Aminobenzazepine compounds, immunoconjugates, and uses thereof

Abstract

To provide a medicament comprising an immunoconjugate for treating cancer.SOLUTION: An immunoconjugate comprising an antibody covalently attached by a linker to one or more aminobenzazepine moieties, having formula I: Ab-[L-Bza]p or a pharmaceutically acceptable salt thereof. (In formula I, Ab is the antibody; p is an integer from 1 to 8; and Bza is the aminobenzazepine moiety having the following formula.)SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This non-provisional application claims the benefit of priority to U.S. Provisional Application No. 62 / 963,884, filed January 21, 2020, and U.S. Provisional Application No. 62 / 861,139, filed June 13, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference herein in its entirety. The ASCII copy, created on June 2, 2020, is designated 17019_002WO1_SL.txt and is 299,523 bytes in size.

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

[0004] New compositions and methods for delivering antibodies and immune adjuvants are needed to reach inaccessible tumors and / or expand treatment options for cancer patients and other subjects. The present invention provides such compositions and methods. Summary of the Invention

[0005] The present disclosure is generally directed to immunoconjugates comprising an antibody linked by conjugation to one or more aminobenzazepine derivatives. The present invention further relates to aminobenzazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions are suitable substrates for the formation of immunoconjugates in which antibodies can be covalently attached to one or more aminobenzazepine derivatives via a linker or linking moiety. The present invention is further directed to the use of such immunoconjugates in the treatment of disease, particularly cancer.

[0006] One aspect of the invention is an immunoconjugate comprising an antibody covalently bound to a linker that is covalently bound to one or more aminobenzazepine moieties.

[0007] Another aspect of the present invention is aminobenzazepine-linker compounds.

[0008] Another aspect of the 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 aminobenzazepine moieties.

[0009] Another aspect of the invention is the use of an immunoconjugate comprising an antibody linked by conjugation to one or more aminobenzazepine moieties to treat cancer.

[0010] Another aspect of the invention is a method for preparing an immunoconjugate by conjugation of one or more aminobenzazepine moieties to an antibody. [Brief explanation of the drawings]

[0011] [Figure 1A] The heavy and light chain CDRs of PD-L1 type A binders 1-42 are shown. [Figure 1B] The heavy and light chain CDRs of PD-L1 type A binders 1-42 are shown. [Figure 1C] The heavy and light chain CDRs of PD-L1 type A binders 1-42 are shown. [Figure 1D] The heavy and light chain CDRs of PD-L1 type A binders 1-42 are shown. [Figure 2A] The first (HFW1), second (HFW2), third (HFW3), and fourth (HFW4) heavy chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 2B] The first (HFW1), second (HFW2), third (HFW3), and fourth (HFW4) heavy chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 2C] The first (HFW1), second (HFW2), third (HFW3), and fourth (HFW4) heavy chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 2D] The first (HFW1), second (HFW2), third (HFW3), and fourth (HFW4) heavy chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 3A] The first (LFW1), second (LFW2), third (LFW3), and fourth (LFW4) light chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 3B] The first (LFW1), second (LFW2), third (LFW3), and fourth (LFW4) light chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 3C] The first (LFW1), second (LFW2), third (LFW3), and fourth (LFW4) light chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 3D] The first (LFW1), second (LFW2), third (LFW3), and fourth (LFW4) light chain framework region polypeptides of PD-L1 type A binders 1-42 are shown. [Figure 4A] The heavy chain variable regions (VH) of PD-L1 type A binders 1-42 are shown. [Figure 4B] The heavy chain variable regions (VH) of PD-L1 type A binders 1-42 are shown. [Figure 4C] The heavy chain variable regions (VH) of PD-L1 type A binders 1-42 are shown. [Figure 4D] The heavy chain variable regions (VH) of PD-L1 type A binders 1-42 are shown. [Figure 4E] The light chain variable region (VL) of PD-L1 type A binder 1-42 is shown. [Figure 4F] The light chain variable region (VL) of PD-L1 type A binder 1-42 is shown. [Figure 4G] The light chain variable region (VL) of PD-L1 type A binder 1-42 is shown. [Figure 5A] The heavy and light chain CDRs of PD-L1 type B binders 1-21 are shown. [Figure 5B] The heavy and light chain CDRs of PD-L1 type B binders 1-21 are shown. [Figure 6A] The first (HFW1), second (HFW2), third (HFW3), and fourth (HFW4) heavy chain framework region polypeptides of PD-L1 type B binders 1-21 are shown. [Figure 6B] The first (HFW1), second (HFW2), third (HFW3), and fourth (HFW4) heavy chain framework region polypeptides of PD-L1 type B binders 1-21 are shown. [Figure 7A] The first (LFW1), second (LFW2), third (LFW3), and fourth (LFW4) light chain framework region polypeptides of PD-L1 type B binders 1-21 are shown. [Figure 7B] The first (LFW1), second (LFW2), third (LFW3), and fourth (LFW4) light chain framework region polypeptides of PD-L1 type B binders 1-21 are shown. [Figure 8A] The heavy chain variable regions (VH) of PD-L1 type B binders 1 to 21 are shown. [Figure 8B] The heavy chain variable regions (VH) of PD-L1 type B binders 1 to 21 are shown. [Figure 8C] The light chain variable region (VL) of PD-L1 type B binder 1-21 is shown. [Figure 8D] The light chain variable region (VL) of PD-L1 type B binder 1-21 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0013] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and the present invention is in no way limited to the methods and materials described.

[0014] definition The term "immunoconjugate" refers to an antibody construct covalently attached to an adjuvant moiety via a linker. The term "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant. The phrase "adjuvant moiety" refers to an adjuvant covalently attached to an antibody construct, e.g., via a linker, as described herein. The adjuvant moiety is capable of eliciting an immune response while attached to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0015] An "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant. The phrase "adjuvant moiety" refers to an adjuvant that is covalently attached to an antibody construct, e.g., via a linker, as described herein. The adjuvant moiety is capable of eliciting an immune response while attached to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0016] 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 key 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.

[0017] 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 No. AAZ99026 for human TLR7 polypeptide or GenBank Accession No. AAK62676 for murine TLR7 polypeptide.

[0018] 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 No. AAZ95441 for human TLR8 polypeptide or GenBank Accession No. AAK62677 for murine TLR8 polypeptide.

[0019] A "TLR agonist" is a substance that directly or indirectly binds 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 a TLR. Differences in signaling can be manifested, for example, as changes in target gene expression, changes in phosphorylation of signaling components, changes in the subcellular localization of downstream elements such as nuclear factor kappa B (NF-κB), changes in the association of certain components (e.g., IL-1 receptor-associated kinase (IRAK)) with other proteins or subcellular structures, or changes in the biochemical activity of components such as kinases (e.g., mitogen-activated protein kinases (MAPKs)).

[0020] "Antibody" refers to a polypeptide comprising 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. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa) connected by a disulfide bond. Each chain is composed of structural domains called immunoglobulin domains. These domains are divided into different categories based on size and function, e.g., variable domains or regions on the light and heavy chains (V and V, respectively). L and V H ) and constant domains or regions on the light and heavy chains (C L and C H), the N-terminus of each chain defines a variable region of approximately 100 to 110 amino acids, called the paratope, which is primarily responsible for antigen recognition, i.e., the antigen-binding domain. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, 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 gamma heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus forming a tetrameric quaternary structure. The two heavy chains are linked to each other and to their respective light chains by disulfide bonds. The resulting tetramer has two identical halves that together form a Y-shape. Each end of the fork contains an identical antigen-binding domain. There are four IgG subclasses in humans (IgG1, IgG2, IgG3, and IgG4), named in order of abundance in serum (i.e., IgG1 is the most abundant). Typically, the antigen-binding domain of an antibody is most important for the specificity and affinity of binding to cancer cells.

[0021] "Antibody construct" refers to an antibody or fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0022] In some embodiments, the binding agent is an antigen-binding antibody "fragment," which is a construct that includes at least the antigen-binding region of an antibody, either alone or together with other components that together form an antigen-binding construct. Many different types of antibody "fragments" are known in the art, including, for example, (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region, and (iii) a V fragment, which is a single-family fragment of an antibody. L and V H(iv) Fab' fragments, which result from cleavage of the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) two domains of the Fv fragment joined by a synthetic linker (i.e., V) that allow the two domains to be synthesized as a single polypeptide chain. L and V H ) and single-chain Fv (scFv), which are monovalent molecules composed of

[0023] An antibody or antibody fragment may be part of a larger construct, e.g., a conjugate or fusion construct of the antibody fragment to additional regions. For example, in some embodiments, an antibody fragment may be fused to an Fc region as described herein. In other embodiments, an antibody fragment (e.g., a Fab or scFv) may be part of a chimeric antigen receptor or chimeric T cell receptor, e.g., by fusing to a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., hinge region)) and optional intercellular signaling domains. For example, an antibody fragment may 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 derivative (BiTE) comprising a CD1- or CD3-binding domain and a linker.

[0024] "Epitope" refers to any antigenic or epitopic 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 groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0025] 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) FcγR, which binds IgG; (2) FcαR, which binds IgA; and (3) FcεR, which binds 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 for IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0026] The "identity" of a nucleic acid or amino acid sequence referred to herein can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of identical (i.e., identical) nucleotides or amino acid residues between optimally aligned sequences of interest and the reference sequence, divided by the length of the longest sequence (i.e., the longer of either the sequence of interest or the reference sequence). Sequence alignment and percent identity calculations can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for nucleic acid and amino acid sequence alignment), BLAST programs (e.g., BLAST2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also described, 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 The percent sequence identity (%) is calculated, for example, by: 100 x [(identical positions) / min(TG A ,TG B)], where TG A and T.G. B , T.G. A and T.G. B is the sum of the number of residues and internal gap positions of peptide sequences A and B in the alignment that minimizes (see, e.g., Russell et al., J. Mol. Biol., 244:332-350 (1994)).

[0027] The binding agent comprises 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 a variety of types of binding agents known in the art, including Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment," or a T-cell receptor.

[0028] "Biosimilar" refers to an approved antibody construct that has similar activity profiles to 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 CEA-targeting antibodies, such as labetuzumab (CEA-CIDE™, MN-14, hMN14, Immunomedics) CAS Registry Number 219649-07-7).

[0029] "Biobetter" refers to an approved antibody construct that is an improvement over a previously approved antibody construct, such as atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and labetuzumab. A biobetter can have one or more modifications (e.g., an altered glycan profile or a unique epitope) relative to the previously approved antibody construct.

[0030] "Amino acid" refers to any monomeric 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-naturally occurring (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to an isomer that has the same molecular formula and intramolecular bond(s) but differs in the three-dimensional arrangement of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glypication) or deglycosylated. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0031] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, D and L stereoisomers of 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. Naturally occurring stereoisomers of 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.

[0032] Naturally occurring amino acids include amino acids formed in proteins by post-translational modifications, such as citrulline (Cit).

[0033] Non-naturally occurring (non-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, which function similarly to naturally occurring amino acids. For example, an "amino acid analog" can be a non-naturally occurring amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to a 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 methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

[0034] "Linker" refers to a functional group that covalently bonds two or more moieties of a compound or material. For example, a linking moiety can function to covalently bond an adjuvant moiety to an antibody construct in an immunoconjugate.

[0035] A "linking moiety" refers to a functional group that covalently bonds two or more portions of a compound or material. For example, a linking moiety can function to covalently bond an adjuvant moiety to an antibody in an immunoconjugate. Useful bonds for connecting linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0036] "Divalent" refers to a chemical moiety containing two points of attachment for linking two functional groups; a polyvalent linking moiety can have additional points of attachment for linking additional functional groups. A divalent radical can be indicated 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 points of attachment 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, amido, acyl, nitro, cyano, and alkoxy.

[0037] Wavy line [ka] Or an asterisk (*) represents the point of attachment of the specified chemical moiety. [ka] When present, it is understood that the divalent chemical moiety can be used on both sides, i.e., reading from left to right or right to left. In some embodiments, two wavy lines [ka] The specified part where is present is considered to be used reading from left to right.

[0038] "Alkyl" refers to a straight- or branched-chain saturated aliphatic radical having the number of carbon atoms indicated. Alkyl can contain any number of carbons. For example, C1-C4 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. Alkyl can also refer to alkyl groups having up to 30 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. "Substituted alkyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0039] The term "alkyldiyl" refers to a divalent alkyl radical.

[0040] "Cycloalkyl" refers 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. Carbocyclic groups can also be partially unsaturated and have one or more double or triple bonds in the ring. Representative partially unsaturated carbocyclic groups 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.

[0041] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0042] "Aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or joined by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl, which has 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.

[0043] "Heterocycloalkyl" and "heteroaryl" refer to a "cycloalkyl" or "aryl" group, as described herein, in which one or more carbon atoms are optionally and independently replaced with a heteroatom selected from N, O, and S. "Heteroaryl," by itself or as part of another substituent, refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, in which 1 to 5 of the ring atoms are heteroatoms such as N, O, or S. Additional heteroatoms, including, but not limited to, B, Al, Si, and P, may also be useful. Heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Any suitable number of heteroatoms can be included in a heteroaryl group, such as 1, 2, 3, 4, or 5, or 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5. Heteroaryl groups can include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Heteroaryl groups can also be fused to aromatic ring systems, such as phenyl rings, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by bonds, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted. A "substituted heteroaryl" group can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0044] The term "heterocycloalkyldiyl" refers to a divalent heterocycloalkyl radical.

[0045] The heteroaryl group can be linked via any position on the ring. For example, pyrrole includes 1-, 2-, and 3-pyrrole, pyridine includes 2-, 3-, and 4-pyridine, imidazole includes 1-, 2-, 4-, and 5-imidazole, pyrazole includes 1-, 3-, 4-, and 5-pyrazole, triazole includes 1-, 4-, and 5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes 2-, 4-, 5-, and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine, 1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-, 5-, and 6-triazine, 1,3,5-triazine includes 2-triazine, thiophene includes 2- and 3-thiophene, and furan includes 2- and 3-furan. oxazoles include 2-, 4-, and 5-oxazoles; isoxazoles include 3-, 4-, and 5-isoxazoles; indoles include 1-, 2-, and 3-indoles; isoindoles include 1- and 2-isoindole; quinolines include 2-, 3-, and 4-quinolines; isoquinolines include 1-, 3-, and 4-isoquinolines; quinazolines include 2- and 4-quinazolines; cinnolines include 3- and 4-cinnolines; benzothiophenes include 2- and 3-benzothiophenes; and benzofurans include 2- and 3-benzofurans.

[0046] The term "heteroaryldiyl" refers to a divalent heteroaryl radical.

[0047] "Heterocycloalkyl," by itself or as part of another substituent, refers to a saturated ring system having 3 to 12 ring members and 1 to 4 heteroatoms of N, O, and S. Additional heteroatoms, including, but not limited to, B, Al, Si, and P, may also be useful. The heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. Heterocycloalkyl groups can contain any number of ring atoms, for example, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in a heterocycloalkyl group, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Heterocycloalkyl groups can include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3-, and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Heterocycloalkyl groups can also be fused to aromatic or non-aromatic ring systems to form members, including, but not limited to, indoline. Heterocycloalkyl groups can be unsubstituted or substituted.

[0048] The heterocycloalkyl group can be linked through any position on the ring. For example, aziridine can be 1- or 2-aziridine, azetidine can be 1- or 2-azetidine, pyrrolidine can be 1-, 2-, or 3-pyrrolidine, piperidine can be 1-, 2-, 3-, or 4-piperidine, pyrazolidine can be 1-, 2-, 3-, or 4-pyrazolidine, imidazolidine can be 1-, 2-, 3-, or 4-imidazolidine, and piperazine can be 1-, 2-, 3-, or 4-piperazine. The tetrahydrofuran may be 1- or 2-tetrahydrofuran, the oxazolidine may be 2-, 3-, 4-, or 5-oxazolidine, the isoxazolidine may be 2-, 3-, 4-, or 5-isoxazolidine, the thiazolidine may be 2-, 3-, 4-, or 5-thiazolidine, the isothiazolidine may be 2-, 3-, 4-, or 5-isothiazolidine, and the morpholine may be 2-, 3-, or 4-morpholine.

[0049] The term "heterocycloalkyldiyl" refers to a divalent heterocycloalkyl radical.

[0050] The terms "halo" and "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0051] 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 the oxygen and to the two other groups of the carbonyl-containing moiety.

[0052] As used herein, the phrase "quaternary ammonium salt" refers to a tertiary amine that is quaternized with an alkyl substituent (e.g., C1-C4 alkyl such as methyl, ethyl, propyl, or butyl).

[0053] The terms "treat," "treatment," and "treating" refer to an indication of success in treating or ameliorating an injury, condition, state (e.g., cancer), or symptom (e.g., cognitive impairment), including any objective or subjective parameter, such as relief; remission; reduction of symptoms or making the symptom, injury, condition, or state more tolerable to the patient; slowing the rate of progression of a symptom; reducing the frequency or duration of a symptom or state; or, in some circumstances, preventing the onset of a symptom. Treatment or amelioration of a symptom can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0054] The terms "cancer," "neoplasm," and "tumor" are used herein to refer to cells exhibiting autonomous, unregulated growth, such that the cells exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells subject to detection, analysis, and / or treatment in the context of the present invention include cancer cells (e.g., cancer cells from an individual with cancer), malignant cancer cells, premetastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. Cancers of virtually all tissues are known. The phrase "cancer burden" refers to the amount of cancer cells or cancer volume in a subject. Thus, reducing cancer burden refers to reducing the number of cancer cells or cancer cell volume in a 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 with cancer) or is derived from a cancer cell, e.g., 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 with cancer, can be a progeny cell from a primary cell isolated from an individual with cancer, etc. 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 skilled in the art and include solid tumors such as carcinoma, sarcoma, glioblastoma, melanoma, lymphoma, and myeloma, as well as circulating cancers such as leukemia.

[0055] As used herein, the term "cancer" includes any form of cancer, including, but not limited to, solid tumor cancers (e.g., skin, lung, prostate, breast, stomach, bladder, colon, ovarian, pancreatic, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head and neck squamous cell carcinoma, melanoma, and neuroendocrine) and liquid cancers (e.g., blood cancers); carcinoma; soft tissue tumors; sarcoma; teratoma; melanoma; leukemia; lymphoma; and brain cancer, e.g., minimal residual disease, including both primary and metastatic tumors.

[0056] "PD-L1 expression" refers to cells that have PD-L1 receptors on their surface. As used herein, "PD-L1 overexpression" refers to cells that have more PD-L1 receptors than corresponding non-cancerous cells.

[0057] "HER2" refers to the protein human epidermal growth factor receptor 2.

[0058] "HER2 expression" refers to a cell having HER2 receptors on its surface. For example, a cell may have about 20,000 to about 50,000 HER2 receptors on its surface. As used herein, "HER2 overexpression" refers to a cell having more than about 50,000 HER2 receptors. For example, the cell has 2, 5, 10, 100, 1,000, 10,000, 100,000, or 10,000,000 times the number of HER2 receptors (e.g., about 1 or 2 million HER2 receptors) compared to a corresponding non-cancerous cell. HER2 is estimated to be overexpressed in about 25% to about 30% of breast cancers.

[0059] The "pathology" of cancer includes all phenomena that compromise the patient's well-being, including, but not limited to, abnormal or uncontrolled cell growth, metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, suppressed or exacerbated inflammatory or immune responses, neoplasia, premalignant tumors, malignant tumors, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0060] As used herein, the phrases "cancer recurrence" and "tumor recurrence," as well as grammatical variations thereof, refer to the further growth of neoplastic or cancerous cells after a cancer diagnosis. In particular, recurrence can occur when further cancerous cell growth occurs in cancerous tissue. Similarly, "tumor spread" occurs when tumor cells spread to local or distant tissues or organs; thus, tumor spread encompasses tumor metastasis. "Tumor invasion" occurs when tumor growth spreads locally and impairs the function of the involved tissue by compressing, destroying, or inhibiting normal organ function.

[0061] 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 undetectable amounts 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 any step in the process, such as the detachment of cancer cells from the original tumor site and the migration and / or invasion of cancer cells to other parts of the body.

[0062] The phrases "effective amount" and "therapeutically effective amount" refer to the dose or amount of a substance, such as an immunoconjugate, that produces the therapeutic effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one of ordinary skill in the art using known techniques (see, 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 ndEdition, (Pharmaceutical Press, London, 2012). In the case of cancer, a therapeutically effective amount of an immunoconjugate may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent the immunoconjugate may prevent the growth of and / or kill existing cancer cells, the immunoconjugate may be cytostatic and / or cytotoxic. With respect to cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0063] "Recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., a human) for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, a "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is a human.

[0064] The phrase "synergistic adjuvant" or "synergistic combination" in the context of the present invention includes a combination of two immune modulators, such as a receptor agonist, a cytokine, and an adjuvant polypeptide, which in combination induce a synergistic effect on immunity compared to either administered alone. In particular, the immunoconjugates disclosed herein comprise synergistic combinations of the claimed adjuvants and antibody constructs. These synergistic combinations, when administered, induce a greater effect on immunity compared to, for example, when the antibody construct or adjuvant is administered in the absence of the other moiety. Furthermore, reduced amounts of immunoconjugate (as measured by the total number of antibody constructs or the total number of adjuvants administered as part of the immunoconjugate) can be administered compared to when either the antibody construct or the adjuvant is administered alone.

[0065] 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, for example, a mini-osmotic pump, into a subject.

[0066] The terms "about" and "approximately" used herein to modify a numerical value indicate an approximate range surrounding the numerical value. Thus, when "X" is a value, "about X" or "approximately X" indicates a value between 0.9X and 1.1X, e.g., between 0.95X and 1.05X or between 0.99X and 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 support for the written description of a claim limitation, e.g., "0.98X."

[0067] antibody The immunoconjugates of the present invention comprise antibodies. 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 with substantial or significant sequence identity or similarity to the parent antibody construct or antigen-binding domain, where 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, variants of the antibody constructs or antigen-binding domains described herein (parent antibody constructs or antigen-binding domains) that retain the ability to recognize target cells that express PD-L1, HER2, or CEA to a similar, comparable, or higher degree than the parent antibody construct or antigen-binding domain.

[0068] With respect to an antibody construct or antigen-binding domain, a functional variant can be, 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 in amino acid sequence to the antibody construct or antigen-binding domain.

[0069] A functional variant can, for example, comprise the amino acid sequence of a parent antibody construct or antigen-binding domain with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant can comprise the amino acid sequence of a parent antibody construct or antigen-binding domain with 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. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the parent antibody construct or antigen-binding domain.

[0070] Antibodies, including immunoconjugates of the invention, include Fc-engineered variants. In some embodiments, mutations in the Fc region that result in modulated binding to one or more Fc receptors include 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), The Fc region may include one or more of 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. Patent Nos. 7,416,726 and 5,624,821, which are incorporated by reference in their entireties.

[0071] Antibodies comprising the immunoconjugates of the invention include glycan variants, such as afucosylation, hi some embodiments, the Fc region of the binding agent is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0072] Amino acid substitutions in the antibody constructs or antigen-binding domains of the present invention are preferably conservative amino acid substitutions, which are known in the art and involve replacing one amino acid with certain physical and / or chemical properties with another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitutions of an amino acid having a nonpolar side chain with an amino acid having another nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitutions of a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitutions of an uncharged amino acid having a polar side chain with an uncharged amino acid having another polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), substitutions of an amino acid with a beta-branched side chain with an amino acid with another beta-branched side chain (e.g., Ile, Thr, and Val), substitutions of an amino acid with an aromatic side chain with an amino acid with another aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0073] An antibody construct or antigen-binding domain can consist essentially of a particular amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the antibody construct or antigen-binding domain functional variant.

[0074] Methods for producing antibodies are described, for example, in Kohler and Milstein, Eur. J. Immunol., 5:511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 9th Ed., Garland Publishing, New York, NY (2017). In certain embodiments, human or chimeric antibodies or antibody fragments can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)).Humanized antibodies can be produced using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, New Jersey (2009)), such as grafting non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1):25-34 (2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)), and using phage display (see, e.g., Fellouse, et al., Journal of Molecular Biology, 373(4):924-940 (2007) and Glanville, et al., PNAS, 106(48):20216-20221 (2009)).

[0075] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to PD-L1.

[0076] 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 suppressing adaptive immune responses. More specifically, binding of PD-L1 to its receptor, PD-1, is thought to transduce signals that inhibit T cell activation and proliferation. Agents that bind to PD-L1 and prevent its ligand from binding to the PD-1 receptor can prevent this immune suppression and thus enhance immune responses when desired, such as in the treatment of cancer or infectious diseases. The PD-L1 / PD-1 pathway also contributes to the prevention of autoimmunity, so agonistic agents against PD-L1 or agents that deliver immunosuppressive payloads may be useful in treating autoimmune diseases.

[0077] 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 need for new PD-L1-binding agents, including agents that bind to PD-L1 with high affinity and effectively prevent PD-L1 / PD-1 signaling, and agents that can deliver therapeutic payloads to PD-L1-expressing cells. Additionally, new PD-L1-binding agents are needed to treat autoimmune and infectious diseases.

[0078] Methods are provided for delivering an aminobenzazepine derivative payload to a cell that expresses PD-L1, comprising administering to the cell, or to a mammal comprising the cell, an immunoconjugate comprising an anti-PD-L1 antibody covalently attached to a linker that is covalently attached to one or more aminobenzazepine moieties.

[0079] Also provided are methods for enhancing, reducing or inhibiting an immune response in a mammal, and methods for treating a disease, disorder, or condition in a mammal that responds to PD-L1 inhibition, comprising administering the PD-L1 immunoconjugate to the mammal.

[0080] The invention provides PD-L1-binding agents comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide.

[0081] PD-L1-binding agents specifically bind to PD-L1, and the binding specificity of the agents allows them to target PD-L1-expressing cells, for example, to deliver a therapeutic payload to such cells.

[0082] In some embodiments, the PD-L1-binding agent (Type A or Type B) binds to human PD-L1, e.g., a protein comprising SEQ ID NO: 307. However, binding agents 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 binding agent binds to human PD-L1 and cynomolgus PD-L1; or human, cynomolgus, and mouse PD-L1. MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET SEQ ID NO: 307

[0083] In some embodiments, the PD-L1-binding agent 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-binding agent can completely or partially block (inhibit or prevent) PD-L1 from binding to its receptor, PD-1, and thus antibodies can be used to inhibit PD-L1 / PD-1 signaling (e.g., for therapeutic purposes).

[0084] 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 be different, targeting different epitopes of the same antigen or targeting different antigens. Methods for constructing multivalent binding constructs are known in the art. Bispecific and multispecific antibodies are known in the art. Additionally, V H and V L V due to a peptide linker that is too short to allow pairing between L V connected to H , which allows for different V H -V LDiabodies, triabodies, or tetrabodies, which are dimers, trimers, or tetramers of polypeptide chains that drive pairing between complementary domains on the polypeptide chains to generate multimeric molecules with two, three, or four functional antigen-binding sites, can be provided. Bis-scFv fragments, which are small scFv fragments with two different variable domains, can also be generated to generate bispecific bis-scFv fragments 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.

[0085] The PD-L1-binding agent may also be an antibody conjugate. In this regard, the PD-L1-binding agent may be a conjugate of (1) an antibody, an alternative scaffold, or a fragment thereof, and (2) a protein or non-protein moiety. For example, the PD-L1-binding agent may be conjugated to a peptide, a fluorescent molecule, a chemotherapeutic agent or other cytotoxic payload, an immunostimulatory agent, or an immunosuppressant.

