ６ is selected from the group consisting of C ６ -C ２０ -aryl diyl and C １ -C ２０ -heteroaryl diyl, -CH ２ O-C(=O)- and optionally: 【Chemical 13】 is substituted with: and Mcgluc is the group: 【Chemical Formula 14】 selected from, where q is from 1 to 8 and AA is an amino acid side chain; Q is selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, which are substituted with one or more groups independently selected from F, Cl, NO ２ and SO ３ － ; Here, alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are F, Cl, Br, I, -CN, -CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH(CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH ３ ) ２ , -CO ２ H, -COCH ３ , -CO ２ CH ３ , -CO ２ C(CH ３ ) ３ , -COCH(OH)CH ３ , -CONH ２ , -CONHCH ３ , -CON(CH ３ ) ２ , -C(CH ３ ) ２ CONH ２ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , -NHCOCH ３ , -N(CH ３ )COCH ３ , -NHSO ２ CH ３ , -N(CH ３ )C(CH ３ ) ２ CONH ２ , -N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ , -NHC(=NH)H, -NHC(=NH)CH ３ , -NHC(=NH)NH ２ , -NHC(=O)NH ２ , -NO ２ , =O, -OH, -OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ , -OCH ２ CH ２ , -N(CH ３ ) ２ , -O(CH ２ CH ２ O) ｎ , -(CH ２ ) ｍ CO ２ H, -O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N(CH ３ ), ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ one or more groups independently selected from H and optionally substituted).

24. PEP has the formula: 【Chemical Formula 15】 and has wherein AA １ and AA ２ is independently selected from the side chains of naturally occurring amino acids, or AA having an adjacent nitrogen atom １ or AA ２ forms a 5-membered ring to form a proline amino acid, the 5-amino-thienoazepine-linker compound according to claim 23.

25. PEP has the formula: 【Chemical Formula 16】 The 5-amino-thienoazepine-linker compound according to claim 23, having

26. MCgluc has the formula: 【Chemical 17】 The 5-amino-thienoazepine-linker compound according to claim 23, having

27. AA １ and AA ２ are independently selected from H, -CH ３ , -CH(CH ３ ) ２ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ NH ２ , -CH ２ CH ２ CH ２ NH C(NH)NH ２、 -CH CH(CH ３ )CH ３ , -CH ２ SO ３ H, and -CH ２ CH ２ CH ２ NH C(O)NH ２ The 5-amino-thienoazepine-linker compound according to claim 23, wherein the compound is independently selected from the above groups.

28. AA １ is -CH(CH ３ ) ２ and AA ２ is -CH ２ CH ２ CH ２ NH C(O)NH ２ The 5-amino-thienoazepine-linker compound according to claim 27, wherein

29. AA １ and AA ２ are independently selected from GlcNAc asparagine, -CH ２ SO ３ H, and -CH ２ OPO ３ H, the 5-amino-thienoazepine-linker compound according to claim 24.

30. X １ is a bond, and R １ is H, the 5-amino-thienoazepine-linker compound according to claim 23.

31. X ２ is a bond, and R ２ is C １ -C ８ alkyl, the 5-amino-thienoazepine-linker compound according to claim 23.

32. X ２ and X ３ are each a bond, R ２ and R ３ is C １ -C ８ alkyl, -O-(C １ -C １２ alkyl), -(C １ -C １２ alkanediyl)-OR ５ -(C １ -C ８ alkanediyl)-N(R ５ ),CO ２ R ５ and -O-(C １ -C １２ alkyl)-N(R ５ ),CO ２ R5, independently selected from, the 5-amino-thienoazepine-linker compound according to claim 23.

33. R ２ and R ３ are each independently selected from -CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -CH ２ CH ２ CF ３ , and -CH ２ CH ２ CH ２ OH, the 5-amino-thienoazepine-linker compound according to claim 32.

34. R ２ is C １ -C ８ alkyl, and R ３ is -(C １ -C ８ alkyldiyl)-N(R ５ )CO ２ R ４ The 5-amino-thienoazepine-linker compound according to claim 32, wherein

35. R ２ is -CH ２ CH ２ CH ３ and R ３ is -CH ２ CH ２ CH ２ NHCO ２ (t-Bu), the 5-amino-thienoazepine-linker compound according to claim 34.

36. R ２ and R ３ are each —CH ２ CH ２ CH ３ The 5-amino-thienoazepine-linker compound according to claim 33, wherein is

37. X ３ -R ３ The 5-amino-thienoazepine-linker compound according to claim 23, wherein X-R is selected from the group consisting of: 【Chemical Formula 18】

38. R ２ and R ３ One of them is: -(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-O-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-N(R ５ )(=NR ５ )-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )- ＊ ; -(C １ -C １２ alkyldiyl)-(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )-C(=NR ５ )N(R ５ )- ＊ ; -(C ２ -C ６ alkynyl diyl)-N(R ５ )- ＊ ; and -(C ２ -C ６ alkynyl diyl)-N(R ５ )(=NR ５ )(R ５ )- ＊ ; selected from X ２ and X ３ are bonded, and the asterisk ＊ indicates the binding site of L. The 5-amino-thienoazepine-linker compound according to claim 23

39. L is: Q-C(=O)-(PEG)-; Q-C(=O)-(PEG)-C(=O)-; Q-C(=O)-(PEG)-O-; Q-C(=O)-(PEG)-N(R ５ )-; and Q-C(=O)-(PEG)-N(R ５ )C(=O)- The 5-amino-thienoazepine-linker compound according to claim 23, selected from the group consisting of

40. Formulas IIa to IIh: 【Chemical 19】 【Chem.】 The 5-amino-thienoazepine-linker compound according to claim 23, selected from

41. R ２ and R ３ are C １ -C ８ alkyl, -O-(C １ -C １２ alkyl), -(C １ -C １２ alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ),CO ２ R ５ , and -O-(C １ -C １２ alkyl)-N(R ５ ),CO ２ R ５ and are independently selected from the 5-amino-thienoazepine-linker compound according to claim 40.

42. R ２ and R ３ are each independently selected from -CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -CH ２ CH ２ CF ３ , and -CH ２ CH ２ CH ２ OH, the 5-amino-thienoazepine-linker compound according to claim 41.

43. Q is selected from the following for the 5-amino-thienoazepine-linker compound according to claim 23: 【Chemical 21】

44. The 5-amino-thienoazepine-linker compound according to claim 23, wherein Q is phenoxy-substituted with one or more Fs

45. The 5-amino-thienoazepine-linker compound according to claim 44, wherein Q is 2,3,5,6-tetrafluorophenoxy

46. The 5-amino-thienoazepine-linker compound according to claim 23, selected from Table 2a and Table 2b below

47. The immunoconjugate according to claim 1, prepared by binding the 5-amino-thienoazepine-linker compound according to claim 46 to an antibody

48. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate according to claim 1 and one or more pharmaceutically acceptable diluents, vehicles, carriers or excipients

49. A pharmaceutical composition for treating cancer, comprising the immunoconjugate according to any one of claims 1 to 3, wherein the cancer is selected from bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer and breast cancer

50. A method for preparing an immunoconjugate of Formula I according to claim 1, comprising conjugating the 5-amino-thienoazepine-linker compound of Formula II according to claim 23 with an antibody.

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