[0086] The PD-L1-binding agent may be or be derived from a human, non-human, humanized, or chimeric antibody, or corresponding antibody fragment. A "chimeric" antibody is typically an antibody or fragment thereof that comprises a human constant region and a non-human variable region. A "humanized" antibody is typically a monoclonal antibody comprising a human antibody scaffold but with amino acids or sequences of non-human origin in at least one CDR (e.g., one, two, three, four, five, or all six CDRs).

[0087] PD-L1 binders - Type A Provided herein are PD-L1-binding agents comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. In some embodiments, the PD-L1-binding agent (Type A) comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, or at least the CDRs thereof; and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, or at least the CDRs thereof. In other embodiments, the PD-L1-binding agent (Type A) comprises an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 223-264, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 265-306. In yet another embodiment, the PD-L1-binding agent (Type A) is an immunoglobulin heavy chain variable region polypeptide comprising a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 1-23, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 24-57, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 58-95; and / or an immunoglobulin light chain variable region polypeptide comprising a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 96-128, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 129-151, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 152-155. Nucleic acids encoding the PD-L1-binding agent, or its individual heavy and light chains; vectors and cells comprising the nucleic acids; and compositions comprising the binding agent or nucleic acids are also provided.

[0088] Furthermore, in some embodiments, the PD-L1-binding agents (Type A) provided herein, upon binding to PD-L1 on the cell surface, cause cellular internalization of PD-L1 or the PD-L1 / PD-L1-binding agent complex. Without wishing to be bound by a particular theory or mechanism of action, it is believed that the PD-L1-binding agent according to this embodiment causes PD-L1 internalization upon binding and remains bound to PD-L1 during internalization, resulting in internalization of the binding agent along with PD-L1. Cellular internalization of PD-L1 and the bound PD-L1-binding agent can be determined by any suitable method, such as by assaying persistence on the cell surface and / or detecting internalizing antibodies. In some embodiments, the PD-L1-binding agent is internalized sufficiently strongly 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-binding agent that binds to PD-L1 on the cell surface is internalized (e.g., using a surface persistence assay, no more than about 75%, no more than about 65%, no more than about 50%, no more than about 25%, or no more than about 10% of the PD-L1-binding agent molecules bound to PD-L1 on the cell surface at the start of the assay remain bound at the end of the assay).

[0089] In one embodiment, the PD-L1-binding agent (Type A) comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs:223-264, a sequence 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:223-264, or at least the CDRs thereof; and / or an immunoglobulin light chain variable region of any one of SEQ ID NOs:265-306, a sequence 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:265-306, or at least the CDRs thereof.

[0090] By way of further example, PD-L1 binding agents (Type A) can include: (1) an immunoglobulin heavy chain variable region of SEQ ID NO: 223, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 265, or at least its CDRs; (2) an immunoglobulin heavy chain variable region of SEQ ID NO: 224, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 266, or at least its CDRs; (3) an immunoglobulin heavy chain variable region of SEQ ID NO: 225, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 267, or at least its CDRs; (4) an immunoglobulin heavy chain variable region of SEQ ID NO: 226, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 268, or at least its CDRs; (5) an immunoglobulin heavy chain variable region of SEQ ID NO: 227, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 269, or at least its CDRs; (6) an immunoglobulin heavy chain variable region of SEQ ID NO: 228, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 270, or at least its CDRs; (7) an immunoglobulin heavy chain variable region of SEQ ID NO: 229, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 271, or at least its CDRs; (8) an immunoglobulin heavy chain variable region of SEQ ID NO: 230, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 272, or at least its CDRs; 2 (9) an immunoglobulin heavy chain variable region of SEQ ID NO: 231, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 273, or at least its CDRs; (10) an immunoglobulin heavy chain variable region of SEQ ID NO: 232, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 274, or at least its CDRs; (11) an immunoglobulin heavy chain variable region of SEQ ID NO: 233, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 275, or at least its CDRs; (12) an immunoglobulin heavy chain variable region of SEQ ID NO: 234, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 276, or at least its CDRs; (13) an immunoglobulin heavy chain variable region of SEQ ID NO: 235, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 277, or at least its CDRs; (14) an immunoglobulin heavy chain variable region of SEQ ID NO: 236, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 278, or at least its CDRs; (15) an immunoglobulin heavy chain variable region of SEQ ID NO: 237, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 279, or at least its CDRs; (16) an immunoglobulin heavy chain variable region of SEQ ID NO: 238, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 280, or at least its CDRs; (17) an immunoglobulin heavy chain variable region of SEQ ID NO: 239, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 281, or at least its CDRs; (18) an immunoglobulin heavy chain variable region of SEQ ID NO: 240, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 282, or at least its CDRs; (19) an immunoglobulin heavy chain variable region of SEQ ID NO: 241, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 283, or at least its CDRs; (20) an immunoglobulin heavy chain variable region of SEQ ID NO: 242, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 284, or at least its CDRs; (21) an immunoglobulin heavy chain variable region of SEQ ID NO: 243, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 285, or at least its CDRs; (22) an immunoglobulin heavy chain variable region of SEQ ID NO: 244, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 286, or at least its CDRs; (23) an immunoglobulin heavy chain variable region of SEQ ID NO: 245, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 287, or at least its CDRs; (24) an immunoglobulin heavy chain variable region of SEQ ID NO: 246, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 288, or at least its CDRs; (25) an immunoglobulin heavy chain variable region of SEQ ID NO: 247, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 289, or at least its CDRs; (26) an immunoglobulin heavy chain variable region of SEQ ID NO: 248, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 290, or at least its CDRs; (27) an immunoglobulin heavy chain variable region of SEQ ID NO: 249, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 291, or at least its CDRs; (28) an immunoglobulin heavy chain variable region of SEQ ID NO: 250, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 292, or at least its CDRs; (29) an immunoglobulin heavy chain variable region of SEQ ID NO: 251, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 293, or at least its CDRs; (30) an immunoglobulin heavy chain variable region of SEQ ID NO: 252, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 294, or at least its CDRs; (31) an immunoglobulin heavy chain variable region of SEQ ID NO: 253, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 295, or at least its CDRs; (32) an immunoglobulin heavy chain variable region of SEQ ID NO: 254, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 296, or at least its CDRs; (33) an immunoglobulin heavy chain variable region of SEQ ID NO: 255, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 297, or at least its CDRs; (34) an immunoglobulin heavy chain variable region of SEQ ID NO: 256, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 298, or at least its CDRs; (35) an immunoglobulin heavy chain variable region of SEQ ID NO: 257, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 299, or at least its CDRs; (36) an immunoglobulin heavy chain variable region of SEQ ID NO: 258, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 300, or at least its CDRs; (37) an immunoglobulin heavy chain variable region of SEQ ID NO: 259, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 301, or at least its CDRs; (38) An immunoglobulin heavy chain variable region of SEQ ID NO: 260, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 302, or at least its CDRs; (39) an immunoglobulin heavy chain variable region of SEQ ID NO: 261, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 303, or at least its CDRs; (40) an immunoglobulin heavy chain variable region of SEQ ID NO: 262, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 304, or at least its CDRs; (41) An immunoglobulin heavy chain variable region of SEQ ID NO: 263, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 305, or at least its CDRs; (42) an immunoglobulin heavy chain variable region of SEQ ID NO: 164, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 306, or at least its CDRs; and / or (43) The immunoglobulin heavy chain variable region of Figures 4A to 4D and / or the immunoglobulin light chain variable region of Figures 4E to 4G, or at least the CDRs thereof.

[0091] The CDRs of a given heavy or light chain Ig sequence can be determined according to any of the various known Ig numbering schemes (e.g., Kabat, Chothia, Martin (extended Chothia), IGMT, AbM). In certain embodiments, the PD-L1-binding agent (Type A) comprises one or more of the following CDRs: an HCDR1 comprising or consisting of any one of SEQ ID NOs: 1-23, or a sequence which 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; an HCDR2 comprising or consisting of any one of SEQ ID NOs: 24-57, or a sequence which 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 an HCDR3 comprising or consisting of any one of SEQ ID NOs: 58-95, or a sequence which 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 / or an immunoglobulin light chain polypeptide, an LCDR1 comprising or consisting of any one of SEQ ID NOs: 96-128, or a sequence which 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: 96-128; an LCDR2 comprising or consisting of any one of SEQ ID NOs: 129-151, or a sequence which 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: 129-151; and and an LCDR3 comprising or consisting of any one of SEQ ID NOs: 152-155, or a sequence which 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: 152-155.

[0092] In certain embodiments, the binding agent (Type A) 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:1, an HCDR2 comprising or consisting of SEQ ID NO:24, and an HCDR3 comprising or consisting of SEQ ID NO:58; and / or the immunoglobulin light chain polypeptide comprises an LCDR1 comprising or consisting of SEQ ID NO:96, an LCDR2 comprising or consisting of SEQ ID NO:129, and an 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 and light chain polypeptides comprise any combination of the CDRs set forth in Figures 1A-D of PD-L1 type A binders 1-42.

[0093] In certain embodiments, the binding agent comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, wherein the immunoglobulin heavy chain polypeptide comprises 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 comprises 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 light chain polypeptide comprise any combination of the framework regions set forth in Figures 2A-D and 3A-D, respectively.

[0094] PD-L1 binders - Type B Provided herein are PD-L1-binding agents (Type B) comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. In some embodiments, the PD-L1-binding agent (Type B) comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 430-450, or at least the CDRs thereof, and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 451-471, or at least the CDRs thereof. In other embodiments, the PD-L1-binding agent comprises an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 430-450, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 451-471. In yet other embodiments, the PD-L1-binding agent, the immunoglobulin heavy chain variable region polypeptide comprises a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 308-321, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 332-338, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 339-359; and / or the immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 360-374, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 131 and 375-386, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 387-398. Nucleic acids encoding the PD-L1-binding agent, or its individual heavy and light chains; vectors and cells comprising the nucleic acids; and compositions comprising the binding agent or nucleic acids are also provided.

[0095] In one embodiment, the PD-L1-binding agent (Type B) comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 430-450, a sequence which 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: 430-450, or at least the CDRs thereof, and / or an immunoglobulin light chain variable region of any one of SEQ ID NOs: 451-471, a sequence which 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: 451-471, or at least the CDRs thereof.

[0096] By way of further example, PD-L1 binding agents (Type B) can include: (1) an immunoglobulin heavy chain variable region of SEQ ID NO: 429, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 450, or at least its CDRs; (2) an immunoglobulin heavy chain variable region of SEQ ID NO: 430, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 451, or at least its CDRs; (3) an immunoglobulin heavy chain variable region of SEQ ID NO: 431, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 452, or at least its CDRs; (4) an immunoglobulin heavy chain variable region of SEQ ID NO: 432, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 453, or at least its CDRs; (5) an immunoglobulin heavy chain variable region of SEQ ID NO: 433, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 454, or at least its CDRs; (6) an immunoglobulin heavy chain variable region of SEQ ID NO: 434, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 455, or at least its CDRs; (7) an immunoglobulin heavy chain variable region of SEQ ID NO: 435, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 456, or at least its CDRs; (8) an immunoglobulin heavy chain variable region of SEQ ID NO: 436, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 457, or at least its CDRs; (9) an immunoglobulin heavy chain variable region of SEQ ID NO: 437, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 458, or at least its CDRs; (10) an immunoglobulin heavy chain variable region of SEQ ID NO: 438, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 459, or at least its CDRs; (11) an immunoglobulin heavy chain variable region of SEQ ID NO: 439, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 460, or at least its CDRs; (12) an immunoglobulin heavy chain variable region of SEQ ID NO: 440, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 461, or at least its CDRs; (13) an immunoglobulin heavy chain variable region of SEQ ID NO: 441, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 462, or at least its CDRs; (14) an immunoglobulin heavy chain variable region of SEQ ID NO: 442, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 463, or at least its CDRs; (15) an immunoglobulin heavy chain variable region of SEQ ID NO: 443, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 464, or at least its CDRs; (16) an immunoglobulin heavy chain variable region of SEQ ID NO: 444, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 465, or at least its CDRs; (17) an immunoglobulin heavy chain variable region of SEQ ID NO: 445, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 466, or at least its CDRs; (18) an immunoglobulin heavy chain variable region of SEQ ID NO: 446, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 467, or at least its CDRs; (19) an immunoglobulin heavy chain variable region of SEQ ID NO: 447, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 468, or at least its CDRs; (20) an immunoglobulin heavy chain variable region of SEQ ID NO: 448, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 469, or at least its CDRs; and / or (21) an immunoglobulin heavy chain variable region of SEQ ID NO: 449, or at least its CDRs, and / or an immunoglobulin light chain variable region of SEQ ID NO: 470, or at least its CDRs; and / or (22) The immunoglobulin heavy chain variable region of Figure 8A-B and / or the immunoglobulin light chain variable region of Figure 8C-D, or at least the CDRs thereof.

[0097] The CDRs of a given heavy or light chain Ig sequence can be determined according to any of the various known Ig numbering schemes (e.g., Kabat, Chothia, Martin (extended Chothia), IGMT, AbM). In certain embodiments, the PD-L1-binding agent comprises one or more of the following CDRs: an HCDR1 comprising or consisting of any one of SEQ ID NOs: 308-321, or a sequence which 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: 308-321; an HCDR2 comprising or consisting of any one of SEQ ID NOs: 322-338, or a sequence which 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: 322-338; and an HCDR3 comprising or consisting of any one of SEQ ID NOs: 339-359, or a sequence which 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: 339-359; and / or an immunoglobulin light chain polypeptide, LCDR1 comprising or consisting of any one of SEQ ID NOs: 360-374, or a sequence which 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: 360-374; an LCDR2 comprising or consisting of any one of SEQ ID NOs: 375-386, or a sequence which 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: 375-386; and an LCDR3 comprising or consisting of any one of SEQ ID NOs: 387-398, or a sequence which 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: 387-398.

[0098] In certain embodiments, the binding agent 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 and light chain polypeptides comprise any combination of the CDRs listed in Figures 5A-B (Type B).

[0099] In certain embodiments, the binding agent comprises an immunoglobulin heavy chain polypeptide and an immunoglobulin light chain polypeptide, wherein the immunoglobulin heavy chain polypeptide comprises 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 comprises 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 light chain polypeptide comprise any combination of the framework regions listed in Figures 6A-B and / or Figures 7A-B (Type B), respectively.

[0100] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to HER2.

[0101] In certain embodiments, the immunoconjugate of the present invention comprises an anti-HER2 antibody. In one embodiment of the present invention, the anti-HER2 antibody of the immunoconjugate of the present invention comprises 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 Pat. No. 5,821,337, which is specifically incorporated herein by reference. These antibodies comprise human framework regions with the complementarity-determining regions of a murine 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.).

[0102] 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 the murine 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. (1985) Science 230:1132-9). al(1989) Science 244:707-12, Slamon et al(2001) New Engl. J. Med. 344:783-792).

[0103] In one embodiment of the present invention, the antibody construct or antigen-binding domain comprises the CDR regions of trastuzumab. In one embodiment of the present invention, the anti-HER2 antibody further comprises the framework regions of trastuzumab. In one embodiment of the present invention, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0104] In another embodiment of the invention, the anti-HER2 antibody of the immunoconjugate of the invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US 7,862,817. An exemplary humanized 2C4 antibody is pertuzumab (CAS Registry Number 380610-27-5), PERJETA™ (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI) that 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, e.g., 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 for the treatment of breast cancer.

[0105] In one embodiment of the invention, the antibody construct or antigen-binding domain comprises the CDR regions of pertuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises the framework regions of pertuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of pertuzumab.

[0106] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to 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-associated protein 1.

[0107] Cytoplasmic activation / proliferation-associated 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 mRNA, accelerating cell progression from G1 to S phase, increasing cell survival, and promoting cell growth, suggesting that caprin-1 may play an important role in tumorigenesis (Wang B, et al. (2005) J Immunol. 175:4274-4282). Caprin-1 acts alone or in combination with other RNA-binding proteins, such as RasGAPSH3 domain-binding protein 1 and fragile X mental retardation protein. In the tumorigenesis process, caprin-1 primarily 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 of tumor cell adaptation to adverse conditions, which contributes to radiation and chemotherapy resistance. Given 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, ZS, et al (2019) Oncology Letters 18:15-21).

[0108] 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).

[0109] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to CEA.

[0110] Increased expression of carcinoembryonic antigens (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of neoplasia, including tumor cell adhesion, metastasis, blockade of cellular immune mechanisms, and anti-apoptotic functions. CEA is also used as a blood marker for many carcinomas. Labetuzumab (CEA-CIDE™, Immunomedics, CAS Registry Number 219649-07-7), also known as MN-14 and hMN14, is a humanized IgG1 monoclonal antibody that is being investigated 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 is being 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).

[0111] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMN-14 / labetuzumab of SEQ ID NO: 472 (US 6,676,924). DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK SEQ ID NO: 472

[0112] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the hMN-14 / labetuzumab light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of SEQ ID NOs: 473-479 (US 6,676,924). [Table 1]

[0113] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of hMN-14 / labetuzumab of SEQ ID NO: 480 (US 6,676,924). EVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPGKGLEWVAEIHPDSSTINYAPSLKDRFTISRDNSKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS SEQ ID NO: 480

[0114] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hMN-14 / labetuzumab in SEQ ID NOs: 481-487 (US 6,676,924). [Table 2]

[0115] In an embodiment of the invention, the CEA-targeting 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

[0116] In an embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hPR1A3 of SEQ ID NOs: 489-495 (US8642742). [Table 3]

[0117] In an embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hPR1A3 of SEQ ID NOs: 496-502 (US8642742). [Table 4]

[0118] In an embodiment of the invention, the CEA-targeting 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

[0119] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hMFE-23 of SEQ ID NOs: 504-510 (US723288). [Table 5]

[0120] In an embodiment of the invention, the CEA-targeting 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

[0121] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hMFE-23 of SEQ ID NOs: 512-518 (US 723288). [Table 6]

[0122] In an embodiment of the invention, the CEA-targeting 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

[0123] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of SM3E of SEQ ID NOs: 520-526 (US 723288). [Table 7]

[0124] In an embodiment of the invention, the CEA-targeting 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

[0125] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of SM3E of SEQ ID NOs: 528-534 (US 723288). [Table 8]

[0126] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the NP-4 / arcitumomab light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of SEQ ID NOs: 535-541. [Table 9]

[0127] In an embodiment of the invention, the CEA-targeting 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.

[0128] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of NP-4 of SEQ ID NOs: 543-549. [Table 10]

[0129] In an embodiment of the invention, the CEA-targeting 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

[0130] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of M5A / hT84.66 of SEQ ID NOs: 551-557 (US7776330). [Table 11]

[0131] In an embodiment of the invention, the CEA-targeting 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)

[0132] In an embodiment of the invention, a CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of M5A / hT84.66 of SEQ ID NOs: 559-565. [Table 12]

[0133] In an embodiment of the invention, the CEA-targeting 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

[0134] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of hAb2-3 of SEQ ID NOs: 567-573 (US9617345). [Table 13]

[0135] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises a variable heavy chain (VH) of SEQ ID NO: 574 (US9617345). EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS SEQ ID NO: 574

[0136] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hAb2-3 of SEQ ID NOs: 575-581. [Table 14]

[0137] In an embodiment of the invention, the CEA-targeting 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

[0138] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity-determining region) or light chain framework (LFR) sequences of A240VL-B9VH / AMG-211 of SEQ ID NOs: 583-589 (US9982063). [Table 15]

[0139] In an embodiment of the invention, the CEA-targeting 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

[0140] In an embodiment of the invention, a CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity 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]

[0141] In an embodiment of the invention, the CEA-targeting 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

[0142] In an embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of SEQ ID NOs: 600-606 (US9982063). [Table 17]

[0143] 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 may be a single-chain variable fragment (scFv). A single-chain variable fragment (scFv) is a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to the variable (V) domain of an antibody light chain via a synthetic peptide, and can be produced using conventional recombinant DNA technology techniques. Similarly, a disulfide-stabilized variable fragment (dsFv) can be prepared by recombinant DNA technology. The antibody construct or antigen-binding domain may comprise one or more variable regions (e.g., two variable regions) of the antigen-binding domain of an anti-PD-L1 antibody, anti-HER2 antibody, or anti-CEA antibody, each variable region comprising CDR1, CDR2, and CDR3.

[0144] In some embodiments, the antibody in the immunoconjugate comprises a modified Fc region, where the modification modulates binding of the Fc region to one or more Fc receptors.

[0145] In some embodiments, the Fc region is modified by including a TGFβ1 receptor or a fragment thereof capable of binding 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. An "Fc linker" can be used to connect the IgG to the TGFβRII extracellular domain, e.g., G4S4G Fc linker (SEQ ID NO: 608). The Fc linker can be a short and flexible peptide that allows 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.

[0146] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0147] In some embodiments, the antibody in the immunoconjugate is a cysteine-engineered antibody, which provides for site-specific conjugation of adjuvants, labels, or drug moieties to the antibody through cysteine substitution at sites where the engineered cysteine is available for conjugation, but does not disrupt immunoglobulin folding and assembly or alter antigen binding and effector function. (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 a cysteine residue. Cysteine engineered antibodies can be conjugated to aminobenzazepine adjuvant moieties as aminobenzazepine-linker compounds with uniform stoichiometry (e.g., up to two aminobenzazepine moieties per antibody for antibodies with a single engineered cysteine site).

[0148] In some embodiments, the cysteine engineered antibody used to prepare the immunoconjugates of Table 3 has a cysteine residue introduced at the 149-lysine position of the light chain (LC K149C). In other embodiments, the cysteine engineered antibody has a cysteine residue introduced at the 118-alanine position (EU numbering) of the heavy chain (HC A118C). This position is numbered 121 in the sequential numbering system and 114 in the Kabat numbering system. In other embodiments, the cysteine engineered antibody has a cysteine residue introduced into the light chain at G64C or R142C according to the Kabat numbering, or into the heavy chain at D101C, V184C, or T205C according to the Kabat numbering system.

[0149] Aminobenzazepine adjuvant compounds The immunoconjugates of the present invention comprise an aminobenzazepine 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 innate immune responses in vertebrates. TLRs recognize various pathogen-associated molecular patterns from bacteria, viruses, and fungi and serve as the first line of defense against invading pathogens. TLRs elicit overlapping yet distinct biological responses due to differences in cellular expression and the signaling pathways they initiate. Once engaged (e.g., by natural stimulation or synthetic TLR agonists), TLRs initiate a signaling cascade that leads to the activation of nuclear factor-κB (NF-κB) via the recruitment of the adaptor proteins myeloid differentiation primary response gene 88 (MyD88) and IL-1 receptor-associated kinase (IRAK). Phosphorylation of IRAK leads to the recruitment of TNF receptor-associated factor 6 (TRAF6), which then phosphorylates the NF-κB inhibitor I-κB. As a result, NF-κB enters the cell nucleus, and its promoter initiates the transcription of genes containing NF-κB binding sites, such as cytokines. Additional modes of regulation of TLR signaling include the TIR domain-containing adaptor-inducing interferon-β (TRIF)-dependent induction of TNF receptor-associated factor 6 (TRAF6) and the activation of a MyD88-independent pathway via TRIF and TRAF3, which leads to 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.

[0150] Typically, the adjuvant moieties described herein are TLR7 and / or TLR8 agonists. Both TLR7 and TLR8 are expressed on myeloid cells (such as monocytes and dendritic cells). In humans, TLR7 is also expressed on plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is primarily expressed on bone marrow-derived cells, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 can detect the presence of "foreign" single-stranded RNA within cells as a means of responding to viral invasion. Treatment of TLR8-expressing cells with a TLR8 agonist can result in the production of high levels of IL-12, IFN-γ, IL-1, TNF-α, IL-6, and other proinflammatory cytokines. Similarly, stimulation of TLR7-expressing cells, such as pDCs, with a TLR7 agonist can result in the production of high levels of IFN-α and other proinflammatory cytokines. TLR7 / TLR8 engagement and resulting cytokine production activates dendritic cells and other antigen-presenting cells, promoting a variety of innate and adaptive immune response mechanisms that lead to tumor destruction.

[0151] Exemplary aminobenzazepine compounds (Bz) of the invention are shown in Tables 1a, 1b, and 1c. Each compound was synthesized and purified by the methods of the Examples provided herein, characterized by mass spectrometry, and shown to have the indicated mass. Activity against HEK293 NFκB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 68. The aminobenzazepine compounds of Tables 1a, 1b, and 1c 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 19-1

Table 19-2

Table 19-3

Table 19-4

Table 19-5

Table 19-6

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

[0152] Aminobenzazepine-linker compounds The immunoconjugates of the present invention are prepared by conjugating an antibody with an aminobenzazepine-linker compound. The aminobenzazepine-linker compound contains an aminobenzazepine moiety covalently attached to a linker unit. The linker unit contains functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. The linker unit contains a reactive functional group that reacts with, i.e., conjugates with, a reactive functional group on an antibody. For example, a nucleophilic group, such as a lysine side chain amino group on an antibody, reacts with an electrophilic reactive functional group on an aminobenzazepine-linker compound to form an immunoconjugate. Alternatively, for example, a cysteine thiol on an antibody reacts with a maleimide or bromoacetamide group on an aminobenzazepine-linker compound to form an immunoconjugate.

[0153] Suitable electrophilic reactive functional groups for aminobenzazepine-linker compounds include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N-hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethylphosphines (amine reactive); maleimides (thiol reactive); halogenated acetamides such as N-iodoacetamide (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (CH) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) esters (amine reactive); imidoesters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Additional reagents include, but are not limited to, those described in Hermanson, Bioconjugate Techniques 2nd Edition, Academic Press, 2008.

[0154] The present invention provides solutions to limitations and challenges in the design, preparation, and use of immunoconjugates. Some linkers are unstable in the bloodstream, which can result in unacceptable amounts of adjuvant / drug released before internalization within target cells (Khot, A. et al. (2015) Bioanalysis 7(13):1633-1648). Other linkers may provide stability in the bloodstream, but the efficacy of intracellular release may be adversely affected. Linkers that provide desired intracellular release typically have poor stability in the bloodstream. In other words, bloodstream stability and intracellular release are typically inversely related. Furthermore, in standard conjugation processes, the amount of adjuvant / drug moiety loaded onto the antibody, i.e., drug loading, 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. Under high drug loading, the aggregates formed must be removed for therapeutic use. As a result, drug-loading-mediated aggregate formation can reduce the yield of immunoconjugates and make the process difficult to scale up.

[0155] An exemplary embodiment is a compound of Formula II: [ka] containing an aminobenzazepine-linker compound of the formula (In the formula, Z is H, —O(C1-C8 alkyl), and N(X 2 R 2 )(X 3 R 3 ) are selected from; R 1 , R 2 , R 3 , and R 4 is H, C1-C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6-C 20 Aryl, C2-C9 heterocyclyl, and C1-C20 heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently selected from the group consisting of: -(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C1-C 12 alkyldiyl)-N(R 5 )2; -(C3-C 12 carbocyclyl); -(C3-C 12 Carbocyclyl)-*; -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*; -(C3-C 12 Carbocyclyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C3-C 12 Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*; -(C6-C 20 aryl); -(C6-C 20 aryl)-*; -(C6-C 20 Aryldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*; -(C2-C 20 heterocyclyl); -(C2-C 20 Heterocyclyl)-*; -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*; -(C2-C9 heterocyclyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*; -(C1-C 20 heteroaryl); -(C1-C 20 Heteroaryl)-*; -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C1-C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*; -C(=O)-*; -C(=O)-(C2-C 20 Heterocyclyldiyl)-*; -C(=O)N(R 5 )2; -C(=O)N(R 5 )-*; -C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)R 5 ; -C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5)C(=O)N(R 5 )2; -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 5 )CO2R 5 ; -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 )2; -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 ; -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 5 (C2-C5 heteroaryl); -C(=O)NR 5 -(C1-C 20 heteroaryldiyl)-N(R 5 )-*; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-*; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)N(R 5 )2; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -*; -N(R 5 )2; -N(R 5 )-*; -N(R 5 )C(=O)R 5 ; -N(R 5)C(=O)-*; -N(R 5 )C(=O)N(R 5 )2; -N(R 5 )C(=O)N(R 5 )-*; -N(R 5 )CO2R 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 )-(C2-C5 heteroaryl); -O-(C1-C 12 alkyl); -O-(C1-C 12 alkyldiyl)-N(R 5 )2; -O-(C1-C 12 alkyldiyl)-N(R 5 )-*; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -*; and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 and optionally substituted independently with one or more groups selected from: (alkyldiyl)-OH; or R 2 and R 3together form a 5- or 6-membered heterocyclyl ring; X 1 , X 2 , X 3 , and X 4 is a bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, and S(O)2N(R 5 ) independently selected from the group consisting of: R 5 H, C6-C 20 Aryl, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 alkyldiyl, or two R 5 the groups taken together form a 5- or 6-membered heterocyclyl ring; R 5a is C6-C 20 Aryl and C1-C 20 selected from the group consisting of heteroaryl; where the asterisk * indicates the binding site of L, and where R 1 , R 2 , R 3 and R 4 one of which is attached to L; L is QC(=O)-(PEG)-; QC(=O)-(PEG)-C(=O)-; QC(=O)-(PEG)-O-; QC(=O)-(PEG)-C(=O)-(PEP)-; QC(=O)-(PEG)-C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-; QC(=O)-(PEG)-C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; QC(=O)-(PEG)-C(=O)N(R 5)-(C1-C 12 Alkyldiyl)-(MCgluc)-; QC(=O)-(PEG)-C(=O)-(MCgluc)-; QC(=O)-(PEG)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 Alkyldiyl)-; QC(=O)-(PEG)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; QC(=O)-(PEG)-N(R 5 )-; QC(=O)-(PEG)-N(R 5 )-(PEG)-C(=O)-(PEP)-; QC(=O)-(PEG)-N + (R 5 )2-(PEG)-C(=O)-(PEP)-; QC(=O)-(PEG)-C(=O)-N(R 5 )CH(AA1)C(=O)-(PEG)-C(=O)-(PEP)-; QC(=O)-(PEG)-C(=O)-N(R 5 )CH(AA1)C(=O)-N(R 5 )-(C1-C 12 Alkyldiyl)-; QC(=O)-(PEG)-SS-(C1-C 12 Alkyldiyl)-OC(=O)-; QC(=O)-(PEG)-SS-(C1-C 12 alkyldiyl)-C(=O)-; QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-(PEP)-; QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 Alkyldiyl)-; QC(=O)-(C1-C12 alkyldiyl)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )-C(=O); QC(=O)-(C1-C 12 alkyldiyl)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; QC(=O)-CH2CH2OCH2CH2-(C1-C 20 Heteroaryldiyl)-CHO-(PEG)-C(=O)-(MCgluc)-; QC(=O)-CH2CH2OCH2CH2-(C1-C 20 heteroaryldiyl)-CHO-(PEG)-C(=O)-(MCgluc)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; and Q-(CH2) m -C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(═O)—(C2-C5 monoheterocyclyldiyl)-; where PEG has the formula: -(CH2CH2O) n -(CH2) m m is an integer from 1 to 5 and n is an integer from 2 to 50; PEP has the formula: [ka] wherein AAl and AA2 are independently selected from amino acid side chains, or AAl or AA2 and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; R 6 is C6-C 20Aryldiyl and C1-C 20 heteroaryldiyl, —CHO—C(═O)—, and optionally: [ka] is replaced by: and MCgluc is a compound of the group: [ka] wherein q is 1 to 8 and AA is an amino acid side chain; Q is F, Cl, NO2 and SO3 - substituted with one or more groups independently selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy; Here, alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3 , -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2O H, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH 2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH) CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -N HS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n -H, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H.

[0156] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is one in which PEP is selected from the group: [ka] (wherein n is 1 or greater and AA is an amino acid side chain). The present invention includes being selected from the following:

[0157] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include those in which AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0158] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include those where AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(CH6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2CH(CH3)2, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2.

[0159] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include where AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0160] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include those in which AA1 and AA2 are independently selected from GlcNAc aspartic acid, —CH2SO3H, and —CH2OPO3H.

[0161] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II are represented by Formulas IIa-d: [ka] is selected from.

[0162] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II are represented by Formulae IIe and IIf: [ka] (Wherein R of formula IIf 5ais phenyl, optionally substituted with one or more groups selected from F, Cl, Br, I, —CN, and —NO2 is selected from.

[0163] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include where L is QC(=O)-(PEG)- or QC(=O)-(PEG)-C(=O)-.

[0164] Exemplary embodiments of aminobenzazepine-linker compounds of formula II are represented by formulae IIg and IIh: [ka] is selected from.

[0165] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include where L is -C(=O)-(PEG)-C(=O)-(PEP)-.

[0166] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is R 2 and R 3 are each C1-C8 alkyl.

[0167] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is R 2 and R 3 are each -CH2CH2CH3.

[0168] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is X 2 and X 3 are bonds, and R 2 or R 3 -O-(C1-C 12 alkyl).

[0169] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is X 2 and X 3are bonds, and R 2 or R 3 is -OCH2CH3.

[0170] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is R 1 and R 4 One of the two is -(C6-C 20 Aryldiyl)-S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2 and -(C6-C 20 Aryldiyl)-S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-OH.

[0171] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include C6-C 20 Aryldiyl is phenyldiyl, C2-C 20 Including heterocyclyldiyl is azetidinediyl.

[0172] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II has the formula: [ka] is selected from.

[0173] An exemplary embodiment of an aminobenzazepine-linker compound of Formula II is R 1 and R 4 One of the groups is -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -L.

[0174] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II include C1-C 20 The heteroaryldiyl is pyridinediyl, and C2-C 20 Heterocyclyldiyl includes piperidiyl.

[0175] An exemplary embodiment of the aminobenzazepine-linker compound of Formula II is where Q is [ka] The present invention includes being selected from the following:

[0176] The present invention includes all reasonable combinations and permutations of features of the embodiments of Formula II.

[0177] Exemplary embodiments of aminobenzazepine-linker compounds of Formula II are selected from the compounds in Tables 2a, 2b, and 2c. Each compound was synthesized and purified by the methods of the Examples provided herein, characterized by mass spectrometry, and shown to have the indicated mass. The aminobenzazepine-linker compounds in Tables 2a, 2b, and 2c exhibit surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5] [Table 21-6] [Table 21-7]

Table 21-8

Table 22-1

Table 22-2

Table 22-3

Table 22-4

Table 22-5

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

[0178] Immunoconjugates An exemplary embodiment of an immunoconjugate comprises an antibody covalently attached to a bivalent linker that covalently attaches one or more aminobenzazepine moieties, and has Formula I: Ab-[L-Bza] p I or a pharmaceutically acceptable salt thereof (In the formula, Ab is antibody; p is an integer from 1 to 8; Bza is formula: [ka] an aminobenzazepine moiety having the formula: R 1 , R 2 , R 3 , and R 4 is H, C1-C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6-C 20 Aryl, C2-C9 heterocyclyl, and C1-C 20 heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently selected from the group consisting of: -(C1-C 12 alkyldiyl)-N(R5 )-*; -(C1-C 12 alkyldiyl)-N(R 5 )2; -(C3-C 12 carbocyclyl); -(C3-C 12 Carbocyclyl)-*; -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*; -(C3-C 12 Carbocyclyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C3-C 12 Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*; -(C6-C 20 aryl); -(C6-C 20 aryl)-*; -(C6-C 20 Aryldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*; -(C2-C 20 heterocyclyl); -(C2-C 20 Heterocyclyl)-*; -(C2-C9 heterocyclyl)-(C1-C 12Alkyldiyl)-NR 5 -*; -(C2-C9 heterocyclyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*; -(C1-C 20 heteroaryl); -(C1-C 20 Heteroaryl)-*; -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C1-C 20 Heteroaryl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -(C1-C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*; -C(=O)-*; -C(=O)-(C2-C 20 Heterocyclyldiyl)-*; -C(=O)N(R 5 )2; -C(=O)N(R 5 )-*; -C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)R 5 ; -C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)N(R 5 )2; -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 5 )CO2R 5 ; -C(=O)NR 5 -(C1-C 12 alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 )2; -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 ; -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 5 (C2-C5 heteroaryl); -C(=O)NR 5 -(C1-C 20 heteroaryldiyl)-N(R 5 )-*; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-*; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -*; -N(R 5 )2; -N(R 5 )-*; -N(R 5 )C(=O)R 5 ; -N(R 5 )C(=O)-*; -N(R 5 )C(=O)N(R 5 )2; -N(R 5 )C(=O)N(R 5 )-*; -N(R 5 )CO2R 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 )-(C2-C5 heteroaryl); -O-(C1-C 12 alkyl); -O-(C1-C 12 alkyldiyl)-N(R 5 )2; -O-(C1-C 12 alkyldiyl)-N(R 5 )-*; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2; -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -*; and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-OH; 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 a bond, C(=O), C(=O)N(R 5), O, N(R 5 ), S, S(O)2, and S(O)2N(R 5 ) independently selected from the group consisting of: R 5 H, C6-C 20 Aryl, C6-C 20 Aryldiyl, C1-C 12 Alkyl and C1-C 12 alkyldiyl, or two R 5 the groups taken together form a 5- or 6-membered heterocyclyl ring; R 5a is C6-C 20 Aryl and C1-C 20 selected from the group consisting of heteroaryl; where the asterisk * indicates the binding site of L, and where R 1 , R 2 , R 3 and R 4 one of which is attached 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 )-(C1-C 12 Alkyldiyl)-; -C(=O)-(PEG)-C(=O)N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; -C(=O)-(PEG)-C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-(MCgluc)-; -C(=O)-(PEG)-C(=O)-(MCgluc)-; -C(=O)-(PEG)-C(=O)-(PEP)-N(R 5 )-(C1-C12 Alkyldiyl)-; -C(=O)-(PEG)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; -C(=O)-(PEG)-N(R 5 )-; -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(AA1)C(=O)-(PEG)-C(=O)-(PEP)-; -C(=O)-(PEG)-C(=O)-N(R 5 )CH(AA1)C(=O)-N(R 5 )-(C1-C 12 Alkyldiyl)-; -C(=O)-(PEG)-SS-(C1-C 12 Alkyldiyl)-OC(=O)-; -C(=O)-(PEG)-SS-(C1-C 12 alkyldiyl)-C(=O)-; -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-(PEP)-; -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 Alkyldiyl)-; -C(=O)-(C1-C 12 alkyldiyl)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )-C(=O); -C(=O)-(C1-C 12alkyldiyl)-C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; -C(=O)-CH2CH2OCH2CH2-(C1-C 20 Heteroaryldiyl)-CHO-(PEG)-C(=O)-(MCgluc)-; -C(=O)-CH2CH2OCH2CH2-(C1-C 20 heteroaryldiyl)-CHO-(PEG)-C(=O)-(MCgluc)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(=O)-(C2-C5 monoheterocyclyldiyl)-; and -(Succinimidyl)-(CH2) m -C(=O)-(PEP)-N(R 5 )-(C1-C 12 alkyldiyl)-N(R 5 )C(═O)—(C2-C5 monoheterocyclyldiyl)-; PEG has the formula: -(CH2CH2O) n -(CH2) m m is an integer from 1 to 5 and n is an integer from 2 to 50; PEP has the formula: [ka] wherein AAl and AA2 are independently selected from amino acid side chains, or AAl or AA2 and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; R 6 is C6-C 20 Aryldiyl and C1-C 20 heteroaryldiyl, -CH2O-C(=O)- and optionally [ka] is substituted with; and MCgluc is a compound of the group: [ka] wherein q is 1 to 8 and AA is an amino acid side chain; Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are selected from the group consisting of F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -CH2CH2CH3, -C H(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, - CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN, -CH2C N, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH 3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NH S(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2, - NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n-H, -OP(O)(OH), -S(O)N(CH), -SCH, -S(O)CH, and -S(O)H.

[0179] 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.

[0180] 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 biobetters thereof.

[0181] 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.

[0182] 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 biobetters thereof.

[0183] 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.

[0184] An exemplary embodiment of the immunoconjugate of Formula I includes where the antibody is labetuzumab, or a biosimilar or biobetter thereof.

[0185] An exemplary embodiment of the immunoconjugate of Formula I is an immunoconjugate wherein the PEP is selected from the group: [ka] (wherein n is 1 or greater and AA is an amino acid side chain). The present invention includes being selected from the following:

[0186] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0187] 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, -CH2CH(CH3)2, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2.

[0188] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0189] 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.

[0190] Exemplary embodiments of the immunoconjugate of Formula I are those in which Bza is of Formula Ia-d: [ka] The present invention includes being selected from the following:

[0191] An exemplary embodiment of the immunoconjugate of Formula I is one in which Bza is of Formula Ie and If: [ka] (Wherein, R in formula If 5a is phenyl, optionally substituted with one or more groups selected from F, Cl, Br, I, —CN, and —NO2 The present invention includes being selected from the following:

[0192] Exemplary embodiments of the immunoconjugate of Formula I include where L is -C(=O)-(PEG)- or -C(=O)-(PEG)-C(=O)-.

[0193] Exemplary embodiments of the immunoconjugate of Formula I include those in which Bza is of Formulas Ig and Ih: [ka] The present invention includes being selected from the following:

[0194] Exemplary embodiments of the immunoconjugate of Formula I include where L is -C(=O)-(PEG)-C(=O)-(PEP)-.

[0195] An exemplary embodiment of the immunoconjugate of Formula I is R 2 and R 3 are each C1-C8 alkyl.

[0196] An exemplary embodiment of the immunoconjugate of Formula I is R 2 and R 3 are each -CH2CH2CH3.

[0197] An exemplary embodiment of the immunoconjugate of Formula I is 2 and X 3 are bonds, and R 2 or R 3 -O-(C1-C 12 alkyl).

[0198] An exemplary embodiment of the immunoconjugate of Formula I is 2 and X 3 are bonds, and R 2 or R 3 is -OCH2CH3.

[0199] An exemplary embodiment of the immunoconjugate of Formula I is R 1 and R 4 One of them is, -(C1-C12 alkyldiyl)-N(R 5 )-*; -(C1-C 12 alkyldiyl)-N(R 5 )C(=NR 5 )N(R 5 )-*; -(C6-C 20 alkyldiyl)-S(=O)2-(C2-C 20 Heterocyclyldiyl)-*; -(C6-C 20 Aryldiyl)-S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-C(=O)-*; -(C6-C 20 Aryldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C6-C 20 Aryldiyl)-C(=O)-(C2-C 20 Heterocyclyldiyl)-*; -C(=O)NR 5 -(C1-C 20 heteroaryldiyl)-*; and -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -*, including being selected from.

[0200] An exemplary embodiment of the immunoconjugate of Formula I is R 2 and R 3 One of them is, -(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C1-C 12Alkyldiyl)-O-(C1-C 12 alkyldiyl)-N(R 5 )-*; -(C1-C 12 alkyldiyl)-N(R 5 )C(=NR 5 )-N(R 5 )-*; -(C1-C 12 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 )-*, including being selected from; X 2 and X 3 is a bond, where the asterisk * indicates the binding site of L.

[0201] An exemplary embodiment of the immunoconjugate of Formula I is R 1 and R 4 One of the two is -(C6-C 20 Aryldiyl)-S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-N(R 5 )2 and -(C6-C 20 Aryldiyl)-S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 alkyldiyl)-OH.

[0202] An exemplary embodiment of the immunoconjugate of Formula I is C6-C 20 Aryldiyl is phenyldiyl, C2-C 20 Including heterocyclyldiyl is azetidinediyl.

[0203] An exemplary embodiment of the immunoconjugate of Formula I is R 1 and R 4 One of the expressions: [ka] The present invention includes being selected from the following:

[0204] An exemplary embodiment of the immunoconjugate of Formula I is R 1 and R 4 One of the groups is -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -L.

[0205] An exemplary embodiment of the immunoconjugate of Formula I is C1-C 20 The heteroaryldiyl is pyridinediyl, and C2-C 20 Heterocyclyldiyl includes piperidiyl.

[0206] In exemplary embodiments, p is 1, 2, 3, or 4.

[0207] An exemplary embodiment of an immunoconjugate comprises an antibody covalently attached to a linker that covalently attaches one or more aminobenzazepine moieties, and has formula III: [ka] a pharmaceutically acceptable salt thereof, or a quaternary ammonium salt thereof, (In the formula, R1 , R 2 , R 3 , and R 4 are independently Y or Z, and R 1 , R 2 , R 3 , and R 4 is Y, and one of the formula: [ka] or [ka] having; Each Z is independently hydrogen or a group of the formula: [ka] Selected from; U is optionally present and is CH, C(=O), CHC(=O), or C(=O)CH; A is optionally present and NR 10 or the expression: [ka] is selected from R 10 and W independently represent hydrogen, Ar 1 , or the expression: [ka] wherein V is optionally present and has the formula: [ka] It is of J 1 and J 2 are independently CH or N, m 1 , m 2 , and m 3 is m 1 , m 2 , and m 3are independently integers between 0 and 25, except that at least one of is a non-zero integer; n 1 , n 2 , n 3 , n 4 , n 5 , and n 6 are independently integers from 0 to 10, t 1 and t 2 are independently an integer from 1 to 3, G 1 , G 2 , G 3 , and G 4 are independently CH, C(=O), CHC(=O), C(=O)CH, or a bond; X 1 , X 2 , X 3 , and X 4 are each optionally present and independently represent O, NR 7 , CHR 7 , SO, S, or one or two cycloalkyldiyl, heterocycloalkyldiyl, aryldiyl, or heteroaryldiyl groups, and when multiple cycloalkyldiyl, heterocycloalkyldiyl, aryldiyl, or heteroaryldiyl groups are present, the multiple cycloalkyldiyl, heterocycloalkyldiyl, aryldiyl, or heteroaryldiyl groups are linked or fused together, and wherein the linked cycloalkyldiyl, heterocycloalkyldiyl, aryldiyl, or heteroaryldiyl groups are linked via a bond or -CO-; R 9 is hydrogen, C1-C4 alkyl, or a group of the formula: [ka] is selected from R 8 are independently hydrogen or C1-C4 alkyl; Ar 1 and Ar 2is independently an aryl or heteroaryl group, optionally substituted with one or more halogens (e.g., fluorine, chlorine, bromine, or iodine), nitrile, hydroxyl, C1-C4 alkyl groups, or combinations thereof; L M is a linking moiety comprising a functional group selected from amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, and thiourea; r is an integer from 1 to 10, Ab is antibody, Each wavy line [ka] represents the point of attachment).

[0208] Exemplary embodiments of the immunoconjugate of Formula III include those in which the subscript r is 1.

[0209] Exemplary embodiments of the immunoconjugate of Formula I or III include those in which the antibody is an antibody construct having an antigen-binding domain that binds to PD-L1.

[0210] Exemplary embodiments of the immunoconjugate of Formula I or III include where the antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab, or biosimilars or biobetters thereof.

[0211] Exemplary embodiments of the immunoconjugate of Formula I or III include those in which the antibody is an antibody construct having an antigen-binding domain that binds to HER2.

[0212] Exemplary embodiments of the immunoconjugate of Formula I or III include where the antibody is selected from the group consisting of trastuzumab and pertuzumab, or a biosimilar or biobetter thereof.

[0213] Exemplary embodiments of the immunoconjugates of Formula I or III include those in which the antibody is an antibody construct having an antigen-binding domain that binds to CEA.

[0214] Exemplary embodiments of the immunoconjugate of Formula I or III include where the antibody is selected from the group consisting of labetuzumab (also known as MN-14, hMN14, or CEA-CIDE™), PR1A3, MFE-23, SM3E, or a biosimilar or biobetter thereof.

[0215] The present invention includes all reasonable combinations and permutations of features of the embodiments of Formulas I and III.

[0216] In certain embodiments, immunoconjugate compounds of the invention include those with immunostimulatory activity. The antibody-drug conjugates of the invention selectively deliver effective doses of aminobenzazepine drugs to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated aminobenzazepines.

[0217] Drug loading is the number of aminobenzazepine moieties per antibody in the immunoconjugate of Formula I or III, represented by p. Drug (aminobenzazepine) loading can range from 1 to 8 drug moieties (D) per antibody. The immunoconjugates of Formula I and III contain mixtures or collections of antibodies conjugated to a range of 1 to about 8 drug moieties. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues, such as lysine and cysteine. In some embodiments, a free cysteine residue is 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 therein, e.g., 1 to 8 or 2 to 5. In such embodiments, p and n are equal (i.e., p = n = 1, 2, 3, 4, 5, 6, 7, or 8, or any range therebetween). Exemplary antibody-drug conjugates of Formula I include, but are not limited to, antibodies with one, two, three, or four 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 to form intrachain disulfide bonds without engineering. In such cases, the existing free cysteine residues may be used to conjugate the antibody to a 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.

[0218] For some anti-immunoconjugates, p may be limited by the number of binding sites on the antibody. For example, if the linkage is a cysteine thiol, as in certain exemplary embodiments described herein, the antibody may have only one or a limited number of cysteine thiol groups, or only one or a limited number of reactive and sufficient thiol groups to which a drug may be attached. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with an aminobenzazepine-linker compound of Formula II. In certain embodiments, higher drug loading, e.g., p greater than 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the average drug loading 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 reveal reactive nucleophilic groups such as lysine or cysteine.

[0219] The loading (drug / antibody ratio) of the immunoconjugate can be controlled in different ways, as well as by, for example, (i) limiting the molar excess of the aminobenzazepine-linker intermediate compound compared to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reductive denaturation conditions for optimized antibody reactivity.

[0220] It is understood that when two or more nucleophilic groups on an antibody react with a drug, the resulting product is a mixture of antibody-drug conjugate compounds with one or more drug moieties distributed among the antibodies. The average number of drugs per antibody may be calculated from the mixture by a double ELISA antibody assay, which is antibody-specific and drug-specific. Individual immunoconjugate molecules may be identified in the mixture by mass spectrometry and separated by HPLC, e.g., 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, KJ, 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, SC, et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract (See, e.g., American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004, No. 627.) In certain embodiments, homogenous immunoconjugates having a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.

[0221] Exemplary embodiments of immunoconjugates of Formula I are selected from the immunoconjugates of Tables 3a, 3b, and 3c. [Table 24-1] [Table 24-2] [Table 25] [Table 26-1] [Table 26-2] [Table 26-3]

[0222] Composition of Immunoconjugates The present invention provides compositions, e.g., pharmaceutically or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates described herein and optionally a carrier therefor, e.g., a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates may be the same or different in composition, i.e., a 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 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.

[0223] In an exemplary embodiment, the composition comprising immunoconjugate compounds comprises a mixture of immunoconjugate compounds, and the average drug loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5.

[0224] Immunoconjugate compositions of the invention can have an average adjuvant to antibody construct ratio of about 0.4 to about 10. One skilled in the art will recognize that the number of aminobenzazepine adjuvants conjugated to an antibody construct may vary among immunoconjugates in a composition comprising multiple immunoconjugates of the invention. Thus, the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average, which is sometimes referred to as the drug-to-antibody ratio (DAR). The adjuvant to antibody construct (e.g., antibody) ratio can be assessed by any suitable means, many of which are known in the art.

[0225] The average number of adjuvant moieties per antibody (DAR) in preparing immunoconjugates from conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of immunoconjugates in the composition in units of p can also be determined. In some cases, separation, purification, and characterization of homogenous immunoconjugates with a particular p value from immunoconjugates with other drug loads may be achieved by means such as reverse-phase HPLC or electrophoresis.

[0226] Pharmaceutical Compositions and Methods of Administration In other embodiments, another aspect of the invention relates to pharmaceutical compositions or dosage forms comprising a therapeutically effective amount of an immunoconjugate of the invention and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

[0227] The pharmaceutical composition may be in any form that allows for administration to a patient. For example, the pharmaceutical composition may be in solid or liquid form. Typical routes of administration include, but are not limited to, parenteral, ocular, and intratumoral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, or intrasternal injection or infusion techniques. In one embodiment, the composition is administered orally. In a specific embodiment, the composition is administered intravenously.

[0228] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically or pharmacologically acceptable additives. 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 lumen of an organ. Alternatively, the immunoconjugates 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 isotonic solutions of water and one or more salts, such as sodium chloride, e.g., Ringer's solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating, fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can also be used in the preparation of injectables. These compositions are desirably 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, etc., required to approximate physiological conditions, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0229] The composition can contain any suitable concentration of immunoconjugate. The concentration of immunoconjugate in the composition can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., in accordance with the particular mode of administration selected and the patient's needs. In certain embodiments, the concentration of immunoconjugate in an injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0230] Methods for treating cancer with immunoconjugates The present invention provides a method for treating cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate described herein (e.g., as a pharmaceutical composition described herein) to a subject in need thereof, e.g., a subject having cancer and in need of cancer treatment. The method comprises administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3.

[0231] It is contemplated that the immunoconjugates of the invention can be used to treat a variety of hyperproliferative diseases or disorders, such as those characterized by overexpression of tumor antigens. Exemplary hyperproliferative disorders include benign or malignant solid tumors, and hematological disorders such as leukemia and lymphoid malignancies.

[0232] In another aspect, immunoconjugates are provided for use as pharmaceuticals. In certain embodiments, the invention provides immunoconjugates for use in methods of treating an individual, comprising administering to the individual an effective amount of the immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0233] In a further aspect, the present invention provides 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 an effective amount of the medicament to an individual with cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0234] Carcinomas are malignant tumors derived from epithelial tissue. Epithelial cells cover the exterior of the body, line internal cavities, and form the lining of glandular tissue. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer arising in glandular (secretory) cells, such as breast cancer, pancreatic cancer, lung cancer, prostate cancer, stomach 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 carcinoma; small cell lung carcinoma; non-small cell lung carcinoma; and others. Carcinomas can 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 containing an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof). In some embodiments, a method for treating breast cancer comprises administering an immunoconjugate containing an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof). In some embodiments, a method for treating triple-negative breast cancer comprises administering an immunoconjugate containing an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof).

[0235] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include alveolar soft part sarcoma; angiomatoid fibrous histiocytoma; chondromyxoid fibroma; fibroma); skeletal chondrosarcoma; extraskeletal myxoid chondrosarcoma; clear cell sarcoma; fibroplastic small round cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing's sarcoma; fibromatosis (desmoid); infantile fibrosarcoma; gastrointestinal stromal tumor; giant cell tumor of bone; giant cell tumor of tendon sheath; inflammatory myofibroblastic tumor; uterine leiomyoma; leiomyosarcoma; lipoblastoma; typical lipoma; spindle cell lipoma or pleomorphic lipoma; atypical lipoma; chondroitinous lipoma; well-differentiated liposarcoma; myxoid / round cell liposarcoma; pleomorphic lipoma; myxoid malignant fibrous histiocytoma; high-grade malignant fibrous histiocytoma; myxofibrosarcoma; malignant peripheral nerve sheath tumor; medium These include, but are not limited to, dermatoma; neuroblastoma; osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolar rhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignant schwannoma; synovial sarcoma; Evans' tumor; nodular fasciitis; desmoid fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; giant cell tumor of tendon sheath (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 / endothelial cells, and nerve sheath cells.

[0236] Sarcoma is a rare type of cancer that begins in the bone or soft tissues of the body, including cells of mesenchymal origin, such as cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissues. The different types of sarcoma are based on where the cancer begins. For example, osteosarcoma begins in the bone, liposarcoma begins in the fat, and rhabdomyosarcoma begins in the muscle. Examples of sarcomas include, but are not limited to, Askin tumor; botryoid sarcoma; chondrosarcoma; Ewing's sarcoma; malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodes; dermatofibrosarcoma protuberances (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangiosarcoma (more commonly called "angiosarcoma"); Kaposi's sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0237] Teratomas are a type of germ cell tumor that may contain several different types of tissue, such as hair, muscle, and bone (e.g., they can include tissue from any and / or all three germ layers: endoderm, mesoderm, and ectoderm). Teratomas most frequently occur in the ovaries in women, the testes in men, and the coccyx in children.

[0238] Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma can start in a mole (cutaneous melanoma) but can also start in other pigmented tissues, such as the eye or intestine.

[0239] Merkel cell carcinoma is a rare type of skin cancer that usually appears as flesh-colored or bluish-red nodules on the face, head, or neck. Merkel cell carcinoma is also known as a neuroendocrine carcinoma of the skin. In some embodiments, a method for treating Merkel cell carcinoma includes administering an immunoconjugate containing an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof). In some embodiments, the Merkel cell carcinoma has metastasized at the time of administration.

[0240] Leukemia is a cancer that originates in hematopoietic tissues, such as the bone marrow, and produces large numbers of abnormal blood cells, which then enter the bloodstream. For example, leukemia can arise in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for the rapidity of the disease's onset and progression (e.g., acute vs. chronic) and the type of white blood cell affected (e.g., myeloid vs. lymphoid). Myeloid leukemia is also called myeloid leukemia or myeloblastic leukemia. Lymphoid leukemia is also called lymphoblastic leukemia or lymphocytic leukemia. Lymphoid leukemia cells may collect in lymph nodes, causing them to swell. 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).

[0241] Lymphoma is a cancer that begins in cells of the immune system. For example, lymphoma can arise 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. There are currently six recognized types of HL. Examples of Hodgkin lymphoma include nodular sclerosing classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-reduced CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0242] Another category of lymphoma is non-Hodgkin's lymphoma (NHL), which includes a large and diverse group of cancers of immune system cells. Non-Hodgkin's lymphomas can be further divided into cancers that have an indolent (slow-growing) course and cancers that have an aggressive (fast-growing) course. There are currently 61 recognized types of NHL. Examples of non-Hodgkin's lymphomas include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, hematologic immunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small non-cleavable 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, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0243] Brain cancer includes any cancer of brain tissue, including, but not limited to, glioma (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningioma, pituitary adenoma, vestibular schwannoma, and primitive neuroectodermal tumor (medulloblastoma).

[0244] The immunoconjugates of the present invention can be used in therapy either alone or in combination with other agents. For example, the immunoconjugates can be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapy includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of the immunoconjugate can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The immunoconjugates can also be used in combination with radiation therapy.

[0245] The immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injections such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0246] Atezolizumab, durvalumab, avelumab, their biosimilars, and their biobetters are known to be useful in treating cancer, particularly breast cancer, especially triple-negative (tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma. The immunoconjugates described herein can be used to treat the same types of cancer as atezolizumab, durvalumab, avelumab, their biosimilars, and their biobetters, especially breast cancer, especially triple-negative (tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma.

[0247] The immunoconjugate is administered to a subject in need thereof in a therapeutically effective amount using any suitable dosing regimen, such as those utilized for atezolizumab, durvalumab, avelumab, their biosimilars, and their biobetters. For example, the method can include administering the immunoconjugate to a subject to provide a dose of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can range from 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 dosage of the immunoconjugate may fall outside these ranges, depending on the specific conjugate and the type and severity of the cancer being treated. The frequency of administration may range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week.

[0248] In another aspect, the present invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to a particular cancer to be prevented. For example, the method can comprise administering the immunoconjugate to the subject to provide a dose of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can range from 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 dosage of the immunoconjugate may fall outside these ranges, depending on the specific conjugate and the type and severity of the cancer being treated. The frequency of administration may range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week.

[0249] 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 in various regions of the breast, and multiple different types of breast cancer are characterized. For example, the immunoconjugates of the present invention can be used to treat other forms of breast cancer, such as ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma of the breast, medullary carcinoma, mucinous carcinoma, papillary carcinoma, or cribriform carcinoma); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and triple-negative (negative for estrogen receptor, progesterone receptor, and excess HER2 protein) breast cancer. In some embodiments, methods for treating breast cancer comprise administering an immunoconjugate containing an antibody construct capable of binding to HER2 (e.g., trastuzumab, pertuzumab, a biosimilar, or a biobetter thereof) and an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar, or a biobetter thereof). In some embodiments, methods for treating colon, lung, renal, pancreatic, gastric, and esophageal cancer comprise administering an immunoconjugate containing an antibody construct capable of binding to CEA or tumors that overexpress CEA (e.g., labetuzumab, a biosimilar, or a biobetter thereof).

[0250] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8. [Example]

[0251] Preparation of aminobenzazepine compounds (Bz) and intermediates Example 1: Synthesis of Bz-1 [ka]

[0252] Synthesis of tert-butyl (3-(benzyl(propyl)amino)propyl)carbamate Bz-1a. tert-Butyl N-(3-aminopropyl)carbamate (10 g, 57.39 mmol, 10.02 mL, 1 equiv.) and benzaldehyde (6.09 g, 57.39 mmol, 5.80 mL, 1 equiv.) in DCE (100 mL) were stirred at 70 °C for 24 h. MeOH (100 mL) and NaBHCN (16.23 g, 258.26 mmol, 4.5 equiv.) were added portionwise to the mixture at 0 °C. The mixture was stirred at 0 °C for 2 h, and then propanal (16.67 g, 286.96 mmol, 20.89 mL, 5 equiv.) was added at 0 °C and stirred for 2 h. LCMS showed the reaction was complete. A few drops of water were added to the mixture, and it was concentrated under reduced pressure at 40 °C. The residue was poured into ice water (200 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (200 mL × 3). The combined organic phase was washed with brine (300 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 10 / 1, 3 / 1) to afford tert-butyl N-[3-[benzyl(propyl)amino]propyl]carbamate, Bz-1a (16 g, 52.21 mmol, 90.98% yield) as a pale yellow oil. 1 H NMR (CDCl3, 400 MHz) δ 7.39-7.29 (m, 5H), 3.60-3.52 (m, 2H), 3.20-3.08 (m, 2H), 2.56-2.45 (m, 2H), 2.39 (s, 2H), 1.73-1.61 (m, 2H), 1.58-1.48 (m, 2H), 1.42 (s, 1H), 1.45 (s, 9H), 0.89 (t, J = 7.2 Hz, 3H).

[0253] Synthesis of tert-butyl N-[3-(propylamino)propyl]carbamate, Bz-1b. To a solution of tert-butyl N-[3-[benzyl(propyl)amino]propyl]carbamate, Bz-1a (10 g, 32.63 mmol, 1 equiv.) in MeOH (150 mL) was added Pd(OH) / C (10%, 3 g) under N. The suspension was degassed under vacuum and purged with H several times. The mixture was stirred at 50 °C for 12 h under H (50 psi). TLC (petroleum ether / ethyl acetate = 3:1) showed complete consumption of the starting material. The reaction mixture was filtered, and the filtrate was concentrated to give tert-butyl N-[3-(propylamino)propyl]carbamate, Bz-1b (5 g, 23.11 mmol, 70.83% yield) as a colorless oil, which was used in the next step without further purification. 1 H NMR (MeOD, 400 MHz) δ 3.13-3.05 (m, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.56-2.50 (m, 2H), 1.66 (m, 2H), 1.58-1.48 (m, 2H), 1.44 (s, 9H), 0.94 (t, J = 7.2 Hz, 3H).

[0254] Synthesis of tert-butyl N-[3-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl]carbamate, Bz-1. To a mixture of tert-butyl N-[3-(propylamino)propyl]carbamate, Bz-1b (202.42 mg, 935.73 μmol (micromoles), 2 equiv.) and 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-10c (0.2 g, 467.87 μmol, 1 equiv.) from Example 6 in DMF (2 mL) was added HATU (213.48 mg, 561.44 μmol, 1.2 equiv.) and EtN (94.69 mg, 935.73 μmol, 130.24 μL (microliters), 2 equiv.) in one portion at 15° C. The mixture was stirred at 15° C. for 30 minutes. LCMS and HPLC indicated the reaction was complete. The mixture was filtered and purified by preparative HPLC (column: Waters Xbridge 150 × 25 mm, 5 micron particle size; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 30%-50%, 20 min) to give tert-butyl N-[3-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl]carbamate, Bz-1 (0.087 g, 139.03 μmol, 29.72% yield) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.07 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.86-7.81 (m, 1H), 7.79-7.73 (m, 1H), 7.50-7.45 (m, 2H), 7.39 (m, 1H), 6.92 (s, 1H), 3.86 (t, J = 8.0 Hz, 2H), 3.61-3.58 (m, 2H), 3.52-3.48 (m, 2H), 3.45-3.41 (m, 4H), 3.10 (s, 4H), 2.62-2.52 (m, 1H), 1.86-1.79 (m, 2H), 1.71-1.65 (m, 2H), 1.42-1.50 (m, 9H), 0.87-0.95 (m, 3H). LC / MS [M+H] 626.30 (calculated value); LC / MS [M+H] 626.40 (measured value).

[0255] Example 2: Synthesis of Bz-3 [ka] Synthesis of tert-butyl (3-(benzyl(propyl)amino)propyl)(methyl)carbamate To a mixture of benzaldehyde (310.02 mg, 2.92 mmol, 295.26 μL, 1 equiv.) in DCE (10 mL) was added tert-butyl N-(3-aminopropyl)-N-methyl-carbamate (0.55 g, 2.92 mmol, 1 equiv.) under N at 25 °C. The mixture was stirred at 60 °C for 12 h, then cooled to 0 °C. MeOH (10 mL) was added to the mixture, and NaBHCN (550.48 mg, 8.76 mmol, 3 equiv.) was added to the mixture and stirred for 1 h. Propanal (339.18 mg, 5.84 mmol, 425.04 μL, 2 equiv.) was added to the mixture and stirred at 0 °C for 1 h. LCMS indicated the reaction was complete. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC column: Luna C18 100 × 30 5u; mobile phase: [water (0.1% TFA)-ACN]; B%: 10% to 40%, 10 min to give tert-butyl N-[3-[benzyl(propyl)amino]propyl]-N-methyl-carbamate (0.4 g, 1.25 mmol, 42.75% yield) as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 7.18-7.37 (m, 5H), 3.57 (s, 2H), 3.20 (t, J = 7.2 Hz, 2H), 2.78 (s, 3H), 2.35-2.52 (m, 4H), 1.70 (quintet, J = 7.2 Hz, 2H), 1.47-1.57 (m, 2H), 1.42 (s, 9H), 0.88 (t, J = 7.2 Hz, 3H)

[0256] Synthesis of tert-butyl methyl (3-(propylamino)propyl)carbamate To a solution of tert-butyl N-[3-[benzyl(propyl)amino]propyl]-N-methyl-carbamate (0.4 g, 1.25 mmol, 1 equiv.) in MeOH (20 mL) was added Pd(OH) / C (0.2 g, 5% purity) under N. The suspension was degassed under vacuum and purged with H several times. The mixture was stirred at 50 °C under H (50 psi) for 12 h. LCMS showed the reactant was consumed and the desired mass was detected. The mixture was filtered and concentrated in vacuo. tert-Butyl N-methyl-N-[3-(propylamino)propyl]carbamate (0.25 g, 1.09 mmol, 86.95% yield) was obtained as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 3.26-3.31 (m, 2H), 2.85 (s, 3H), 2.56 (q, J = 8.0 Hz, 4H), 1.74 (quintet, J = 7.2 Hz, 2H), 1.48-1.59 (m, 2H), 1.46 (s, 9H), 0.94 (t, J = 7.2 Hz, 3H)

[0257] Synthesis of tert-butyl(3-(2-amino-8-bromo-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)(methyl)carbamate, Bz-3b To a mixture of 2-amino-8-bromo-3H-1-benzazepine-4-carboxylic acid, Bz-3a (80 mg, 284.59 μmol, 1 equiv.) and tert-butyl N-methyl-N-[3-(propylamino)propyl]carbamate (78.67 mg, 341.51 μmol, 1.2 equiv.) in DMF (1 mL), HATU (162.32 mg, 426.89 μmol, 1.5 equiv.) and EtN (57.60 mg, 569.18 μmol, 79.22 μL, 2 equiv.) were added at 25°C under N2. The mixture was stirred at 25°C for 1 h. LCMS showed the desired product. The mixture was poured into water (20 mL). The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phase was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether / ethyl acetate = 0 / 1) to give Bz-3b (60 mg, 121.60 μmol, 42.73% yield) as a yellow oil.

[0258] Synthesis of tert-butyl(3-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)(methyl)carbamate, Bz-3 To a mixture of [1-(3-bromophenyl)sulfonylazetidin-3-yl]methanol (155.12 mg, 506.65 μmol, 1 equiv.), Pin2B2 (154.39 mg, 607.98 μmol, 1.2 equiv.), potassium acetate, and KOAc (124.31 mg, 1.27 mmol, 2.5 equiv.) in dioxane (30 mL) was added Pd(dppf)Cl·CHCl (41.38 mg, 50.67 μmol, 0.1 equiv.) under N at 25 °C. The mixture was stirred at 90 °C for 2 h. tert-Butyl N-[3-[(2-amino-8-bromo-3H-1-benzazepine-4-carbonyl)-propyl-amino]propyl]-N-methyl-carbamate, Bz-3b (0.25 g, 506.65 μmol, 1 equiv.), KCO (140.04 mg, 1.01 mmol, 2 equiv.) in HO (2 mL) was added to the mixture and stirred at 90 °C for 2 h (h) under nitrogen gas N. LCMS showed the reaction was complete. The mixture was filtered and concentrated in vacuo. The residue was purified by preparative TLC (EtOAc / MeOH = 7:1) to give Bz-3 (112 mg, 175.05 μmol, 34.55% yield) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.07 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.85 (br d, J = 7.6 Hz, 1H), 7.73-7.79 (m, 1H), 7.41-7.54 (m, 3H), 6.95 (s, 1H), 3.86 (t, J = 8.2 Hz, 2H), 3.60 (dd,J = 8.0, 6.0 Hz, 2H), 3.39-3.52 (m, 6H), 3.17-3.29 (m, 2H), 2.82-2.90 (m, 4H), 2.53-2.67 (m, 1H), 1.89-1.92 (m, 2H), 1.66-1.72 (m, 2H), 1.42-1.46 (m, 9H), 0.80-1.05 (m, 3H). LC / MS [M+H] 640.32 (calculated value); LC / MS [M+H] 640.30 (measured value).

[0259] Example 3: Synthesis of Bz-5 [ka] [ka]

[0260] Synthesis of 5-bromo-1-iodo-2-methyl-3-nitrobenzene, Bz-5b To a mixture of 4-bromo-1-methyl-2-nitrobenzene, Bz-5a (20 g, 92.58 mmol, 20.00 mL, 1 equiv.) in H2SO4 (20 mL) was added NIS (37.49 g, 166.64 mmol, 1.8 equiv.) under N2 at 0 °C. The mixture was stirred at 0 °C for 1 h. TLC showed that the reactant was consumed and two points had formed. The mixture was slowly poured into ice water (200 mL). The aqueous phase was extracted with ethyl acetate (150 mL x 2). The combined organic phase was washed with brine (150 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 = 100 / 1, 20 / 1) to give Bz-5b (14 g, 40.94 mmol, 44.23% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 8.20 (d, J= 2.0 Hz, 1H), 7.87 (d, J=2.0 Hz, 1H), 2.55 (s, 3H).

[0261] Synthesis of 5-bromo-2-(bromomethyl)-1-iodo-3-nitrobenzene, Bz-5c To a mixture of 5-bromo-1-iodo-2-methyl-3-nitrobenzene, Bz-5b (13 g, 38.02 mmol, 1 equiv.) in CCl (100 mL) was added NBS (10.15 g, 57.03 mmol, 1.5 equiv.), BPO (920.94 mg, 3.80 mmol, 0.1 equiv.) under N at 25 °C. The mixture was stirred at 90 °C for 12 h. TLC showed one new point formed, and HPLC and LCMS showed approximately 50% as desired, with approximately 50% reaction remaining. The mixture 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 = 50 / 1, 10 / 1) to give Bz-5c (7 g, 16.63 mmol, 43.75% yield) as a white solid. 1 H NMR (CDCl3-d6, 400 MHz) δ 8.29 (d, J= 2.0 Hz, 1H), 8.02 (d, J= 2.0 Hz, 1H), 4.82 (s, 3H).

[0262] Synthesis of 4-bromo-2-iodo-6-nitrobenzaldehyde, Bz-5d To a mixture of 5-bromo-2-(bromomethyl)-1-iodo-3-nitrobenzene, Bz-5c (7 g, 16.63 mmol, 1 equiv.) in CHCN (10 mL) was added NMO (3.90 g, 33.27 mmol, 3.51 mL, 2 equiv.) under N at 25 °C. The mixture was stirred at 25 °C for 2 h. TLC showed the reaction was complete. The mixture 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 = 20 / 1, 4 / 1) to give Bz-5d (5 g, 14.05 mmol, 84.46% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 10.00 (s, 1H), 8.37 (d, J= 1.6 Hz, 1H), 8.15 (d, J= 1.6 Hz, 1H).

[0263] Synthesis of (E)-ethyl 3-(4-bromo-2-iodo-6-nitrophenyl)-2-(cyanomethyl)acrylate, Bz-5e To a mixture of 4-bromo-2-iodo-6-nitro-benzaldehyde, Bz-5d (3.5 g, 9.83 mmol, 1 equiv.) in toluene (30 mL), ethyl 3-cyano-2-(triphenylphosphanylidene)propanoate (5.71 g, 14.75 mmol, 1.5 equiv.) was added at 25 °C under N. The mixture was stirred at 85 °C for 12 h. TLC showed the desired product. The mixture 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 give Bz-5e (2 g, 4.30 mmol, 43.73% yield) as a yellow oil. 1 H NMR (CDCl3, 400 MHz) δ 8.62 (d, J= 1.8 Hz, 1H), 8.42 (d, J= 1.8 Hz, 1H), 7.74 (s, 1H), 4.32 (q, J= 7.2 Hz, 2H), 3.33 (s, 2H), 1.31 (t, J= 7.2Hz, 3H)

[0264] Synthesis of ethyl 2-amino-8-bromo-6-iodo-3H-benzo[b]azepine-4-carboxylate, Bz-5f To a mixture of ethyl (E)-3-(4-bromo-2-iodo-6-nitro-phenyl)-2-(cyanomethyl)prop-2-enoate, Bz-5e (2 g, 4.30 mmol, 1 equiv.) in acetic acid, AcOH (20 mL), Fe (1.20 g, 21.50 mmol, 5 equiv.) was added at 25 °C under N. The mixture was stirred at 80 °C for 5 h. LCMS showed a measure of desired product, indicating that the reactant had been consumed. The reaction 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 = 1 / 1, 0 / 1) to give Bz-5f (1.8 g, 4.14 mmol, 96.20% yield) as an off-white solid. 1H NMR (DMSO-d6, 400 MHz) δ 7.71 (s, 1H), 7.69 (d, J= 2.0 Hz, 1H), 7.22 (br d, J= 2.0 Hz, 1H), 4.26 (q, J= 7.0 Hz, 3H), 2.83 (s, 2H), 1.30 (t, J= 7.2 Hz, 3H).

[0265] Synthesis of 2-amino-8-bromo-6-iodo-3H-benzo[b]azepine-4-carboxylic acid, Bz-5g To a mixture of ethyl 2-amino-8-bromo-6-iodo-3H-1-benzazepine-4-carboxylate, Bz-5f (1.8 g, 4.14 mmol, 1 equiv.) in EtOH (40 mL) was added LiOH HO (1.04 g, 24.82 mmol, 6 equiv.) in HO (10 mL) at 25 °C under N. The mixture was stirred at 35 °C for 2 h. LCMS showed the reaction was complete. The mixture was concentrated to remove EtOH, then the pH was adjusted to 5 with aqueous HCl (4 M) and filtered to give the desired solid, Bz-5g (1.2 g, 2.95 mmol, 71.26% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.77 (s, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 2.92 (s, 2H)

[0266] Synthesis of 2-amino-8-bromo-6-iodo-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, Bz-5h To a mixture of N-propylpropan-1-amine (186.47 mg, 1.84 mmol, 254.04 μL, 1.5 equiv) and 2-amino-8-bromo-6-iodo-3H-1-benzazepine-4-carboxylic acid, Bz-5g (0.5 g, 1.23 mmol, 1 equiv) in DMF (10 mL) was added HATU (700.67 mg, 1.84 mmol, 1.5 equiv), EtN (186.47 mg, 1.84 mmol, 256.49 μL, 1.5 equiv) at 25 °C. The mixture was stirred at 25 °C for 30 min. LCMS indicated the reaction was complete. The mixture was poured into water (50 mL), separated from the mixture, and filtered to give Bz-5h (0.55 g, 1.12 mmol, 91.33% yield) as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 7.74 (d, J= 2.0 Hz, 1H), 7.33 (d, J= 2.0 Hz, 1H), 6.81 (s, 1H), 3.43-3.47 (m, 4H), 1.66-1.72 (m, 4H), 0.93 (s, 6H)

[0267] Synthesis of tert-butyl (4-(2-amino-8-bromo-4-(dipropylcarbamoyl)-3H-benzo[b]azepin-6-yl)but-3-yn-1-yl)carbamate, Bz-5i To a mixture of 2-amino-8-bromo-6-iodo-N,N-dipropyl-3H-1-benzazepine-4-carboxamide, Bz-5h (200 mg, 408.02 μmol, 1 equiv.), and tert-butyl N-but-3-ynylcarbamate (72.50 mg, 428.42 μmol, 1.05 equiv.) in DMF (5 mL), EtN (1 mL), Pd(PPh)Cl (14.32 mg, 20.40 μmol, 0.05 equiv.), EtN (0.5 mL), and CuI (15.54 mg, 81.60 μmol, 0.2 equiv.) were added at 25 °C under N. The mixture was stirred at 80 °C for 1 h. LCMS showed a major reaction as desired. The mixture was poured into water (20 mL). The aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phase was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (petroleum ether / ethyl acetate = 0 / 1) to give Bz-5i (0.2 g, 376.31 μmol, 92.23% yield) as a yellow solid. 1 H NMR (CDCl3, 400 MHz) δ 7.40 (s, 1H), 7.35 (s, 1H), 7.13 (s, 1H), 3.46-3.52 (m, 4H), 3.35-3.40 (m, 2H), 2.65 (s, 2H), 1.58-1.78 (m, 4H), 1.46 (s, 9H), 0.93 (t, J= 7.2 Hz, 6H)

[0268] Synthesis of tert-butyl (4-(2-amino-4-(dipropylcarbamoyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepin-6-yl)but-3-yn-1-yl)carbamate, Bz-5j tert-Butyl N-[4-[2-amino-8-bromo-4-(dipropylcarbamoyl)-3H-1-benzazepin-6-yl]but-3-ynyl]carbamate, Bz-5i (0.18 g, 338.67 μmol, 1 equiv.) and [1-[3-(4,4,5,5-tetramethyl-1,3,2- To a mixture of [(dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methanol (179.45 mg, 508.01 μmol, 1.5 equiv.), Pd(dppf)Cl (12.39 mg, 16.93 μmol, 0.05 equiv.), KCO (93.61 mg, 677.35 μmol, 2 equiv.) was added under N at 25 °C. The mixture was stirred at 90 °C for 2 h. LCMS showed that the desired mass was detected. The mixture was concentrated in vacuo to give Bz-5j (0.2 g, crude) as a yellow solid.

[0269] Synthesis of tert-butyl (4-(2-amino-4-(dipropylcarbamoyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepin-6-yl)butyl)carbamate, Bz-5 To a solution of tert-butyl N-[4-[2-amino-4-(dipropylcarbamoyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepin-6-yl]but-3-ynyl]carbamate, Bz-5j (140 mg, 206.53 μmol, 1 equiv.) in MeOH (20 mL) was added Pd(OH) / C (0.1 g, 5% purity) under N. The suspension was degassed under vacuum and purged with H several times. The mixture was stirred for 2 h at 25 °C under H (50 psi). LCMS indicated the reaction was complete. The mixture was filtered and concentrated in vacuo. The residue was purified by preparative HPLC column: Xtimate C18 150 × 25 mm, 5 micron particle size; mobile phase: [water (0.04% NH3H2O + 10 mM NH4HCO3)-ACN]; B%: 50%–60%, 10.5 min. Bz-5 (45 mg, 65.99 μmol, 31.95% yield) was obtained as a white solid. 1H NMR (MeOD, 400 MHz) δ 8.00-8.08 (m, 2H), 7.83 (d, J= 7.6 Hz, 1H), 7.71-7.79 (m, 1H), 7.33 (s, 1H), 7.28 (s, 1H), 6.99 (s, 1H), 3.86 (t, J= 8.0 Hz, 2H), 3.57-3.66 (m, 2H), 3.38-3.51 (m, 6H), 3.06 (t, J= 6.4 Hz, 2H), 2.84 (t, J= 7.6 Hz, 2H), 2.52-2.63 (m, 1H), 1.50-1.77 (m, 8H), 1.41 (s, 9H), 0.94 (s, 6H). LC / MS [M+H] 682.36 (calculated); LC / MS [M+H] 682.40 (observed).

[0270] Example 4: Synthesis of Bz-6 [ka]

[0271] Synthesis of tert-butyl ((1-((3-bromophenyl)sulfonyl)azetidin-3-yl)methyl)carbamate, Bz-6a To a mixture of tert-butyl N-(azetidin-3-ylmethyl)carbamate (1.6 g, 8.59 mmol, 1.2 equiv) in DCM (5 mL) was added TEA (1.45 g, 14.32 mmol, 1.99 mL, 2 equiv) and 3-bromobenzenesulfonyl chloride (1.83 g, 7.16 mmol, 1.03 mL, 1 equiv) at 0 °C. The mixture was stirred at 20 °C for 1 h. The mixture was diluted with water (50 mL) and extracted with DCM (25 mL × 3). The organic layer was washed with brine (25 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® silica flash column, elution with a 0–100% ethyl acetate / petroleum ether gradient at 35 mL / min). The compound tert-butyl N-[[1-(3-bromophenyl)sulfonylazetidin-3-yl]methyl]carbamate, Bz-6a (2.5 g, 6.17 mmol, 86.16% yield) was obtained as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 7.99 (t, J= 4.0 Hz, 1H), 7.74-7.81 (m, 2H), 7.47 (t, J = 8.0 Hz, 1H), 4.61 (s, 1H), 3.86 (t, J = 8.0 Hz, 2H), 3.50-3.58 (m, 2H), 3.19 (t, J = 4.0 2H), 2.58-2.70 (m, 1H), 1.42 (s, 9H).

[0272] Preparation of tert-butyl N-[[1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methyl]carbamate, Bz-6b To a mixture of tert-butyl-N-[[1-(3-bromophenyl)sulfonylazetidin-3-yl]methyl]carbamate, Bz-6a (1 g, 2.47 mmol, 1 equiv.) in dioxane (10 mL), Pin2B2 (939.80 mg, 3.70 mmol, 1.5 equiv.), KOAc (484.29 mg, 4.93 mmol, 2 equiv.), and Pd(dppf)Cl (90.27 mg, 123.36 μmol, 0.05 equiv.) were added under N at 15° C. The mixture was stirred at 110° C. for 2 h. The product tert-butyl N-[[1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methyl]carbamate, Bz-6b, was not isolated and was used in the next step.

[0273] Synthesis of tert-butyl ((1-((3-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepin-8-yl)phenyl)sulfonyl)azetidin-3-yl)methyl)carbamate, Bz-6 To a mixture of tert-butyl N-[[1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methyl]carbamate, Bz-6b (1.12 g, 2.48 mmol, 1 equiv.) and 2-amino-8-bromo-N,N-dipropyl-3H-1-benzazepine-4-carboxamide, Bz-6c (901.90 mg, 2.48 mmol, 1 equiv.) in dioxane (3 mL), KCO (684.35 mg, 4.95 mmol, 2 equiv.) and Pd(dppf)Cl (90.58 mg, 123.79 μmol, 0.05 equiv.) were added under N at 15 °C. The mixture was stirred at 120 °C for 2 h. The mixture was filtered and concentrated, and the residue was purified by flash silica gel chromatography (ISCO®; 2 g SepaFlash® silica flash column, 0–100% ethyl acetate / petroleum ether gradient elution at 60 mL / min) to afford Bz-6 (600 mg, 983.97 μmol, 39.74% yield, 100% purity) as a yellow solid. 1H NMR (MeOD-d4, 400 MHz) δ 7.99-8.10 (m, 2H), 7.74-7.86 (m, 2H), 7.36-7.52 (m, 3H), 6.89 (s, 1H), 3.83 (t, J = 8.0 Hz, 2H), 3.54 (t, J = 8.0 Hz, 2H), 3.34-3.48 (m, 6H), 3.02 (d, J = 8.0 Hz, 2H), 2.48-2.64 (m, 1H), 1.59-1.76 (m, 4H), 1.37 (s, 9H), 0.96-0.89 (m, 6H). LC / MS [M+H] 610.31 (calculated); LC / MS [M+H] 610.40 (observed).

[0274] Example 5: Synthesis of Bz-9 [ka] Synthesis of tert-butyl (5-(benzyl(propyl)amino)pentyl)carbamate Bz-9a A mixture of tert-butyl N-(5-aminopentyl)carbamate (1 g, 4.94 mmol, 1.03 mL, 1 equiv.) and benzaldehyde (524.59 mg, 4.94 mmol, 499.61 μL, 1 equiv.) in DCE (10 mL) was stirred at 60 °C for 12 h. The mixture was then cooled to 0 °C, and MeOH (10 mL) was added to the mixture. NaBHCN (931.94 mg, 14.83 mmol, 3 equiv.) was added to the mixture and stirred at 0 °C for 1 h. Propanal (574.20 mg, 9.89 mmol, 719.55 μL, 2 equiv.) was added to the mixture and stirred for 1 h. LCMS indicated the reaction was complete. The mixture was concentrated. The residue was further purified by preparative HPLC (column: Luna C18 100 × 30, 5 micron particle size; mobile phase: [water (0.1% TFA)-ACN]; B%: 25% to 40%, 10 min) to afford tert-butyl-N-[5-[benzyl(propyl)amino]pentyl]carbamate Bz-9a (0.5 g, 1.49 mmol, 30.24% yield) as a yellow oil. 1H NMR (400MHz, methanol-d4) δ = 7.33-7.28 (m, 3H), 7.27-7.19 (m, 1H), 3.58 (s, 2H), 3.00 (t, J =7.2 Hz, 2H), 2.47-2.37 (m, 4H), 1.58-1.46 (m, 6H), 1.47 (s, 9H) 1.37-1.20 (m, 3H), 0.87 (t, J =7.6 Hz, 3H)

[0275] Synthesis of tert-butyl (5-(propylamino)pentyl)carbamate Bz-9b To a solution of tert-butyl N-[5-[benzyl(propyl)amino]pentyl]carbamate Bz-9a (0.5 g, 1.49 mmol, 1 equiv.) in MeOH (20 mL) was added Pd(OH)2 / C (0.2 g, 5% purity) at 25 °C under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred for 12 h at 50 °C under H2 (50 psi). LCMS showed the reaction was complete. The mixture was filtered and concentrated. The product, tert-butyl N-[5-(propylamino)pentyl]carbamate Bz-9b (0.3 g, crude), was obtained as a colorless oil. 1 H NMR (400MHz, methanol-d4) δ = 3.03 (t, J = 6.8 Hz, 2H), 2.55 (d, J = 7.6, 13.6 Hz, 4H), 1.59-1.44 (m, 6H), 1.47 (s. 9H)1.43-1.20 (m, 2H), 0.97-0.88 (m, 3H).

[0276] To a mixture of tert-butyl N-[5-(propylamino)pentyl]carbamate Bz-9b (57.17 mg, 233.93 μmol, 1 equiv.) and 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid Bz-10c (0.1 g, 233.93 μmol, 1 equiv.) in DMF (4 mL) was added HATU (133.42 mg, 350.90 μmol, 1.5 equiv.) and EtN (71.02 mg, 701.80 μmol, 97.68 μL, 3 equiv.) in one portion at 25 °C. The mixture was stirred at 25 °C for 0.5 h. LCMS indicated the reaction was complete. The mixture was diluted with water and extracted with EA (30 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was further purified by preparative HPLC (column: Xtimate C18 150 × 25 mm, 5 micron particle size; mobile phase: [water (0.1% TFA)-ACN]; B%: 32% to 62%, 10.5 min) to give tert-butyl N-[5-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]pentyl]carbamate Bz-9 (0.128 g, 179.48 μmol, 76.72% yield, 91.68% purity) as a yellow solid. 1H NMR (400MHz, methanol-d4) δ = 8.10 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.83-7.78 (m, 1H), 7.77-7.65 (m, 3H), 7.09 (s, 1H), 3.86 (t, J = 8.2 Hz, 2H), 3.61 (J = 5.6, 8.0 Hz, 2H), 3.56-3.35 (m, 8H), 3.31 (s, 2H), 3.10-2.99 (m, 2H), 2.64-2.53 (m, 1H), 1.80-1.59 (m, 4H), 1.57-1.47 (m, 2H), 1.40 (s, 9H), 1.03-0.86 (m, 3H). LC / MS [M+H] 654.33 (calculated value); LC / MS [M+H] 654.50 (measured value).

[0277] Example 6: Synthesis of Bz-10 [ka] Preparation of Bz-10c: To a mixture of tert-butyl 3-(hydroxymethyl)azetidine-1-carboxylate Bz-10d (15 g, 80.11 mmol) in DCM (100 mL) was added TFA (63.94 g, 560.79 mmol, 41.52 mL, 7 equiv) at 15 °C. The mixture was stirred at 15 °C for 1 h. The mixture was concentrated to give azetidin-3-ylmethanol Bz-10e (36 g, crude, TFA) as a yellow oil. 1 H NMR (DMSO-d6, 400 MHz) δ 4.50-4.56 (m, 2H), 3.94-4.10 (m, 2H), 3.80-3.93 (m, 2H), 3.15-3.30 (m, 1H).

[0278] Preparation of [1-(3-bromophenyl)sulfonylazetidin-3-yl]methanol, Bz-10f: To a mixture of azetidin-3-ylmethanol (33.06 g, 164.37 mmol, 2 equiv., TFA) and 3-bromobenzenesulfonyl chloride (21 g, 82.19 mmol, 11.86 mL, 1 equiv.) in DCM (200 mL) was added TEA (33.27 g, 328.75 mmol, 45.76 mL, 4 equiv.) at 0 °C. The mixture was stirred at 15 °C for 1 h. The residue was poured into saturated sodium bicarbonate in water (200 mL) and stirred for 10 min. The aqueous phase was extracted with DCM (100 mL × 3). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 1 g SepaFlash® silica flash column, elution with a 0-100% ethyl acetate / petroleum ether gradient at 50 mL / min) to give the compound [1-(3-bromophenyl)sulfonylazetidin-3-yl]methanol Bz-10f (21 g, 68.59 mmol, 83.45% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 7.89-8.11 (m, 1H), 7.78 (dd, J = 8.0, 2.0 Hz, 2H), 7.39-7.54 (m, 1H), 3.78-3.97 (m, 2H), 3.49-3.74 (m, 4H), 2.41-2.77 (m, 1H).

[0279] Preparation of [1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaneboron-2-yl)phenyl]sulfonylazetidin-3-yl]methanol, Bz-10g: To a mixture of [1-(3-bromophenyl)sulfonylazetidin-3-yl]methanol (8 g, 26.13 mmol, 1 equiv.) in dioxane (10 mL), Pin2B2 (9.95 g, 39.19 mmol, 1.5 equiv.), KOAc (5.13 g, 52.26 mmol, 2 equiv.), and Pd(dppf)Cl2 (1.91 g, 2.61 mmol, 0.1 equiv.) were added at 15 °C. The mixture was stirred at 110 °C for 3 h. LC-MS showed that reactant 1 was completely consumed and one major peak with the desired mass was detected. The mixture was filtered and washed with ethyl acetate. The filtrate was then concentrated under vacuum. 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 / 1, 0 / 1) to obtain 12 g of crude product. The crude product was triturated with heptane / methyl tert-butyl ether = 5 / 1 (50 mL), filtered, and the filter cake was dried under vacuum. The compound [1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methanol (8.2 g, 23.21 mmol, 88.84% yield) was obtained as a pink solid. 1 H NMR (CDCl3, 400 MHz) δ 8.28 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.89-7.95 (m, 1H), 7.58 (t, J = 8.0 Hz, 1H), 3.87 (t, J = 8.0 Hz, 2H), 3.62-3.68 (m, 4H), 2.55-2.65 (m, 1H), 1.37 (s, 12H).

[0280] Preparation of ethyl 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylate, Bz-10h: [1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methanol in dioxane (40 mL) and HO (3 mL). To a mixture of Bz-10g (4.11 g, 11.64 mmol, 1.2 equiv.) and ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate (3 g, 9.70 mmol, 1 equiv.) was added K2CO3 (2.68 g, 19.41 mmol, 2 equiv.) and Pd(dppf)Cl2 (355.02 mg, 485.19 μmol, 0.05 equiv.) under N2 at 15 °C. The mixture was stirred at 110 °C for 3 h. LC-MS showed that reactant 1 was completely consumed and one major peak with the desired mass was detected. The mixture was concentrated. The crude product was triturated with 1:1 EtOAc / H2O (200 mL) at 0 °C for 10 min, filtered, and the filter cake was dried in vacuo. Compound ethyl 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylate, Bz-10h (4 g, crude) was obtained as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.06-8.15 (m, 1H), 7.96 (s, 1H), 7.71-7.85 (m, 3H), 7.57 (d, J = 8.0 Hz, 1H), 7.29-7.38 (m, 2H), 6.94 (s, 2H), 4.17-4.30 (m, 2H), 3.77 (t, J = 8.0 Hz, 2H), 3.49 (t, J = 8.0 Hz, 2H), 3.2 (d, J = 8.0 Hz, 2H), 2.93 (s, 2H), 2.43-2.49 (m, 1H), 1.31 (t, J = 8.0 Hz, 3H).

[0281] 2-Amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-10c To a solution of ethyl 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylate, Bz-10h (4 g, 8.78 mmol, 1 equiv.) in MeOH (50 mL) and HO (10 mL) was added LiOH·HO (1.84 g, 43.91 mmol, 5 equiv.). The mixture was stirred at 30 °C for 12 h. LC-MS showed that reactant 1 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove MeOH. The mixture was filtered. The pH of the filtrate was adjusted to approximately 6 by slowly adding a solution of HCl (1 M) and then filtered to obtain the crude product. The crude product was triturated with CHCN (100 mL) at 0 °C for 10 min. The product was dried in vacuo. The compound 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-10c (2.51 g, 5.72 mmol, 65.11% yield, 97.375% purity) was obtained as a gray solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.11-8.16 (m, 1H), 8.02 (s, 1H), 7.92 (s, 1H), 7.78-7.88 (m, 4H), 7.75 (s, 1H), 3.76 (t, J = 8.0 Hz, 2H), 3.45-3.54 (m, 4H), 3.20 (d, J = 4.0 Hz, 2H), 2.45-2.49 (m, 1H). LC / MS [M+H] 428.13 (calculated value); LC / MS [M+H] 428.20 (measured value).

[0282] [ka] Synthesis of tert-butyl N-[2-[benzyl(propyl)amino]ethyl]carbamate Bz-10a To a mixture of benzaldehyde (2 g, 18.85 mmol, 1.90 mL, 1 equiv.) and tert-butyl N-(2-aminoethyl)carbamate (3.32 g, 20.73 mmol, 3.26 mL, 1.1 equiv.) in DCE (30 mL) was added NaBHCN (2.37 g, 37.69 mmol, 2 equiv.) at 0 °C. The mixture was stirred at 0 °C for 30 min, and then propanal (5.47 g, 94.23 mmol, 6.86 mL, 5 equiv.) was added to the mixture and stirred at 25 °C for 1 h. The mixture was poured into ice-water (50 mL), and the aqueous phase was extracted with ethyl acetate (50 mL × 3). The combined organic phase was washed with brine (50 mL), dried over anhydrous NaSO, 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, 1 / 1) to give tert-butyl N-[2-[benzyl(propyl)amino]ethyl]carbamate Bz-10a (3 g, 10.26 mmol, 54.44% yield) as a colorless oil.

[0283] Synthesis of tert-butyl N-[2-(propylamino)ethyl]carbamate Bz-10b To a solution of tert-butyl N-[2-[benzyl(propyl)amino]ethyl]carbamate (2 g, 6.84 mmol, 1 equiv.) in MeOH (50 mL) was added Pd(OH) / C (10%, 1 g) under N. The suspension was degassed under vacuum and purged with H several times. The mixture was stirred at 50 °C for 12 h under H (50 psi). TLC (petroleum ether / ethyl acetate = 3:1) showed complete consumption of the starting material. The reaction mixture was filtered, and the filtrate was concentrated to give the crude product tert-butyl N-[2-(propylamino)ethyl]carbamate (1.3 g, 6.43 mmol, 93.96% yield) as a colorless oil, which was used in the next step without further purification. 1H NMR (MeOD, 400MHz) δ 3.18 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H), 2.56 (t, J = 8.0 Hz, 2H), 1.58-1.48 (m, 2H), 1.44 (s, 9H), 0.94 (t, J = 8.0 Hz, 3H).

[0284] Synthesis of tert-butyl (2-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)ethyl)carbamate, Bz-10 To a mixture of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-10c (0.15 g, 350.90 μmol, 1 equiv.) and tert-butyl-N-[2-(propylamino)ethyl]carbamate (141.97 mg, 701.80 μmol, 2 equiv.) in DMF (4 mL) was added HATU (160.11 mg, 421.08 μmol, 1.2 equiv.) and EtN (106.52 mg, 1.05 mmol, 146.52 μL, 3 equiv.) in one portion at 25° C. The mixture was stirred at 25° C. for 12 h. LCMS indicated the reaction was complete. The mixture was filtered and purified by preparative HPLC (column: Waters Xbridge 150 × 25 5u; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 25%-45%, 20 min) to give tert-butyl N-[2-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]ethyl]carbamate (0.036 g, 55.05 μmol, 15.69% yield, 93.54% purity) as a yellow solid. 1H NMR (MeOD, 400MHz) δ 8.07 (s, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.86-7.81 (d, J = 8.0 Hz, 1H), 7.78-7.73 (m, 1H), 7.47 (s, 2H), 7.41-7.36 (m, 1H), 6.95 (s, 1H), 3.86 (t, J = 8.4 Hz, 2H), 3.62-3.53 (m, 4H), 3.49-3.44 (m, 2H), 3.41 (d, J = 6.4 Hz, 2H), 3.32-3.29 (m, 3H), 2.63-2.51 (m, 1H), 1.68 (d, J = 7.2 Hz, 2H), 1.43 (s, 9H), 0.98-0.83 (m, 3H). LC / MS [M+H] 612.29 (calculated value); LC / MS [M+H] 612.40 (measured value).

[0285] Example 7: Synthesis of Bz-11 Synthesis of 2-amino-N-(3-aminopropyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-11a. [ka] To a mixture of tert-butyl N-[3-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl]carbamate, Bz-1 (0.5 g, 799.01 μmol, 1 equiv.) in DCM (20 mL), TFA (1.82 g, 15.98 mmol, 1.18 mL, 20 equiv.) was added in one portion at 15 °C. The mixture was stirred at 15 °C for 3 h. LCMS showed that the reactants were consumed. The mixture was concentrated in vacuo, and the residue was poured into ice water (30 mL) and adjusted to pH = 11 with aqueous Na2CO3. The aqueous phase was extracted with DCM / i-PrOH = 3 / 1 (20 mL × 3). The combined organic phase was washed with brine (10 mL), dried over NaSO, filtered, and concentrated in vacuo to give the crude product 2-amino-N-(3-aminopropyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-11a (0.4 g, crude) as a yellow oil, which was used in the next step without further purification.

[0286] Synthesis of 2-amino-N-[3-(tert-butylcarbamoylamino)propyl]-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-11 [ka] To a solution of 2-amino-N-(3-aminopropyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-11a (0.1 g, 190.24 μmol, 1 equiv) in DMF (2 mL) was added 2-isocyanato-2-methyl-propane (18.86 mg, 190.24 μmol, 22.45 μL, 1 equiv) in one portion at 15° C. The mixture was stirred at 15° C. for 12 h. LCMS showed the reaction was complete. The mixture was filtered and purified by preparative HPLC (column: Nano-micro Kromasil® (Nouryon) C18 100 × 30 mm, 5 micron particle size; mobile phase: [water (0.1% TFA)-ACN]; B%: 25% to 45%, 10 min) to give the crude product, which was then purified by preparative HPLC (column: Welch Xtimate C18 150 × 25 mm, 5 micron particle size; mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 25% to 65%, 10.5 min) to give Bz-11 (0.007 g, 11.20 μmol, 5.89% yield) as a pale yellow solid. 1 H NMR (MeOD, 400 MHz) δ 8.09 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.87-7.85 (m, 1H), 7.80-7.76 (m, 1H), 7.51-7.49 (m, 2H), 7.43-7.41 (m, 1H), 6.94 (s, 1H), 3.88 (t, J = 8.0 Hz, 2H), 3.63-3.60 (m, 2H), 3.54-3.50 (m, 2H), 3.44-3.43 (m, 4H), 3.15-2.91 (m, 4H), 2.67-2.58 (m, 1H), 1.84-1.79 (m, 2H), 1.73-1.66 (m, 2H), 1.40-1.14 (m, 9H), 1.00-0.90 (m, 3H).

[0287] Example 8 Synthesis of Bz-12 [ka] To a solution of 2-amino-N-(3-aminopropyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-11a (0.1 g, 190.24 μmol, 1 equiv.) in DMF (0.3 mL) was added 3-isocyanatobenzonitrile (27.42 mg, 190.24 μmol, 1 equiv.) in one portion at 15° C. The mixture was stirred at 15° C. for 12 h. LCMS showed the reaction was complete. The mixture was filtered and purified by preparative HPLC (column: Nano-micro Kromasil C18 100 × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%–45%, 10 min) to give 2-amino-N-[3-[(3-cyanophenyl)carbamoylamino]propyl]-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-12 (10 mg, 14.93 μmol, 7.85% yield) as a yellow solid. 1 H NMR (CD3OD, 400 MHz) δ 8.21-7.88 (m, 4H), 7.86-7.80 (m, 1H), 7.68 (s, 3H), 7.59-7.24 (m, 3H), 7.15 (s, 1H), 3.89 (t, J = 8.0 Hz, 2H), 3.64 (m, 4H), 3.51 (s, 2H), 3.46 (d, J = 6.0 Hz, 2H), 3.40 (s, 2H), 3.30-3.19 (m, 2H), 2.63-2.60 (m, 1H), 1.96-1.92 (m, 2H), 1.77-1.71 (m, 2H), 1.07-0.86 (m, 3H).

[0288] Example 9 Synthesis of Bz-13 [ka] To a mixture of 2-amino-N-(3-aminopropyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-11a (0.1 g, 190.24 μmol, 1 equiv.) in DMF (2 mL) was added ethyl carbonochloridate (61.94 mg, 570.72 μmol, 54.33 μL, 3 equiv.) in one portion at 15° C. The mixture was stirred at 15° C. for 1 h. LCMS and HPLC showed the desired product was detected. The mixture was filtered and purified by preparative HPLC (column: Waters Xbridge BEH C18 100 × 25 mm, 5 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-45%, 20 min) to give ethyl N-[3-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl]carbamate, Bz-13 (0.018 g, 30.11 μmol, 15.83% yield) as a pale yellow solid. 1 H NMR (CD3OD, 400 MHz) δ 8.11 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.81-7.75 (m, 1H), 7.74-7.68 (m, 2H), 7.12 (s, 1H), 4.07 (brs, 2H), 3.87 (t, J = 8.0 Hz, 2H), 3.61 (m, 2H), 3.55 (m, 2H), 3.48 (m, 2H), 3.42 (d, J = 6.4 Hz, 2H), 3.37 (s, 2H), 3.14 (m, 2H), 2.67-2.51 (m, 1H), 1.93-1.80 (m, 2H), 1.77-1.64 (m, 2H), 1.33-1.06 (m, 3H), 0.95 (s, 3H).

[0289] Example 10 Synthesis of Bz-14 [ka] 2-Amino-6-(4-aminobutyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, Bz-14, was synthesized from Bz-5 according to the procedure described for Bz-11a. LC / MS [M+H] 582.31 (calculated); LC / MS [M+H] 582.57 (observed).

[0290] Example 11: Synthesis of Bz-15 [ka] To a solution of tert-butyl N-[[1-[3-[2-amino-4-(dipropylcarbamoyl)-3H-1-benzazepin-8-yl]phenyl]sulfonylazetidin-3-yl]methyl]carbamate, Bz-6 (0.15 g, 245.99 μmol, 1 equiv.) in DCM (20 mL) was added TFA (56.10 mg, 491.98 μmol, 36.43 μL, 2 equiv.) at 25° C. and stirred for 1 h. The mixture was concentrated under reduced pressure at 40° C. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 25% to 50%, 10 min) to give 2-amino-8-[3-[3-(aminomethyl)azetidin-1-yl]sulfonylphenyl]-N,N-dipropyl-3H-1-benzazepine-4-carboxamide, Bz-15 (0.0546 g, 105.69 μmol, 42.97% yield, 98.66% purity) as a yellow solid. 1H NMR (MeOD-d4, 400 MHz) δ 8.16-8.07 (m, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.79-7.72 (m, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.09 (s, 1H), 3.96 (t, J = 8.4 Hz, 2H), 3.67-3.63 (m, 2H), 3.50-3.42 (m, 4H), 3.37 (s, 2H), 3.05 (d, J = 7.4 Hz, 2H), 2.78-2.65 (m, 1H), 1.75-1.66 (m, 4H), 1.08-0.82 (m, 6H). LC / MS [M+H] 510.25 (calculated); LC / MS [M+H] 510.10 (observed).

[0291] Example 12: Synthesis of Bz-16 [ka] Synthesis of N-(2-acetamidoethyl)-1-(5-nitropyridin-2-yl)piperidine-4-carboxamide, Bz-16a. To a mixture of acetyl chloride (142.82 mg, 1.82 mmol, 129.83 μL, 3 equiv.) and N-(2-aminoethyl)-1-(5-nitro-2-pyridyl)piperidine-4-carboxamide, BzL-23b (0.2 g, 606.46 μmol, 1 equiv., HCl) in THF (10 mL) was added EtN (245.47 mg, 2.43 mmol, 337.65 μL, 4 equiv.) under N at 25° C. The mixture was stirred at 25° C. for 1 h. LCMS showed the reaction was complete. The mixture was poured into water (20 mL). The mixture was filtered to give Bz-16a (0.2 g, 596.38 μmol, 98.34% yield) as a yellow solid. 1H NMR (DMSO-d6, 400 MHz) δ 8.95 (d, J = 2.4 Hz, 1H), 8.19 (dd, J = 9.6, 2.4 Hz, 1H), 7.78-7.98 (m, 2H), 6.95 (d, J = 9.6 Hz, 1H), 4.50 (d, J = 9.6 Hz, 2H), 2.93-3.15 (m, 7H), 1.73-1.80 (m, 5H), 1.43-1.62 (m, 2H), 1.07-1.28 (m, 3H).

[0292] Synthesis of N-(2-acetamidoethyl)-1-(5-aminopyridin-2-yl)piperidine-4-carboxamide, Bz-16b. To a solution of N-(2-acetamidoethyl)-1-(5-nitro-2-pyridyl)piperidine-4-carboxamide, Bz-16a (0.2, 596.38 μmol, 1 equiv.) in MeOH (20 mL) was added Pd / C (0.2 g, 5% purity) under N. The suspension was degassed under vacuum and purged with H several times. The mixture was stirred for 4 h at 25 °C under H (15 psi). LCMS showed the reaction was complete. The mixture was filtered and concentrated to give Bz-16b (0.18 g, 589.44 μmol, 98.84% yield) as a yellow solid.

[0293] Synthesis of tert-butyl (3-(8-((6-(4-((2-acetamidoethyl)carbamoyl)piperidin-1-yl)pyridin-3-yl)carbamoyl)-2-amino-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, Bz-16. To a mixture of 2-amino-4-[3-(tert-butoxycarbonylamino)propyl-propyl-carbamoyl]-3H-1-benzazepine-8-carboxylic acid, Bz-16c (0.22 g, 494.91 μmol, 1 equiv.), HATU (225.82 mg, 593.90 μmol, 1.2 equiv.) in DMF (5 mL) was added EtN (150.24 mg, 1.48 mmol, 206.66 μL, 3 equiv.) at 25° C. The mixture was stirred at 25° C. for 5 min, and then N-(2-acetamidoethyl)-1-(5-amino-2-pyridyl)piperidine-4-carboxamide, Bz-16b (151.13 mg, 494.91 μmol, 1 equiv.) was added to the mixture and stirred for 30 min. The mixture was poured into water (50 mL). The aqueous phase was extracted with ethyl acetate (50 mL). The combined organic phase was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. 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% to 50%, 10.5 min to give Bz-16 (96 mg, 131.17 μmol, 26.50% yield) as an off-white solid. 1 H NMR (MeOD, 400 MHz) δ 8.39 (d, J = 2.6 Hz, 1H), 7.90 (dd, J = 9.2, 2.6 Hz, 1H), 7.69 (d, J = 1.2 Hz, 1H), 7.54-7.60 (m, 1H), 7.46 (br d, J = 8.0 Hz, 1H), 6.85-6.95 (m, 2H), 4.30 (d, J = 13.6 Hz, 2H), 3.39-3.53 (m, 4H), 3.28 (s, 2H), 3.08-3.12 (m, 2H), 2.83-2.93 (m, 2H), 2.37-2.47 (m, 1H), 1.94 (s, 3H), 1.60-1.90 (m, 8H), 1.24-1.50 (m, 9H). LC / MS [M+H] 732.42 (calculated value); LC / MS [M+H] 732.40 (measured value).

[0294] Example 13 Synthesis of Bz-17 [ka] To a solution of tert-butyl N-[3-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]propyl]carbamate, Bz-1 (1.5 g, 2.40 mmol, 1 equiv.) in DCM (20 mL) was added TFA (6.16 g, 54.03 mmol, 4 mL, 22.54 equiv.) at 25 °C under N and then stirred at this temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was diluted with CHCN (30 mL) and HO (10 mL) and adjusted to pH = 8-9 with aqueous NaHCO at 0 °C. The mixture was stirred at 25 °C for 30 min and then concentrated under reduced pressure to remove CHCN. The aqueous phase was extracted with DCM / i-PrOH = 3 / 1 (20 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA condition; column: luna® (Phenomenex) C18 250*80 mm*10 μm (micron); mobile phase: [water (0.1% TFA)-ACN]; B%: 10% to 40%, 20 min) to give 2-amino-N-(3-aminopropyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-17 (1.00 g, 1.57 mmol, 65.48% yield, TFA salt) as a white solid. 1H NMR (MeOD-d4, 400 MHz) δ8.14-8.05 (m, 2H), 7.91 (d, J = 7.6 Hz, 1H), 7.86-7.81 (m, 1H), 7.80-7.72 (m, 2H), 7.71-7.67 (m, 1H), 7.15 (s, 1H), 3.87 (t, J = 8.0 Hz, 2H), 3.65-3.57 (m, 4H), 3.55-3.52 (m, 2H), 3.45-3.36 (m, 4H), 3.04-3.01 (m, 2H), 2.63-2.53 (m, 1H), 2.04 (quintet, J = 7.2 Hz, 2H), 1.77-1.70 (m, 2H), 0.94 (br t, J = 6.8 Hz, 3H). LC / MS [M+H] 526.2 (calculated value); LC / MS [M+H] 526.2 (measured value).

[0295] Example 14 Synthesis of Bz-18 [ka] Preparation of tert-butyl (3-(3-((N-benzyl-2-nitrophenyl)sulfonamido)propoxy)propyl)carbamate, Bz-18a. 3,3'-Oxybis(propan-1-amine) (0.5 g, 3.8 mmol, 1 equiv.) and potassium carbonate (1.3 g, 9.5 mmol, 2.5 equiv.) were taken up in 10 mL of DMF. 2-Nitrophenylsulfonyl chloride (0.84 g, 3.8 mmol, 1 equiv.) was added, and the reaction was monitored by LCMS. Di-tert-butyl dicarbonate (0.87 mL, 3.8 mmol, 1 equiv.) was then added. After approximately another hour, benzyl bromide (0.45 mL, 3.8 mmol, 1 equiv.) was added, and the reaction was heated to 75 °C. Upon completion, the reaction was filtered, concentrated, and purified by flash chromatography to give Bz-18a (0.47 g, 0.93 mmol, 25%). LC / MS [M+H] 508.21 (calculated); LC / MS [M+H] 508.43 (observed).

[0296] Preparation of tert-butyl (3-(3-(benzylamino)propoxy)propyl)carbamate, Bz-18b Bz-18a (0.47 g, 0.93 mmol, 1 equiv.) was dissolved in DMF. Potassium carbonate (0.19 g, 1.4 mmol, 1.5 equiv.) was added, followed by dodecanethiol (0.33 mL, 1.4 mmol, 1.5 equiv.). The reaction was stirred at 60° C. overnight and then purified by column chromatography to give Bz-18b (0.18 g, 0.57 mmol, 61%). LC / MS [M+H] 323.23 (calculated); LC / MS [M+H] 323.38 (observed).

[0297] Preparation of tert-butyl (3-(3-(benzyl(propyl)amino)propoxy)propyl)carbamate, Bz-18c Bz-18b (0.183 g, 0.57 mmol, 1 equiv.) was dissolved in DCM. Propionaldehyde (0.1 mL, 1.4 mmol, 2.5 equiv.) and sodium triacetoxyborohydride (0.3 g, 1.4 mmol, 2.5 equiv.) were added. The reaction was stirred at room temperature, then concentrated and purified by HPLC to give Bz-18c (0.058 g, 0.159 mmol, 31%). LC / MS [M+H] 365.28 (calculated); LC / MS [M+H] 365.44 (observed).

[0298] Preparation of tert-butyl (3-(3-(propylamino)propoxy)propyl)carbamate, Bz-18d Bz-18c (0.058 g, 0.159 mmol, 1 equiv.) was dissolved in 4 mL of methanol. To this solution was added triethylamine (0.067 mL, 0.48 mmol, 3 equiv.), followed by formic acid (0.015 mL, 0.40 mmol, 2.5 equiv.), and then Pd / C (5 mg, 10 wt%). The mixture was heated to 60° C. Once the starting material was consumed, the reaction mixture was filtered and concentrated to give Bz-18d (0.007 g, 0.0092 mmol, 26%). LC / MS [M+H] 275.23 (calculated); LC / MS [M+H] 275.27 (observed).

[0299] Preparation of Bz-18 2-Amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carboxylic acid, Bz-18e (0.025 g, 0.075 mmol, 1 equiv.), Bz-18d (0.02 g, 0.075 mmol, 1 equiv.), and diisopropylethylamine (0.065 mL, 0.38 mmol, 5 equiv.) were dissolved in DMF. HATU (0.043 g, 0.113 mmol, 1.5 equiv.) was added, and the mixture was stirred at room temperature. Upon completion, the reaction mixture was concentrated and purified by RP-HPLC. The isolated product was concentrated, dissolved in minimal TFA, and allowed to stand at room temperature for 15 min. The solution was then concentrated, purified by RP-HPLC, and lyophilized to give 2-amino-N-(3-(3-aminopropoxy)propyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, Bz-18 (1.2 mg, 0.002 mmol, 3%) as a white powder. LC / MS [M+H] 584.29 (calculated); LC / MS [M+H] 584.50 (observed).

[0300] Example 15 Synthesis of Bz-19 [ka] A vial was charged with Bz-17 (0.0275 mmol), diisopropylethylamine (15 μL, 0.0825 mmol), tert-butylacetyl chloride (0.0275 mmol), 250 μL of DCM, and 250 μL of DMF. The reaction was maintained for 3 h and purified by normal-phase chromatography using a 0-10% MeOH:DCM gradient to afford 6.6 mg of 2-amino-N-(3-(3,3-dimethylbutanamido)propyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, Bz-19, in 39% yield. LC / MS [M+H] 624.3 (calculated); LC / MS [M+H] 624.3 (observed).

[0301] Example 16 Synthesis of Bz-20 [ka] A vial was charged with Bz-9 (28 mg, 0.043 mmol), 300 μL of DCM, and 100 μL of trifluoroacetic acid. The reaction was maintained for 1 h before being concentrated under reduced pressure. The resulting oil was azeotroped three times with 1 mL of toluene, followed by the addition of 1 mL of methanol and K2CO3 (38 mg, 0.28 mmol). After stirring for 16 h, the reaction was filtered, concentrated under reduced pressure, and then purified by reverse-phase preparative HPLC using a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to afford 5.8 mg of 2-amino-N-(5-aminopentyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, Bz-20, in 24% yield. LC / MS [M+H] 554.28 (calculated); LC / MS [M+H] 554.47 (observed).

[0302] Example 17 Synthesis of Bz-21 [ka] Preparation of tert-butyl (2-(2-(3-hydroxypropoxy)ethoxy)ethyl)carbamate, Bz-21a tert-Butyl 3-(2-(2-aminoethoxy)ethoxy)propanoate (0.5 g, 2.1 mmol, 1 equiv.) was dissolved in THF. Lithium aluminum hydride (0.244 g, 6.4 mmol, 3 equiv.) was added, and the reaction was heated to 60 °C. Upon complete reduction of the ester, the reaction was cooled on ice, and saturated aqueous sodium bicarbonate was added. The mixture was stirred for 10 minutes, and then di-tert-butyl dicarbonate (0.49 ml, 2.1 mmol, 1 equiv.) was added. The reaction was stirred at room temperature, then concentrated to remove THF before HPLC purification to give Bz-21a (0.205 g, 0.78 mmol, 36%). LC / MS [M+H] 264.18 (calculated); LC / MS [M+H] 264.27 (observed).

[0303] Preparation of tert-butyl (2-(2-(3-(benzyl(propyl)amino)propoxy)ethoxy)ethyl)carbamate, Bz-21b Oxalyl chloride (0.205 ml, 2.4 mmol, 3 equiv) was dissolved in 0.5 ml of DCM at −78° C. DMSO (0.34 ml, 4.8 mmol, 6 equiv) was added dropwise. The reaction was stirred at −78° C. for 15 minutes, then Bz-21a (0.21 g, 0.80 mmol, 1 equiv) was added dropwise as a solution in 0.5 ml of DCM. The reaction was stirred at −78° C. for 30 minutes, then triethylamine (1 ml, 7.2 mmol, 9 equiv) was added dropwise. The reaction was stirred at −78° C. for an additional 30 minutes, then removed from cooling and allowed to warm to ambient temperature over 30 minutes. N-benzylpropan-1-amine (0.119 g, 0.80 mmol, 1 equiv.) and sodium triacetoxyborohydride, STAB (0.845 g, 4.0 mmol, 5 equiv.) were suspended in 2 mL of DCM. The crude aldehyde solution was added to the stirring amine solution. After 30 min, the reaction was added to a separatory funnel and washed with saturated NaHCO3, water, and then brine. The organic fraction was dried over sodium sulfate, filtered, concentrated, and then purified by RP-HPLC to give Bz-21b (0.228 g, 0.58 mmol, 73%). LC / MS [M+H] 395.29 (calculated); LC / MS [M+H] 395.44 (observed).

[0304] Preparation of tert-butyl (2-(2-(3-(propylamino)propoxy)ethoxy)ethyl)carbamate, Bz-21c Bz-21b (0.228 g, 0.58 mmol, 1 equiv.) was dissolved in methanol. Formic acid (0.033 mol, 0.87 mmol, 1.5 equiv.) was added, followed by 10 wt. % Pd / C (0.02 g). The reaction was stirred at 60 °C, then filtered, concentrated, and purified by HPLC to give Bz-21c as the TFA salt (0.193 g, 0.46 mmol, 80%). LC / MS [M+H] 305.24 (calculated); LC / MS [M+H] 305.38 (observed).

[0305] Preparation of Bz-21: 2-Amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carboxylic acid, Bz-21d (0.042 g, 0.099 mmol, 1 equiv.), Bz-21c (0.03 g, 0.099 mmol, 1 equiv.), and diisopropylethylamine (0.1 mL, 0.57 mmol, 5.8 equiv.) were dissolved in DMF. 7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, PyAOP, CAS Registry Number 156311-83-0 (0.077 g, 0.15 mmol, 1.5 equiv.) was added, and the mixture was stirred at room temperature. Upon completion, the reaction mixture was concentrated and purified by HPLC. The isolated product was concentrated, dissolved in minimal TFA, and allowed to stand at room temperature for 15 minutes. The solution was then concentrated and purified by HPLC to give an oil that was triturated with diethyl ether to give 2-amino-N-(3-(2-(2-aminoethoxy)ethoxy)propyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, Bz-21 (0.037 g, 0.060 mmol, 61%) as a white solid. LC / MS [M+H] 614.30 (calculated); LC / MS [M+H] 614.58 (observed).

[0306] Example 18 Synthesis of Bz-22 [ka] Preparation of (E)-2-(4-bromobut-2-en-1-yl)isoindoline-1,3-dione, Bz-22a To a solution of (1,3-dioxoisoindolin-2-yl)potassium (7.5 g, 40.5 mmol, 1 equiv.) in DMF (100 mL) was added (E)-1,4-dibromobut-2-ene (17.3 g, 80.9 mmol, 2 equiv.). The mixture was stirred at 20 °C for 12 h, then diluted with water (200 mL) and extracted with EtOAc (80 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®; 12 g SepaFlash® silica flash column, eluent: 0–60% ethyl acetate / petroleum ether gradient at 60 mL / min) to afford Bz-22a (8.6 g, 30.7 mmol, 75.82% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ7.90-7.83 (m, 2H), 7.78-7.70 (m, 2H), 6.01-5.90 (m, 1H), 5.89-5.79 (m, 1H), 4.32 (d, J = 5.6 Hz, 2H), 3.92 (d, J = 7.2 Hz, 2H).

[0307] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-(1,3-dioxoisoindolin-2-yl)but-2-enyl]carbamate, Bz-22b To a solution of Bz-22a (11 g, 39.3 mmol, 1 equiv.) in DMF (200 mL) was added CsCO (19.2 g, 58.9 mmol, 1.5 equiv.) and tert-butyl N-tert-butoxycarbonylcarbamate (11.1 g, 51.1 mmol, 1.3 equiv.). The mixture was stirred at 20 °C for 12 h, then diluted with water (400 mL) and extracted with EtOAc (100 mL × 3). The organic layer was washed with brine (80 mL × 3), dried over NaSO, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 5 g SepaFlash® silica flash column, elution with a 0-70% ethyl acetate / petroleum ether gradient @ 65 mL / min) to afford Bz-22b (16 g, 38.4 mmol, 97.83% yield) as a white solid. 1 H NMR (DMSO-d6, 400 MHz) δ7.90-7.83 (m, 4H), 5.63-5.53 (m, 2H), 4.20-4.12 (m, 2H), 4.05-3.99 (m, 2H), 1.36 (s, 18H)

[0308] Preparation of tert-butyl N-[(E)-4-aminobut-2-enyl]-N-tert-butoxycarbonyl-carbamate, Bz-22c To a solution of Bz-22b (18 g, 43.2 mmol, 1 equiv.) in MeOH (200 mL) at 20 °C was added hydrazine hydrate (10.2 g, 173 mmol, 9.90 mL 85% purity, 4 equiv.) and then stirred at 70 °C for 3 h. The mixture was filtered, and the filtrate was concentrated. The crude product was triturated with CHCN at 20 °C for 20 min, filtered, and the filtrate was concentrated to give Bz-22c (10 g, 34.9 mmol, 80.80% yield) as a pale yellow oil. 1 H NMR (CDCl3, 400 MHz) δ5.78-5.69 (m, 1H), 5.64-5.54 (m, 1H), 4.17-4.09 (m, 2H), 3.31-3.23 (m, 2H), 1.49 (s, 18H)

[0309] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-[(4-nitrophenyl)sulfonylamino]but-2-enyl]carbamate, Bz-22d To a solution of Bz-22c (1 g, 3.49 mmol, 1 equiv.) in DCM (10 mL) was added TEA (706.72 mg, 6.98 mmol, 972.10 μL (microliters), 2 equiv.) and 4-nitrobenzenesulfonyl chloride (851.29 mg, 3.84 mmol, 1.1 equiv.) under N at 0 °C. The mixture was stirred at 25 °C for 1 h and then quenched by the addition of HO (20 mL) at 0 °C, followed by extraction with EtOAc (10 mL × 3). The combined organic layers were washed with brine (5 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 1) to give Bz-22d (1.2 g, 2.54 mmol, 72.74% yield) as a pale yellow oil. 1 H NMR (CDCl3, 400 MHz) δ8.41-8.35 (m, 2H), 8.05 (d, J = 9.2 Hz, 2H), 5.71-5.61 (m, 1H), 5.57-5.47 (m, 1H), 4.61 (t, J = 5.6 Hz, 1H), 4.10 (d, J = 5.6 Hz, 2H), 3.67 (t, J = 6.0 Hz, 2H), 1.49 (s, 18H).

[0310] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-[(4-nitrophenyl)sulfonyl-propyl-amino]but-2-enyl]carbamate, Bz-22e To a solution of Bz-22d (1 g, 2.12 mmol, 1 equiv.) in DMF (10 mL) was added CsCO (1.38 g, 4.24 mmol, 2 equiv.) and 1-iodopropane (360.52 mg, 2.12 mmol, 207.19 μL, 1 equiv.) at 25 °C, followed by stirring at this temperature for 12 h. The reaction mixture was quenched by adding HO (50 mL) at 0 °C and then extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to afford Bz-22e (0.89 g, 1.73 mmol, 81.71% yield) as a pale yellow oil. 1 H NMR (CDCl3, 400 MHz) δ8.36 (d, J = 8.8 Hz, 2H), 7.99 (d, J = 8.8 Hz, 2H), 5.74-5.60 (m, 1H), 5.51-5.37 (m, 1H), 4.11 (d, J = 7.2 Hz, 2H), 3.86 (d, J = 6.4 Hz, 2H), 3.16-3.07 (m, 2H), 1.55-1.46 (m, 20H), 0.86 (t, J = 7.6 Hz, 3H)

[0311] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-(propylamino)but-2-enyl]carbamate, Bz-22f To a solution of Bz-22e (0.79 g, 1.54 mmol, 1 equiv.) in CH3CN (10 mL) was added LiOH·HO (387.25 mg, 9.23 mmol, 6 equiv.) and methyl 2-sulfanylacetate (490 mg, 4.61 mmol, 419 µL, 3 equiv.) at 0 °C. The resulting mixture was stirred at 25 °C for 12 h, then filtered and concentrated under reduced pressure. The residue was diluted with HO (20 mL) at 0 °C, then adjusted to pH = 2–3 with 1 M HCl and extracted with MTBE (10 mL × 3). The pH of the aqueous phase was adjusted to approximately 10 with aqueous K2CO3 and extracted with (10 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give Bz-22f (0.35 g, 1.07 mmol, 69.28% yield) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ5.79-5.58 (m, 2H), 4.15 (d, J = 5.2 Hz, 2H), 3.23 (d, J = 5.6 Hz, 2H), 2.56 (t, J = 6.8 Hz, 2H), 1.56-1.42 (m, 20H), 0.92 (t, J = 7.6 Hz, 3H)

[0312] Preparation of tert-butyl N-[(E)-4-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]but-2-enyl]-N-tert-butoxycarbonyl-carbamate, Bz-22g To a mixture of 2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carboxylic acid, Bz-21d (0.45 g, 1.05 mmol, 1 equiv.) in DMF (5 mL), HATU (440 mg, 1.16 mmol, 1.1 equiv.) and DIPEA (408 mg, 3.16 mmol, 550 μL, 3 equiv.) were added at 25 °C. After 10 min, Bz-22f (345.75 mg, 1.05 mmol, 1 equiv.) was added to the mixture at 25 °C, followed by stirring at this temperature for 1 h. The reaction mixture was poured into ice water (30 mL) at 0 °C and extracted with DCM / i-PrOH = 3 / 1 (20 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give Bz-22g (0.41 g, crude) as a brown solid.

[0313] Preparation of Bz-22: To a solution of tert-butyl N-[(E)-4-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]but-2-enyl]-N-tert-butoxycarbonyl-carbamate (13 mg, 17.6 μmol, 1 equiv.) in DCM (1 mL) was added TFA (154 mg, 1.35 mmol, 0.1 mL, 76.7 equiv.) at 25 °C, followed by stirring at this temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in CHCN (10 mL) and HO (1 mL) and adjusted to pH = 9 with aqueous LiOH at 0 °C. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA condition; column: Welch Xtimate C18 100*25mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%~35%, 12 min) to give 2-amino-N-[(E)-4-aminobut-2-enyl]-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-22 (7 mg, 10.74 umol, 60.97% yield, TFA) as a white solid. 1H NMR (MeOD-d4, 400 MHz) δ8.15-8.04 (m, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.86-7.72 (m, 3H), 7.68 (d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.07-5.94 (m, 1H), 5.89-5.77 (m, 1H), 4.21 (br s, 2H), 3.87 (t, J = 8.4 Hz, 2H), 3.67-3.56 (m, 4H), 3.48 (br s, 2H), 3.45-3.37 (m, 4H), 2.68-2.50 (m, 1H), 1.77-1.61 (m, 2H), 0.95-0.93 (m, 3H). LC / MS [M+H] 538.2 (calculated value); LC / MS [M+H] 538.3 (measured value).

[0314] Example 19 Synthesis of Bz-23 [ka] Preparation of N'-benzyl-N'-propyl-N-pyrimidin-2-yl-propane-1,3-diamine, Bz-23b A mixture of N'-benzyl-N'-propyl-propane-1,3-diamine, Bz-23a (0.2 g, 823.77 umol, 1 equiv, HCl), DIEA (426 mg, 3.30 mmol, 574 uL, 4 equiv) in dioxane (4 mL) was stirred at 25 °C for 10 min, then 2-chloropyrimidine (188.70 mg, 1.65 mmol, 2 equiv) was added, and the mixture was stirred at 25 °C for 16 h. The reaction was quenched with HO (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic phase was washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by preparative TLC (SiO 2 , DCM:MeOH=7:1) to give Bz-23b (130 mg, 457 μmol, 55.49% yield) as a yellow oil. 1H NMR (CDCl3, 400 MHz) δ8.26 (d, J = 4.8 Hz, 2H), 7.38-7.32 (m, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.26-7.20 (m, 1H), 6.49 (t, J = 5.2 Hz, 1H), 5.74 (br s, 1H), 3.58 (s, 2H), 3.47-3.39 (m, 2H), 2.54 (t, J = 6.8 Hz, 2H), 2.44-2.38 (m, 2H), 1.77 (quintet, J = 6.4 Hz, 2H), 1.57-1.50 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H)

[0315] Preparation of N-propyl-N'-pyrimidin-2-yl-propane-1,3-diamine, Bz-23c To a solution of Bz-23b (130 mg, 457 μmol, 1 equiv.) in MeOH (10 mL) was added Pd / C (0.1 g, 10% purity) under a N atmosphere. The suspension was degassed and purged with hydrogen gas (H) three times, and the mixture was stirred at 25 °C for 16 h, then filtered and concentrated under reduced pressure. The residue was purified by preparative TLC ((SiO, DCM:MeOH = 5:1)) to give Bz-23c (80 mg, 412 μmol, 90.09% yield) as a brown oil.

[0316] Preparation of Bz-23: To a solution of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-21d (264 mg, 618 μmol, 1 equiv.) in DMF (2 mL) was added DIEA (240 mg, 1.85 mmol, 323 μL, 3 equiv.), 7-aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, PYAOP (483 mg, 927 μmol, 1.5 equiv.), and Bz-23c (120 mg, 618 μmol, 1 equiv.). The mixture was stirred at 25° C. for 1 h, then filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (Welch Xtimate C18 100 x 25 mm x 3 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15% to 35%, 12 min) to give 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-N-[3-(pyrimidin-2-ylamino)propyl]-3H-1-benzazepine-4-carboxamide, Bz-23 (16 mg, 26.5 μmol, 4.29% yield) as a white solid. 1 H NMR (MeOD-d4, 400 MHz) δ8.38 (br s, 1H), 8.15 (s, 1H), 8.11 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.85-7.79 (m, 1H), 7.75 (br s, 1H), 7.71 (br s, 1H), 7.53 (s, 1H), 7.11 (br s, 1H), 6.74 (br s, 1H), 3.87 (t, J = 8.0 Hz, 2H), 3.62 (dd, J = 6.0, 8.0 Hz, 4H), 3.54-3.49 (m, 2H), 3.42 (d, J = 6.8 Hz, 2H), 3.35 (br s, 2H), 2.64-2.51 (m, 1H), 2.08-1.95 (m, 2H), 1.77-1.66 (m, 2H), 0.99-0.94 (m, 3H). LC / MS [M+H] 604.3 (calculated value); LC / MS [M+H] 604.3 (measured value).

[0317] Example 20 Synthesis of Bz-24 [ka] Preparation of tert-butyl N-[4-[(4-nitrophenyl)sulfonylamino]butyl]carbamate, Bz-24a To a solution of tert-butyl N-(4-aminobutyl)carbamate (0.5 g, 2.66 mmol, 1 equiv) and EtN (537 mg, 5.31 mmol, 739 uL, 2 equiv) in DCM (5 mL) was added 4-nitrobenzenesulfonyl chloride (647 mg, 2.92 mmol, 1.1 equiv) at 0 °C. After the addition, the resulting mixture was stirred at 25 °C for 1 h, then quenched by the addition of HO (20 mL) at 0 °C, and then extracted with DCM (10 mL × 3). The combined organic layers were washed with brine (5 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was triturated with PE / MTBE=10 / 1 (20 mL), stirred for 30 min, filtered, and the filter cake was dried under reduced pressure to give Bz-24a (0.99 g, 2.65 mmol, 99.82% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ8.37 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H), 5.28 (br s, 1H), 4.59 (br s, 1H), 3.12-3.03 (m, 4H), 1.58-1.48 (m, 4H), 1.44 (s, 9H)

[0318] Preparation of tert-butyl N-[4-[(4-nitrophenyl)sulfonyl-propyl-amino]butyl]carbamate, Bz-24b To a solution of Bz-24a (0.99 g, 2.65 mmol, 1 equiv.) in DMF (7 mL) was added CsCO (1.73 g, 5.30 mmol, 2 equiv.) and 1-iodopropane (451 mg, 2.65 mmol, 259 μL, 1 equiv.) at 0 °C. The mixture was stirred at 25 °C for 12 h, then poured into ice-water (30 mL) at 0 °C, and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was triturated with PE / MTBE = 10 / 1 (20 mL), stirred at 25 °C for 30 min, filtered, and the filter cake was dried under reduced pressure to give Bz-24b (0.97 g, 2.33 mmol, 88.06% yield) as a pale yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ8.39 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 6.79 (br t, J = 6.0 Hz, 1H), 3.13-3.05 (m, 4H), 2.88 (q, J = 6.4 Hz, 2H), 1.54-1.40 (m, 4H), 1.39-1.27 (m, 11H), 0.81 (t, J = 7.2 Hz, 3H).

[0319] Preparation of tert-butyl N-[4-(propylamino)butyl]carbamate, Bz-24c To a solution of Bz-24b (0.97 g, 2.33 mmol, 1 equiv) in CHCN (10 mL) was added LiOH·HO (587.74 mg, 14.01 mmol, 6 equiv) and methyl 2-sulfanylacetate (744 mg, 7.00 mmol, 635 μL, 3 equiv) at 0 °C. The resulting mixture was stirred at 25 °C for 12 h, then filtered and concentrated under reduced pressure. The residue was diluted with HO (20 mL) at 0 °C, then adjusted to pH = 2–3 with 1 M HCl and extracted with MTBE (10 mL × 3). The pH of the aqueous phase was adjusted to approximately 10 with aqueous KCO solution and extracted with EtOAc (10 mL × 3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to afford Bz-24c (445 mg, 1.93 mmol, 82.75% yield) as a brown oil. 1H NMR (DMSO-d6, 400 MHz) δ6.81 (br s, 1H), 2.89 (q, J = 6.4 Hz, 2H), 2.47-2.39 (m, 4H), 1.44-1.31 (m, 15H), 0.85 (t, J = 7.6 Hz, 3H).

[0320] Preparation of tert-butyl N-[4-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]butyl]carbamate, Bz-24d To a solution of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-21d (100 mg, 234 μmol, 1 equiv.), and DIPEA (90.7 mg, 702 μmol, 122.24 μL, 3 equiv.) in DMF (1 mL) was added HATU (97.8 mg, 257 μmol, 1.1 equiv.) at 25° C. After 10 min, Bz-24c (64.66 mg, 280.72 μmol, 1.2 equiv.) was added at 25° C., followed by stirring at this temperature for 1 h. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA condition; column: Welch Xtimate C18 100*25mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-45%, 12 min) to give Bz-24d (8 mg, 12.50 umol, 5.35% yield) as a yellow solid. 1H NMR (MeOD-d4, 400 MHz) δ8.14-8.04 (m, 2H), 7.92 (d, J = 8.0 Hz, 1H), 7.85-7.81 (m, 1H), 7.81-7.76 (m, 1H), 7.73-7.68 (m, 2H), 7.11 (s, 1H), 3.87 (t, J = 7.6 Hz, 2H), 3.61 (dd, J = 6.0Hz, 7.6 Hz, 2H), 3.58-3.45 (m, 4H), 3.44-3.35 (m, 4H), 3.12-3.04 (m, 2H), 2.65-2.52 (m, 1H), 1.78-1.63 (m, 4H), 1.55-1.40 (m, 11H), 0.95-0.93 (m, 3H). LC / MS [M+H] 640.3 (calculated value); LC / MS [M+H] 640.3 (measured value).

[0321] Preparation of Bz-24: To a solution of Bz-24d (0.1 g, 156 μmol, 1 equiv) in DCM (2 mL) was added TFA (308 mg, 2.70 mmol, 0.2 mL, 17.28 equiv) at 25 °C, followed by stirring at this temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in CHCN (10 mL) and HO (1 mL) and adjusted to pH = 9 with aqueous LiOH at 0 °C. The mixture was stirred at 25 °C for 1 h, then filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA condition; column: Welch Xtimate C18 100*25mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%~30%, 12 min) to give 2-amino-N-(4-aminobutyl)-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-24 (34 mg, 52.01 umol, 33.28% yield, TFA) as a white solid. 1H NMR (MeOD-d4, 400 MHz) δ8.13-8.05 (m, 2H), 7.90 (d, J = 8.0 Hz, 1H), 7.85-7.78 (m, 1H), 7.77-7.72 (m, 2H), 7.71-7.65 (m, 1H), 7.10 (s, 1H), 3.86 (t, J = 8.4 Hz, 2H), 3.61 (dd, J = 5.6 Hz, 7.6 Hz, 2H), 3.58-3.46 (m, 4H), 3.44-3.36 (m, 4H), 3.05-2.94 (m, 2H), 2.64-2.52 (m, 1H), 1.84-1.62 (m, 6H), 1.03-0.85 (m, 3H). LC / MS [M+H] 540.3 (calculated); LC / MS [M+H] 540.3 (observed).

[0322] Example 21 Synthesis of Bz-25 [ka] Preparation of tert-butyl N-[2-(4-methoxyphenyl)ethyl]carbamate, Bz-25a To a mixture of 2-(4-methoxyphenyl)ethanamine (1 g, 6.61 mmol, 970.87 μL, 1 equiv.) in THF and HO (10 mL) was added BocO (2.17 g, 9.92 mmol, 2.28 mL, 1.5 equiv.), followed by stirring at 25 °C for 30 min under a N atmosphere. The mixture was diluted with water and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, dried over NaSO, 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 = 5 / 1 to 1 / 1) to give Bz-25a (1.60 g, 6.37 mmol, 96.26% yield) as a white solid. 1H NMR (CDCl3, 400 MHz) δ7.12 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 4.53(br s, 1H), 3.80 (s, 3H), 3.37-3.33 (m, 2H), 2.74 (br t, J = 6.4Hz, 2H), 1.44 (s, 9H)

[0323] Preparation of tert-butyl 4-methoxyphenethyl(propyl)carbamate, Bz-25b To a mixture of Bz-25a (0.8 g, 3.18 mmol, 1 equiv.) and 1-iodopropane (1.08 g, 6.37 mmol, 621 uL, 2 equiv.) in DMF (8 mL) was added NaH (191 mg, 4.77 mmol, 60% purity, 1.5 equiv.) at 0 °C, followed by stirring at 25 °C for 2 h. The mixture was poured into water (20 mL). The aqueous phase was extracted with ethyl acetate (15 mL × 3). The combined organic phase was washed with brine (10 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure 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 = 5 / 1, 1 / 1) to give Bz-25b (365 mg, 1.24 mmol, 39.08% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ7.11 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H), 3.36-3.30 (m, 2H), 3.15-3.09 (m, 2H), 2.79-2.71 (m, 2H), 1.57-1.50 (m, 2H), 1.46 (s, 9H), 0.87 (t, J = 7.6 Hz, 3H)

[0324] Preparation of N-[2-(4-methoxyphenyl)ethyl]propan-1-amine, Bz-25c To a solution of Bz-25b (365 mg, 1.24 mmol, 1 equiv) in EtOAc (5 mL) was added HCl / EtOAc (5 mL). The mixture was stirred at 25° C. for 3 h and then concentrated in vacuo to give Bz-25c.

[0325] Preparation of Bz-25: To a solution of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-21d (186 mg, 435 μmol, 1 equiv.) in DMF (1.00 mL), PYAOP (340 mg, 653 μmol, 1.5 equiv.) and DIEA (393 mg, 3.05 mmol, 531 μL, 7 equiv.) were added, followed by Bz-25c (100 mg, 435 μmol, 1 equiv., HCl). The mixture was stirred at 25° C. for 3 h, then filtered and concentrated. The residue was purified by preparative HPLC (column: Nano-micro Kromasil® C18 100*30mm 8um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%~55%, 10min]) to give 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-[2-(4-methoxyphenyl)ethyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-25 (14mg, 23.23umol, 5.34% yield) as a pale yellow solid. 1H NMR (MeOD-d4, 400 MHz) δ8.13-8.03 (m, 2H), 7.93-7.87 (m, 1H), 7.84-7.80 (m, 1H), 7.79-7.74 (m, 1H), 7.69 (br s, 1H), 7.60 (br d, J = 8.0 Hz, 1H), 7.08 -6.51 (m, 5H), 3.86 (t, J = 8.4 Hz, 2H), 3.75 (s, 4H), 3.61 (dd, J = 5.8, 8.1 Hz, 2H), 3.56-3.45 (m, 1H), 3.54-3.49 (m, 1H), 3.42 (d, J = 6.2 Hz, 2H), 2.93-2.87 (m, 2H), 2.65-2.47 (m, 1H), 1.75-1.68 (m, 2H), 1.03-0.94 (m, 3H). LC / MS [M+H] 603.3 (calcd); LC / MS [M+H] 603.3 (observed).

[0326] Example 22 Synthesis of Bz-26.

change

[0327] Preparation of Bz-26: To a mixture of [1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methanol (1.73 g, 4.90 mmol, 1.2 equiv.), Bz-26b (2.0 g, 4.09 mmol, 1.0 equiv.), and Pd(dppf)Cl (150 mg, 204 µmol, 0.05 equiv.) in dioxane (40 mL) was added KCO (1.13 g, 8.17 mmol, 2 equiv.) in HO (5 mL) at 25 °C under N, followed by stirring at 100 °C for 1 h. The mixture was 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=1 / 1, 0 / 1) to give tert-butyl N-[4-[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]but-2-ynyl]carbamate, Bz-26 (2.0 g, 3.15 mmol, 76.9% yield) as a pale yellow solid. 1 H NMR (MeOD, 400MHz) δ8 .07 (s, 1H), 8.04 (br d, J = 7.6 Hz, 1H), 7.88-7.82 (m, 1H), 7.79-7.73 (m, 1H), 7.53-7.46 (m, 2H), 7.43-7.37 (m, 1H), 7.12 (s, 1H), 4.29 (s, 2H), 3.93-3.82 (m, 4H), 3.62-3.50 (m, 4H), 3.42 (d, J = 6.4 Hz, 2H), 3.31 (s, 2H), 2.64-2.52 (m, 1H), 1.76-1.70 (m, 2H), 1.43 (s, 9H), 0.99-0.91 (m, 3H). LC / MS [M+H] 636.3 (calculated value); LC / MS [M+H] 636.3 (measured value). LCMS (ESI): C 33 H 41 Calculated mass for N5O6S 635.28, measured m / z 636.3 [M+H] +

[0328] Example 23 Synthesis of Bz-27: [ka] Preparation of Bz-27a: To a solution of tert-butyl N-[(4-formylphenyl)methyl]carbamate (400 mg, 1.70 mmol, 1 equiv.), propan-1-amine (1.00 g, 17.0 mmol, 1.40 mL, 10 equiv.), and AcOH (10 mg, 170 umol, 9.72 uL, 0.1 equiv.) in MeOH (1 mL), NaBHCN (213 mg, 3.40 mmol, 2 equiv.) was added and the mixture was stirred at 25 °C for 3 h. The reaction mixture was poured into water (10 mL) and then extracted with EtOAc (5 mL × 3). The combined organic layers were washed with brine (5 mL × 1), dried, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, EtOAC:MeOH=5:1) to give tert-butyl-N-[[4-(propylaminomethyl)phenyl]methyl]carbamate, Bz-27a (200 mg, 718 μmol, 42.26% yield) as a colorless oil. 1 H NMR (MeOD-d4, 400 MHz) δ7.43 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 4.24 (s, 2H), 4.17 (s, 2H), 3.00-2.96 (m, 2H), 1.77-1.67 (m, 2H), 1.44 (s, 9H), 1.01 (t, J = 7.6 Hz, 3H).

[0329] Preparation of Bz-27b: To a solution of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-21d (122 mg, 287 μmol, 1 equiv.) in DMF (0.80 mL), PYAOP (224 mg, 431.05 μmol, 1.5 equiv.) and DIEA (111 mg, 862.10 μmol, 150.16 μL, 3 equiv.) were added. Next, tert-butyl N-[[4-(propylaminomethyl)phenyl]methyl]carbamate (80 mg, 287 μmol, 1 equiv.) was added. The mixture was stirred at 25° C. for 3 hours, filtered, and concentrated. The residue was purified by preparative HPLC (column: Welch Xtimate C18 100*25mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%~50%, 12 min) to obtain the compound tert-butyl N-[[4-[[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]methyl]phenyl]methyl]carbamate (27 mg, 39.3 umol, 13.66% yield) as a pale yellow solid. 1 H NMR (MeOD-d4, 400 MHz) δ8.08 (t, J = 9.6Hz, 2H), 7.92-7.90 (m, 1H),7.82 (t, J = 8.4 Hz, 1H), 7.81-7.79 (m, 1H), 7.69-7.64 (m, 4H), 7.57(s, 1H), 7.30-7.29 (m, 4H), 7.13 (s, 1H), 4.23 (s, 2H), 3.87 (t, J = 8.4Hz, 2H), 3.61 (t, J = 6.0Hz, 2H), 3.42-3.41 (m, 2H), 3.31 (t, J = 1.6Hz, 2H), 2.60-2.55 (m, 1H), 1.71-1.70 (m, 2H), 1.44 (s, 9H), 0.99-0.90(m, 3H). LC / MS [M+H] 688.3 (calculated value); LC / MS [M+H] 688.3 (measured value).

[0330] Preparation of Bz-27: To a solution of Bz-27b (50 mg, 72.7 umol, 1 equiv) in DCM (1 mL) was added TFA (165 mg, 1.45 mmol, 108 uL, 20 equiv) and stirred for 2 h at 25° C. The mixture was filtered and concentrated. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100*30mm 8um; mobile phase: [water (0.1% TFA)-CAN]; B%: 5%~30%, 10min) to give 2-amino-N-[[4-(aminomethyl)phenyl]methyl]-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-27 (4mg, 6.81umol, 9.36% yield) as a white solid. 1 H NMR (MeOH-d4, 400 MHz) δ8.13-8.03 (m, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.85-7.78 (m, 1H), 7.75-7.70 (m, 2H), 7.59-7.33 (m, 5H), 7.15 (s, 1H), 4.13 (s, 2H), 3.86 (t, J = 8.4 Hz, 2H), 3.61 (dd, J = 6.1, 7.8 Hz, 2H), 3.48 (br d, J = 7.6 Hz, 2H), 3.42 (d, J = 6.2 Hz, 4H), 3.32 (br s, 1H), 3.31-3.31 (m, 1H), 3.31-3.30 (m, 2H), 2.63-2.52 (m, 1H), 1.76-1.61 (m, 2H), 0.91 (br s, 3H). LC / MS [M+H] 588.3 (calculated value); LC / MS [M+H] 588.3 (measured value).

[0331] Example 24 Synthesis of Bz-28 [ka] Preparation of Bz-28b: A mixture of 1-[1-(3-bromophenyl)sulfonylazetidin-3-yl]-N,N-dimethyl-methanamine, Bz-28a (0.3 g, 900.24 μmol, 1 equiv.), Pin2B2 (342.91 mg, 1.35 mmol, 1.5 equiv.), Pd(dppf)Cl (32.94 mg, 45.01 μmol, 0.05 equiv.), and KOAc (176.70 mg, 1.80 mmol, 2 equiv.) in dioxane (6 mL) was degassed and purged with N three times, then stirred at 90 °C under a N atmosphere for 2 h. The reaction mixture was cooled to 25 °C, and de-Pd silica gel (1 g) was added, followed by stirring at 25 °C for 30 min. The mixture was filtered, washed with EtOAc (10 mL × 5), and concentrated under reduced pressure to give N,N-dimethyl-1-[1-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfonylazetidin-3-yl]methanamine, Bz-28b (0.6 g, crude) as a yellow oil.

[0332] Preparation of Bz-28: A mixture of Bz-28b (699 mg, 920 μmol, 1.5 equiv), tert-butyl N-[4-[(2-amino-8-bromo-3H-1-benzazepine-4-carbonyl)-propyl-amino]but-2-ynyl]carbamate, Bz-26b (300 mg, 613 μmol, 1 equiv), Pd(dppf)Cl (22.4 mg, 30.6 μmol, 0.05 equiv), and KCO (169 mg, 1.23 mmol, 2 equiv) in dioxane (20 mL) and HO (2 mL) was degassed and purged with N three times, then stirred at 90 °C under a N atmosphere for 2 h. The reaction mixture was quenched by adding HO (60 mL) at 0 °C and then extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:0 to 0:1) and then (SiO2, EtOAc:MeOH = 1:0 to 1:1) to afford tert-butyl N-[4-[[2-amino-8-[3-[3-[(dimethylamino)methyl]azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]but-2-ynyl]carbamate, Bz-28 (230 mg crude product, 347 μmol, 56.61% yield) as a brown solid. 1 H NMR (MeOD-d4, 400 MHz) δ8.16-8.06 (m, 2H), 7.97-7.90 (m, 1H), 7.89-7.65 (m, 4H), 7.34 (br s, 1H), 4.34 (s, 2H), 4.01 (t, J = 8.4 Hz, 2H), 3.87 (s, 2H), 3.69 (dd, J = 5.6, 8.4 Hz, 2H), 3.56 (br s, 2H), 3.39 (s, 2H), 3.33 (s, 2H), 3.03-2.89 (m, 1H), 2.82 (s, 6H), 1.81-1.67 (m, 2H), 1.43 (s, 9H), 0.97 (br t, J = 6.8 Hz, 3H). LC / MS [M+H] 663.3 (calculated value); LC / MS [M+H] 663.3 (measured value).

[0333] Example 25 Synthesis of Bz-29 [ka] Preparation of Bz-29a: To a mixture of O-ethylhydroxylamine (3 g, 30.8 mmol, 1 equiv., HCl) and Na2CO3 (32.6 g, 307.55 mmol, 10 equiv.) in DCM (30 mL) and water (30 mL), tert-butoxycarbonyl tert-butyl carbonate (8.05 g, 36.9 mmol, 8.48 mL, 1.2 equiv.) was added at 25 °C and then stirred for 3 h. The mixture was separated, and the organic layer was dried over Na2SO4 and concentrated to a residue. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1:0 to 5:1) to afford tert-butyl N-ethoxycarbamate, Bz-29a (4 g, 24.81 mmol, 80.68% yield) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 3.87 (q, J = 7.2 Hz, 2H), 1.45 (s, 9H), 1.20 (t, J = 7.2 Hz, 3H).

[0334] Preparation of Bz-29b: To a mixture of Bz-29a (1 g, 6.20 mmol, 1 equiv) in DMF (10 mL) was added NaH (298 mg, 7.44 mmol, 60% purity, 1.2 equiv) at 0 °C, followed by stirring at 0 °C for 0.5 h. 1-Iodopropane (1.16 g, 6.82 mmol, 666.67 uL, 1.1 equiv) was added to the mixture at 0 °C, which was stirred at 25 °C for 10 h. The mixture was quenched with saturated aqueous NH4Cl (10 ml) and extracted with EtOAc (3 * 10 ml). The organic layer was dried over Na2SO4 and concentrated to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1:0 to 5:1) to afford tert-butyl N-ethoxy-N-propyl-carbamate, Bz-29b (0.84 g, 4.13 mmol, 66.61% yield) as a colorless oil. 1H NMR (400 MHz, chloroform-d) δ 3.89 (q, J = 7.2 Hz, 2H), 3.47-3.25 (m, 2H), 1.69-1.59 (m, 2H), 1.49 (s, 9H), 1.23 (t, J = 7.2 Hz, 3H), 0.91 (t, J = 7.2 Hz, 3H).

[0335] Preparation of Bz-29c: To a mixture of Bz-29b (0.84 g, 4.13 mmol, 1 equiv.) in EtOAc (10 mL) was added HCl / EtOAc (4 M, 5 mL, 4.84 equiv.). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated to give N-ethoxypropan-1-amine, Bz-29c (0.4 g, 2.86 mmol, 69.33% yield, HCl) as a white solid. 1 H NMR (400MHz, methanol-d4) δ4.16 (dq, J = 2.0, 7.2 Hz, 2H), 3.29-3.23 (m, 2H), 1.76 (sxt, J = 7.6 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.05 (t, J = 7.2 Hz, 3H).

[0336] Preparation of Bz-29: To a mixture of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid (200 mg, 468 μmol, 1 equiv.) in DMF (2 mL) was added PYAOP (293 mg, 561 μmol, 1.2 equiv.) and DIEA (181 mg, 1.40 mmol, 245 μL, 3 equiv.), followed 3 min later by the addition of N-ethoxypropan-1-amine (71.86 mg, 514.65 μmol, 1.1 equiv., HCl). The mixture was stirred at 25° C. for 1 h and then concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 30%~60%, 10.5 min) to give 2-amino-N-ethoxy-8-[3-[3-(hydroxylmethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-3H-1-benzazepine-4-carboxamide, Bz-29 (3.5 mg, 6.36 umol, 1.36% yield, 93.17% purity) as a white solid. 1 H NMR (400MHz, methanol-d4) δ8.10-8.02 (m, 2H), 7.89-7.73 (m, 2H), 7.53-7.48 (m, 2H), 7.46-7.40 (m, 1H), 7.31 (s, 1H), 3.95 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 8.4 Hz, 2H), 3.74 (t, J = 7.2 Hz, 2H), 3.60 (dd, J = 6.4, 8.2 Hz, 2H), 3.41 (d, J = 6.4 Hz, 2H), 3.34-3.31 (m, 2H), 2.67-2.43 (m, 1H), 1.77 (sxt, J = 7.2 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H), 0.99 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 513.2 (calculated value); LC / MS [M+H] 513.4 (measured value).

[0337] Example 26 Synthesis of Bz-30 [ka] Preparation of Bz-30a: To a mixture of 1,4-bis(bromomethyl)benzene (6.48 g, 24.6 mmol, 2.0 equiv.) and 4-nitro-N-propyl-benzenesulfonamide (3.0 g, 12.3 mmol, 1.0 equiv.) in DMF (40 mL) was added CsCO (4.80 g, 14.7 mmol, 1.2 equiv.) in one portion at 25 °C, followed by stirring for 12 h. The reaction was diluted with water (100 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate=1 / 0, 3 / 1) to give N-[[4-(bromomethyl)phenyl]methyl]-4-nitro-N-propyl-benzenesulfonamide, Bz-30a (1.5 g, 3.51 mmol, 28.6% yield) as a white solid. 1 H NMR (CDCl3, 400MHz) δ8.35 (d, J = 8.8 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 4.48 (s, 2H), 4.40 (s, 2H), 3.19-3.11 (m, 2H), 1.42 (m, 2H), 0.76 (t, J = 7.6 Hz, 3H).

[0338] Preparation of Bz-30b: To a mixture of Bz-30a (1.3 g, 3.04 mmol, 1.0 equiv) and tert-butyl piperazine-1-carboxylate (2.27 g, 12.2 mmol, 4.0 equiv) in DMF (15 mL) was added EtN (1.23 g, 12.2 mmol, 1.69 mL, 4.0 equiv) at 25 °C, followed by stirring at 80 °C for 12 h. The mixture was diluted with water (50 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, dried over NaSO, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate=1 / 0, 3 / 1) to give tert-butyl 4-[[4-[[(4-nitrophenyl)sulfonyl-propyl-amino]methyl]phenyl]methyl]piperazine-1-carboxylate, Bz-30b (1.7 g, crude) as a yellow solid. 1 H NMR (DMSO, 400MHz) δ8.39 (d, J = 8.8 Hz, 2H), 8.11 (d, J = 8.8 Hz, 2H), 7.21 (s, 4H),4.36 (s, 2H), 3.45 (s, 2H), 3.31-2.27 (m, 4H), 3.12-3.05 (m, 2H), 2.28-2.26 (m, 4H), 1.38 (s, 9H), 1.33-1.25 (m, 2H), 0.65 (t, J = 7.6 Hz, 3H).

[0339] Preparation of Bz-30c: To a solution of Bz-30b (1.0 g, 1.88 mmol, 1.0 equiv) in CH3CN (6 mL) was added LiOH·HO (473 mg, 11.3 mmol, 6.0 equiv) in one portion at 0 °C. Next, methyl 2-sulfanylacetate (598 mg, 5.63 mmol, 511 µL, 3.0 equiv) was added, and the mixture was stirred at 25 °C for 2 h. The mixture was filtered and concentrated. The residue was diluted with MTBE (5 mL), and the pH of the mixture was adjusted to approximately 2 with aqueous HCl (1 M) and extracted with MTBE (20 mL) (discarded). The aqueous phase was adjusted to pH = 9 with aqueous NaHCO3 and then extracted with EtOAc (30 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give tert-butyl 4-[[4-(propylaminomethyl)phenyl]methyl]piperazine-1-carboxylate, Bz-30c (0.5 g, crude) as a yellow oil. 1 H NMR (MeOD, 400MHz) δ7.32-7.30 (m, 4H), 3.73 (s, 2H), 3.53 (s, 2H), 3.43-3.40 (m, 4H), 2.57-2.50 (m, 2H), 2.41-2.48 (m, 4H), 1.58-1.51 (m, 2H), 1.45 (s, 9H), 0.92 (t, J = 7.6 Hz, 3H).

[0340] Preparation of Bz-30: To a mixture of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-21d (400 mg, 936 μmol, 1.0 equiv.) in DMF (8 mL), PYAOP (585 mg, 1.12 mmol, 1.2 equiv.), DIEA (363 mg, 2.81 mmol, 489 μL, 3.0 equiv.), and Bz-30c (358 mg, 1.03 mmol, 1.1 equiv.) were added in one portion at 25° C., followed by stirring for 1 h. The mixture was filtered and concentrated. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100*30mm*5um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%~45%, 10 min) to give tert-butyl 4-[[4-[[[2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]methyl]phenyl]methyl]piperazine-1-carboxylate, Bz-30 (0.35g, 462umol, 49.4% yield) as a white solid. 1 H NMR (MeOD, 400MHz) δ 8.14-8.05 (m, 2H), 7.92 (d, J = 7.6 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.78-7.69 (m, 2H), 7.63-7.42 (m, 5H), 7.17 (s, 1H), 4.37 (s, 2H), 3.86 (t, J = 8.0 Hz, 2H), 3.61 (dd, J = 6.0, 8.0 Hz, 2H), 3.53-3.49 (m, 2H), 3.43-3.41 (m, 6H), 3.31-3.29 (m, 8H), 2.63-2.54 (m, 1H), 1.76-1.65 (m, 2H), 1.47 (s, 9H), 0.95-0.89 (m, 3H). LC / MS [M+H] 757.4 (calculated value); LC / MS [M+H] 757.4 (measured value).

[0341] Example 27 Synthesis of Bz-31 [ka] Preparation of Bz-31a: To a mixture of 3,3,3-trifluoropropan-1-amine (0.5 g, 3.34 mmol, 1 equiv., HCl) and NaHCO (842.64 mg, 10.03 mmol, 390.11 uL, 3 equiv.) in THF (3 mL) and HO (3 mL), tert-butoxycarbonyl tert-butyl carbonate (730 mg, 3.34 mmol, 768 uL, 1 equiv.) was added, followed by stirring at 25 °C for 1 h under a N atmosphere. The mixture was poured into HO (15 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic phase was washed with brine (15 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with (petroleum ether:ethyl acetate=5:0 to 1:1) to give tert-butyl N-(3,3,3-trifluoropropyl)carbamate, Bz-31a (500 mg, 2.35 mmol, 70.14% yield) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ4.75 (br s, 1H), 3.40 (q, J = 6.4 Hz, 2H), 2.40-2.27 (m, 2H), 1.45 (s, 9H).

[0342] Preparation of Bz-31b: To a solution of Bz-31a (400 mg, 1.88 mmol, 1 equiv.) in DMF (5 mL) was added NaH (113 mg, 2.81 mmol, 60% purity, 1.5 equiv.) at 0 °C. After 30 min, 1-iodopropane (637.88 mg, 3.75 mmol, 366 μL, 2 equiv.) was added to the mixture and stirred at 20 °C for 2 h. The reaction mixture was quenched at 0 °C by adding saturated NH Cl (10 mL) and then extracted with EtOAc (10 mL × 3). The organic phase was dried over anhydrous Na SO , filtered, and concentrated in vacuo. The reaction mixture was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 1:1). Compound tert-butyl N-propyl-N-(3,3,3-trifluoropropyl)carbamate, Bz-31b (400 mg, 1.57 mmol, 83.52% yield) was obtained as a colorless oil. 1H NMR (CDCl3, 400 MHz) δ3.41 (t, J = 7.2 Hz, 2H), 3.19-3.12 (m, 1H), 2.40-2.32 (m, 2H), 1.58-1.50 (m, 2H), 1.47 (s, 9H), 0.89 (t, J = 7.6Hz, 3H)

[0343] Preparation of Bz-31c: To a solution of tert-butyl N-propyl-N-(3,3,3-trifluoropropyl)carbamate (400 mg, 1.57 mmol, 1 equiv) in EtOAc (3 mL) was added HCl / EtOAc (4 M, 5.88 mL, 15 equiv), followed by stirring for 2 h at 20° C. The mixture was filtered and concentrated in vacuo to afford 3,3,3-trifluoro-N-propyl-propan-1-amine, Bz-31c (240 mg, crude, HCl) as a white solid. 1 H NMR (MeOD-d4, 400 MHz) δ3.34-3.31 (m, 2H), 3.06-3.00 (m,

[0344] Preparation of Bz-31: To a solution of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carboxylic acid, Bz-21d (100 mg, 233 μmol, 1 equiv.), DIEA (90.7 mg, 702 μmol, 122 μL, 3 equiv.), and PYAOP (183 mg, 351 μmol, 1.5 equiv.) in DMF (1 mL) was added Bz-31c (44.8 mg, 234 μmol, 1 equiv., HCl), followed by stirring at 20° C. for 1 h. The mixture was filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 30%~60%, 8 min) to give 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonylphenyl]-N-propyl-N-(3,3,3-trifluoropropyl)-3H-1-benzazepine-4-carboxamide, Bz-31 (7 mg, 12.40umol, 5.30% yield) as a white solid. 1H NMR (MeOD-d4,400MHz) δ8.07 (s, 1H), 8.04 (br d, J = 7.6 Hz, 1H), 7.86-7.81 (m, 1H), 7.80-7.73 (m, 1H), 7.49-7.44 (m, 2H), 7.42-7.37 (m, 1H), 6.94 (s, 1H), 3.86 (t, J = 8.4 Hz, 2H), 3.73 (br s, 2H), 3.60 (dd, J = 6.0, 8.0 Hz, 2H), 3.52-3.45 (m, 2H), 3.42 (d, J = 6.4 Hz, 2H), 3.33-3.32 (m, δ 5.77-5.93 (m, 2H), 2.68-2.53 (m, 3H), 1.74-1.64 (m, 2H), 0.91 (br s, 3H). LC / MS [M+H] 565.2 (calcd); LC / MS [M+H] 565.3 (observed).

[0345] Example 28 Synthesis of Bz-32

change

[0346] Example 29 Synthesis of Bz-33 [ka] 2-Amino-N-(3-aminopropyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, Bz-17 (0.01 g, 0.019 mmol, 1 equiv.) was dissolved in DCM. Triethylamine (4 μL, 0.029 mmol, 1.5 equiv.) was added, followed by 4-ethoxybenzoyl chloride (0.004 g, 0.019 mmol, 1 equiv.). The reaction was stirred at room temperature, then concentrated and purified by HPLC to give 2-amino-N-(3-(4-ethoxybenzamido)propyl)-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, Bz-33 (0.0028 g, 0.0042 mmol, 22%). LC / MS [M+H] 674.30 (calculated); LC / MS [M+H] 674.74 (observed).

[0347] Example 30 Synthesis of Bz-34 [ka] 2-Amino-N 4 -(3-aminopropyl)-N 8 -phenyl-N 4 4-Propyl-3H-benzo[b]azepine-4,8-dicarboxamide, Bz-34a (0.01 g, 0.024 mmol, 1 equiv.) was dissolved in DCM. Triethylamine (5 μl, 0.036 mmol, 1.5 equiv.) was added, followed by 4-ethoxybenzoyl chloride (0.004 g, 0.024 mmol, 1 equiv.). The reaction was stirred at room temperature, then concentrated and purified by HPLC to give 2-amino-N 4 -(3-(4-ethoxybenzamido)propyl)-N 8 -phenyl-N 4 To give 3,4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, Bz-34 (0.005 g, 0.009 mmol, 38%): LC / MS [M+H] 568.29 (calculated); LC / MS [M+H] 568.50 (observed).

[0348] Preparation of Aminobenzazepine-Linker Formula II Compounds (BzL) and Intermediates Example 31 Synthesis of BzL-1 Ethyl 2-amino-8-(3-((2-(2-(3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy)ethoxy)ethyl)carbamoyl)phenyl)-3H-benzo[b]azepine-4-carboxylate, BzL-1, was prepared and characterized according to the procedures described herein.

[0349] Example 32 Synthesis of BzL-2 [ka] [ka] Synthesis of 2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-(3-(methylamino)propyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, BzL-2a BzL-2a was synthesized from Bz-3 according to the procedure described for Bz-11a: LC / MS [M+H] 540.26 (calculated); LC / MS [M+H] 540.53 (observed).

[0350] Synthesis of tert-butyl 80-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carbonyl)-76-methyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetracosaoxa-76,80-diazatrioctacontanoate, BzL-2b. A vial was charged with BzL-2a (15.1 mg, 0.028 mmol), tert-butyl 1-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxapentaheptacontane-75-oate (0.042 mmol), and sodium triacetoxyborohydride (30 mg, 0.14 mmol) in 100 µL of DMF. The reaction was stirred for 5 h, then 100 µL of 10% sodium carbonate was added and stirred for 1 h. The mixture was filtered and purified by reverse-phase preparative HPLC using a 25-75% gradient of acetonitrile:water containing 0.1% trifluoroacetic acid. The purified fractions were combined and lyophilized to give 40.7 mg of BzL-2b in 84% yield: LC / MS [M+H] 1724.98 (calculated); LC / MS [M+H] 1726.52 (observed).

[0351] Synthesis of 80-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carbonyl)-76-methyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetracosaoxa-76,80-diazatrioctacontanoic acid, BzL-2c. A vial was charged with BzL-2b (18 mg, 0.010 mmol), 300 μL of DCM, and 100 μL of trifluoroacetic acid. The reaction was maintained for 45 min, concentrated in vacuo, and azeotroped three times with 1 mL of toluene. The reaction was used further without further purification.

[0352] 2,3,5,6-Tetrafluorophenyl 80-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carbonyl)-76-methyl-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73-tetracosaoxa-76,80-diazatrioctacontanoate, BzL-2, was synthesized according to the procedure described for BzL-22. LC / MS [M+H] 1816.91 (calculated); LC / MS [M+H] 1818.51 (observed).

[0353] Example 33 Synthesis of BzL-3 [ka] Synthesis of 2-benzylsulfanyl-4-bromo-benzonitrile, BzL-3b To a mixture of phenylmethanethiol (3.10 g, 25.00 mmol, 2.93 mL, 1 equiv.) and 4-bromo-2-fluoro-benzonitrile, BzL-3a (5 g, 25.00 mmol, 1 equiv.) in DMF (10 mL) was added CsCO (12.22 g, 37.50 mmol, 1.5 equiv.) at 25 °C. The mixture was stirred at 25 °C for 1 h. TLC and LCMS showed the reaction was complete. The mixture was poured into ice water (100 mL), stirred for 5 min, and filtered to give BzL-3b (4 g, 13.15 mmol, 52.60% yield) as a white solid, which was used in the next step without further purification. 1 H NMR (CDCl3, 400 MHz) δ 7.50 (d, J = 2.0 Hz, 1H), 7.47-7.43 (m, 1H), 7.41-7.38 (m, 1H), 7.35-7.28 (m, 5H), 4.23 (s, 2H).

[0354] Synthesis of 5-bromo-2-cyano-benzenesulfonyl chloride, BzL-3c To a mixture of 2-benzylsulfanyl-4-bromo-benzonitrile (1 g, 3.29 mmol, 1 equiv.), AcOH (0.7 mL), and HO (0.5 mL) in CHCN (20 mL) was added 1,3-dichloro-5,5-imethyl-imidazolidine-2,4-dione (1.30 g, 6.57 mmol, 2 equiv.) portionwise at 0 °C. The mixture was stirred at 0 °C for 30 min. TLC and LCMS showed the reaction was complete. The mixture was poured into ice water (50 mL) and stirred for 2 min. The aqueous phase was extracted with DCM (20 mL × 2). The combined organic phase was washed with brine (30 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate=20 / 1, 10 / 1) to give BzL-3c (0.8 g, 2.85 mmol, 86.75% yield) as a white solid. 1 H NMR (CDCl3, 400 MHz) δ 8.34 (d, J = 2.0 Hz, 1H), 7.99 (dd, J = 8.4, 2.0 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H)

[0355] Synthesis of 4-bromo-2-[3-(hydroxymethyl)azetidin-1-yl]sulfonyl-benzonitrile, BzL-3d To a mixture of azetidin-3-ylmethanol (1.54 g, 12.48 mmol, 1 equiv., HCl) in DCM (100 mL), DBU (3.80 g, 24.95 mmol, 3.76 mL, 2 equiv.) was added dropwise at 0 °C and stirred for 10 min. 5-Bromo-2-cyano-benzenesulfonyl chloride, BzL-3c (3.5 g, 12.48 mmol, 1 equiv.) was added to the mixture and stirred at 0 °C for 30 min. TLC showed the reaction was complete. The mixture was poured into ice water (100 mL) and stirred for 2 min. The aqueous phase was extracted with DCM (50 mL × 3). The combined organic phase was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated to give BzL-3d (3.5 g, crude) as a colorless oil, which was used in the next step without further purification.

[0356] Synthesis of 4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-benzonitrile, BzL-3e To a mixture of 4-bromo-2-[3-(hydroxymethyl)azetidin-1-yl]sulfonyl-benzonitrile, BzL-3d (3.5 g, 10.57 mmol, 1 equiv.) and tert-butyldimethylsilyl chloride, TBSCl (1.91 g, 12.68 mmol, 1.55 mL, 1.2 equiv.) in DCM (30 mL), imidazole (1.08 g, 15.85 mmol, 1.5 equiv.) was added in one portion at 25 °C. The mixture was stirred at 25 °C for 2 h. LCMS showed the reaction was complete. The mixture was poured into ice water (200 mL) and stirred for 2 min. The aqueous phase was extracted with DCM (100 mL × 3). 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 (petroleum ether / ethyl acetate = 20 / 1, 10 / 1) to give BzL-3e (3.8 g, 8.53 mmol, 80.72% yield) as a colorless oil. 1 H NMR (CDCl3, 400 MHz) δ 8.20 (d, J = 2.0 Hz, 1H), 7.82 (dd, J = 8.4, 2.0 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 4.10-4.06 (m, 2H), 3.96-3.93 (m, 2H), 3.68 (d, J = 5.2 Hz, 2H), 2.82-2.76 (m, 1H), 0.86 (s, 9H), 0.00 (s, 6H).

[0357] [ka] Synthesis of 4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-benzaldehyde, BzL-3f To a solution of 4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-benzonitrile, BzL-3e (3.8 g, 8.53 mmol, 1 equiv.) in DCM (100 mL) was added diisobutylaluminum hydride, DIBAL-H (1 M, 9.38 mL, 1.1 equiv.) dropwise at 0° C. under N. The mixture was stirred at 0° C. for 1 h. LCMS showed the reaction was complete. To the mixture was added saturated aqueous NH4Cl (3 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 = 20 / 1, 5 / 1) to give BzL-3f (3.5 g, 7.80 mmol, 91.49% yield) as a pale yellow oil. 1 H NMR (CDCl3, 400 MHz) δ 10.69 (s, 1H), 8.16 (d, J = 1.6 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.86 (dd, J = 1.6, 8.4 Hz, 1H), 3.95-3.88 (m, 2H), 3.81-3.76 (m, 2H), 3.65-3.64 (m, 2H), 2.85-2.71 (m, 1H), 0.85 (s, 8H), 0.03 (s, 6H).

[0358] Synthesis of 1-[4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-phenyl]-N-methyl-methanamine, BzL-3g To a solution of methanamine (4.16 g, 40.14 mmol, 5 equiv) (30% in MeOH) and 4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-benzaldehyde, BzL-3f (3.6 g, 8.03 mmol, 1 equiv) in MeOH (15 mL) and DCE (15 mL) was added AcOH (482.08 mg, 8.03 mmol, 459.12 μL, 1 equiv) and NaBHCN (1.26 g, 20.07 mmol, 2.5 equiv). The mixture was stirred at 25 °C for 18 h. A few drops of water were added to the mixture and it was concentrated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1:1) to give BzL-3g (2 g, 4.31 mmol, 53.75% yield) as a colorless oil. 1 H NMR (DMSO-d6, 400 MHz) δ 8.09-8.06 (m, 1H), 8.01-7.99 (m, 1H), 7.71 (d, J = 8.4 Hz, 1H), 4.27 (s, 2H), 3.85-3.80 (m, 2H), 3.62-3.58 (m, 2H), 3.55 (d, J = 5.2 Hz, 2H), 2.69-2.75 (m, 1H), 2.56 (s, 3H), 0.82 (s, 9H),0.00 (s, 6H).

[0359] Synthesis of tert-butyl N-[[4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-phenyl]methyl]-N-methyl-carbamate, BzL-3h To a mixture of 1-[4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-phenyl]-N-methyl-methanamine, BzL-3g (2 g, 4.31 mmol, 1 equiv.) in THF (15 mL) and HO (3 mL), NaCO (914.68 mg, 8.63 mmol, 2 equiv.) and BocO (1.41 g, 6.47 mmol, 1.49 mL, 1.5 equiv.) were added in one portion at 25 °C. The mixture was stirred at 25 °C for 1 h. The mixture was poured into ice water (10 mL) and stirred for 1 min. The aqueous phase was extracted with ethyl acetate (10 mL × 3). The combined organic phase was washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 2 g SepaFlash® silica flash column, eluent of 0–50% ethyl acetate / petroleum ether gradient at 45 mL / min) to afford BzL-3h (1.4 g, 2.48 mmol, 57.57% yield) as a colorless oil. 1 H NMR (DMSO-d6, 400 MHz) δ 8.00-7.99 (m, 2H), 7.23 (d, J = 8.4 Hz, 1H), 4.66 (s, 2H), 3.85-3.79 (m, 2H), 3.61-3.57 (m, 4H), 2.85 (s, 3H), 2.51-2.49 (m, 1H), 1.47-1.31 (m, 9H), 0.81 (s, 9H), -0.01 (s, 6H)

[0360] Synthesis of tert-butyl N-[[4-[2-amino-4-(dipropylcarbamoyl)-3H-1-benzazepin-8-yl]-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-phenyl]methyl]-N-methyl-carbamate, BzL-3i To a mixture of [2-amino-4-(dipropylcarbamoyl)-3H-1-benzazepin-8-yl]boronic acid (360 mg, 1.09 mmol, 1 equiv.) and tert-butyl N-[[4-bromo-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-phenyl]methyl]-N-methyl-carbamate, BzL-3h (616.35 mg, 1.09 mmol, 1 equiv.) in dioxane (3 mL) and HO (0.5 mL), Pd(dppf)Cl (80.02 mg, 109.36 μmol, 0.1 equiv.) and NaCO (231.81 mg, 2.19 mmol, 2 equiv.) were added in one portion under N at 25 °C. The mixture was stirred at 90 °C for 2 h. The mixture was filtered and concentrated. The residue was poured into HO (20 mL) and extracted with ethyl acetate (20 mL × 2). The combined organic phases were washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 1 g SepaFlash® silica flash column, eluent: 0–100% ethyl acetate / petroleum ether gradient at 75 mL / min) to afford BzL-3i (360 mg, 468.69 μmol, 42.86% yield) as a yellow solid.

[0361] [ka] Synthesis of 2-amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonyl-4-(methylamineomethyl)phenyl]-N,N-dipropyl-3H-1-benzazepine-4-carboxamide, BzL-3j To a mixture of tert-butyl N-[[4-[2-amino-4-(dipropylcarbamoyl)-3H-1-benzazepin-8-yl]-2-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]azetidin-1-yl]sulfonyl-phenyl]methyl]-N-methyl-carbamate, BzL-3i (170 mg, 221.33 μmol, 1 equiv.) in THF (5 mL) and HO (1 mL), TFA (504.72 mg, 4.43 mmol, 327.74 μL, 20 equiv.) was added, and the mixture was stirred at 50 °C for 12 h. LC-MS showed that reactant 1 was completely consumed and one major peak with the desired mass was detected. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Nano-micro Kromasil C18 100 × 30 mm 5 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 20% to 45%, 10 min) to give BzL-3j (crude 95 mg) product as a yellow solid. 1 H NMR (DMSO-d6, 400 MHz) δ 12.49 (s, 1H), 9.88 (s, 1H), 9.50 (s, 1H), 8.87 (s, 2H), 8.24-8.22 (m, 1H), 8.17-8.16 (m, 1H), 7.92-7.90 (m, 1H), 7.74-7.71 (m, 1H), 7.67-7.70 (m, 2H), 7.06 (s, 1H), 4.79 (s, 1H), 4.46 (s, 2H), 3.85 (t, J = 8.0 Hz, 2H), 3.61 (t, J = 4.0 Hz, 2H), 3.35 (s, 4H), 2.67 (s, 3H), 2.64-2.55 (m, 2H), 1.74-1.39 (m, 4H), 0.86-0.80 (m, 6H). LC / MS [M+H] 554.28 (calculated value); LC / MS [M+H] 554.40 (measured value).

[0362] Synthesis of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-(dipropylcarbamoyl)-3H-1-benzazepin-8-yl]-2-[3-(hydroxymethyl)azetidin-1-yl]sulfonyl-phenyl]methyl-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, BzL-3k 2-Amino-8-[3-[3-(hydroxymethyl)azetidin-1-yl]sulfonyl-4-(methylaminomethyl)phenyl]-N,N-dipropyl-3H-1-benzazepine-4-carboxamide, BzL-3j (0.05 g, 90.30 μmol, 1 equiv.) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy)) in MeOH (2 mL). To a mixture of [ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, t-BuOOC-PEG10-CHO (52.80 mg, 90.30 μmol, 1 equiv.), EtN (27.41 mg, 270.90 μmol, 37.71 μL, 3 equiv.), AcOH (5.42 mg, 90.30 μmol, 5.16 μL, 1 equiv.), and NaBHCN (14.19 mg, 225.75 μmol, 2.5 equiv.) was added at 25 °C. The mixture was stirred for 12 h. The mixture was concentrated in vacuo to give BzL-3k (crude 100 mg) as a yellow oil.

[0363] Synthesis of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-(dipropylcarbamoyl)-3H-1-benzazepin-8-yl]-2-[3-(hydroxymethyl)azetidin-1-yl]sulfonyl-phenyl]methyl-methyl-amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, BzL-3l To a solution of BzL-3k (100 mg, 89.09 μmol, 1 equiv.) in HO (1 mL) was added TFA (203.18 mg, 1.78 mmol, 131.93 μL, 20 equiv.). The mixture was stirred at 60 °C for 12 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Luna C18 100 × 30 5 μL; liquid phase: [A - TFA / HO = 0.075% v / v; B - ACN], B%: 20% to 45%, 10 min) to give BzL-3l (20 mg, 18.38 μmol, 20.63% yield, 97.989% purity) as a colorless oil. 1 H NMR (MeOD, 400 MHz) δ 8.39-8.38 (m, 1H), 8.23-8.20 (m, 1H), 7.98-7.96 (m, 1H), 7.83-7.81 (m, 2H), 7.73-7.71 (m, 1H), 7.11 (s, 1H), 4.02-4.00 (m, 2H), 3.94-3.88 (m, 2H), 3.79-3.74 (m, 2H), 3.74-3.40 (m, 45H), 3.40-3.35 (m, 2H), 2.98-2.94 (m, 3H), 2.79-2.71 (m, 2H), 2.56-2.51 (m, 2H), 1.80-1.66 (m, 5H), 0.95 (s, 6H). LC / MS [M+2H / 2] 533.78 (calculated value); LC / MS [M+2H / 2] 534.20 (measured value).

[0364] 2,3,5,6-Tetrafluorophenyl 1-(4-(2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepin-8-yl)-2-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-2-methyl-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azapentatriacontan-35-oate, BzL-3, was synthesized according to the procedure described for BzL-22. LC / MS [M+H] 1214.56 (calculated); LC / MS [M+H] 1214.97 (observed).

[0365] Example 34 Synthesis of BzL-4 [ka] 2,3,5,6-Tetrafluorophenyl 84-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-3H-benzo[b]azepine-4-carbonyl)-80-methyl-79-oxo-4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-pentacosaoxa-80,84-diazaheptaoctacontanoate, BzL-4, was synthesized according to the procedure described for BzL-15. LC / MS [M+H] 1888.93 (calculated); LC / MS [M+H] 1889.53 (observed).

[0366] Example 35 Synthesis of BzL-5 [ka] 4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl (3-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)(methyl)carbamate, BzL-5a, was synthesized according to the procedure described for BzL-26a.

[0367] 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (3-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)(methyl)carbamate, BzL-5b, was synthesized according to the procedure described for BzL-26. LC / MS [M+H] 945.47 (calculated); LC / MS [M+H] 945.82 (observed).

[0368] 2,3,5,6-Tetrafluorophenyl(6S,9S)-1-amino-6-((4-((((3-(2-amino-8-(3-((3-(hydroxymethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)(methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-9- Isopropyl-1,8,11-trioxo-14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,...

Claims

1. and comprising an antibody covalently linked by a linker to one or more aminobenzazepine moieties, said antibody having formula I: I ｐ 9 or a pharmaceutically acceptable salt thereof. (In the formula, Ab is the antibody; p is an integer from 1 to 8; Bza has the formula: 【Chemical 1】 wherein the aminobenzazepine moiety has the formula: R １ , R ２ , R ３ , and R ４ is H, C １ -C １２ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryl, C ２ -C ９ Heterocyclyl, and C １ -C ２０ heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently selected from the group consisting of: -(C １ -C １２ alkyldiyl)-N(R ５ )-*; -(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C ３ -C １２ carbocyclyl); -(C ３ -C １２ carbocyclyl)-*; -(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)-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 alkyldiyl)-NR ５ - *; -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C ２ -C ９ Heterocyclyl)-NR ５ -C(=NR ５ａ ) NR ５ - *; -(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(=O)-*; -C(=O)-(C ２ -C ２０ Heterocyclyldiyl)-*; -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 )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 )C(=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 ２ -C ５ heteroaryl); -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 heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-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 heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-OH; or R ２ and R ３ taken together form a 5- or 6-membered heterocyclyl ring; X １ , X ２ , X ３ , and X ４ represents a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N (R ５ ) independently selected from the group consisting of: R ５ is H, C ６ -C ２０ Aryl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups taken together form a 5- or 6-membered heterocyclyl ring; R ５ａ is C ６ -C ２０ Aryl and C １ -C ２０ selected from the group consisting of heteroaryl; where the asterisk * indicates the binding site of L, and where R １ , R ２ , R ３ and R ４ is attached 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 alkyldiyl)-; -C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )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 alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-; -C(=O)-(PEG)-N(R ５ )-; -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 alkyldiyl)-OC(═O)-; -C(=O)-(PEG)-SS-(C １ -C １２ alkyldiyl)-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 the linker is selected from the group consisting of: PEG is a group represented by 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 has the formula: 【Chemistry 2】 wherein AA １ and A.A. ２ are independently selected from amino acid side chains, or AA １ Or AA ２ and the adjacent nitrogen atom form a five-membered ring proline amino acid, the wavy line indicating the point of attachment; R ６ is C ６ -C ２０ Aryldiyl and C １ -C ２０ heteroaryldiyl, —CH ２ O—C(═O)— and optionally 【Chemistry 3】 is substituted with; and MCgluc is a group: 【Chemistry 4】 wherein q is 1 to 8 and AA is an amino acid side chain; Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are selected from the group consisting of 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 P. ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ -NHCOCH ３ 、-N(CH ３ )COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ P. 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 SO ３ 、-OCH ２ CH ２ H 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, -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H).

2. The immunoconjugate of claim 1, wherein the antibody is an antibody construct having an antigen-binding domain that binds to PD-L1.

3. 3. The immunoconjugate of claim 2, wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, and avelumab, or a biosimilar or biobetter thereof.

4. The immunoconjugate of claim 1 , wherein the antibody is an antibody construct having an antigen-binding domain that binds to HER2.

5. 5. The immunoconjugate of claim 4, wherein the antibody is selected from the group consisting of trastuzumab and pertuzumab, or a biosimilar or biobetter thereof.

6. The immunoconjugate of claim 1 , wherein the antibody is an antibody construct having an antigen-binding domain that binds to CEA.

7. The immunoconjugate of claim 6 , wherein the antibody is labetuzumab, or a biosimilar or biobetter thereof.

8. PEP has the formula: 【Chemistry 5】 The immunoconjugate of any one of claims 1 to 7, having the formula:

9. PEP is a group: 【Chemistry 6】 8. The immunoconjugate of any one of claims 1 to 7, wherein n is 1 or more and AA is an amino acid side chain.

10. A.A. １ and A.A. ２ The immunoconjugate of any one of claims 1 to 9, wherein are independently selected from the side chains of naturally occurring amino acids.

11. A.A. １ and A.A. ２ is 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 NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ SO ３ H, and -CH ２ CH ２ CH ２ NHC(O)NH ２ The immunoconjugate of any one of claims 1 to 9, wherein the immunoconjugate is independently selected from:

12. A.A. １ But -CH(CH ３ ) ２ and AA ２ But -CH ２ CH ２ CH ２ NHC(O)NH ２ The immunoconjugate of claim 11, wherein:

13. A.A. １ and A.A. ２ GlcNAc aspartic acid, -CH ２ SO ３ H, and -CH ２ OPO ３ H. The immunoconjugate of any one of claims 1 to 7, wherein

14. Bza is a compound of formula Ia-d: 【Chemistry 7】 The immunoconjugate of any one of claims 1 to 13, selected from:

15. Bza is a compound of formula Ie and If: 【Chemistry 8】 wherein R of formula If is selected from ５ａ is selected from the group consisting of phenyl and pyridyl, and is selected from the group consisting of F, Cl, Br, I, —CN, —NO ２ and -OCH ３ The immunoconjugate of any one of claims 1 to 13, optionally substituted with one or more groups selected from:

16. The immunoconjugate of claim 15, wherein L is -C(=O)-(PEG)-.

17. Bza is a compound of formula Ig and Ih: 【Chemistry 9】 The immunoconjugate of any one of claims 1 to 13, selected from:

18. 18. The immunoconjugate of claim 17, wherein L is -C(=O)-(PEG)- or -C(=O)-(PEG)-C(=O)-.

19. R ２ and R ３ However, each １ -C ８ 19. The immunoconjugate of claim 18, wherein the aryl group is alkyl.

20. R ２ and R ３ are -CH ２ CH ２ CH ３ 20. The immunoconjugate of claim 19, wherein:

21. X ２ and X ３ are bonds, and R ２ or R ３ But -O-(C １ -C １２ The immunoconjugate of claim 1 , wherein the aryl group is alkyl.

22. R ２ or R ３ But, -OCH ２ CH ３ 22. The immunoconjugate of claim 21, wherein:

23. R １ and R ４ One of them is, -(C １ -C １２ alkyldiyl)-N(R ５ )-*; -(C １ -C １２ alkyldiyl)-N(R ５ ) C(=NR ５ ) N (R ５ )-*; -(C ６ -C ２０ alkyldiyl)-S(=O) ２ -(C ２ -C ２０ Heterocyclyldiyl)-*; -(C ６ -C ２０ Aryldiyl)-S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )-*; -(C ６ -C ２０ aryldiyl)-C(═O)-*; -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )-*; -(C ６ -C ２０ aryldiyl)-C(═O)-(C ２ -C ２０ Heterocyclyldiyl)-*; -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-*; and -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 -*; X １ and X ４ The immunoconjugate of any one of claims 1 to 7, wherein: is a bond, and the asterisk * indicates the binding site of L.

24. 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 ) C(=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 alkynyldiyl)-N(R ５ )-*; and -(C ２ -C ６ alkynyldiyl)-N(R ５ ) C(=NR ５ ) N (R ５ )-*; X ２ and X ３ The immunoconjugate of any one of claims 1 to 7, wherein: is a bond, and the asterisk * indicates the binding site of L.

25. R １ and R ４ One of the two is -(C ６ -C ２０ Aryldiyl)-S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ and -(C ６ -C ２０ Aryldiyl)-S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ The immunoconjugate of any one of claims 1 to 7, wherein the aryl group is selected from the group consisting of alkyldiyl)-OH.

26. C ６ -C ２０ Aryldiyl is phenyldiyl, ２ -C ２０ 26. The immunoconjugate of claim 25, wherein the heterocyclyldiyl is azetidinediyl.

27. R １ and R ４ is of the formula: 【Chemistry 10】 27. The immunoconjugate of claim 26, selected from:

28. R １ and R ４ One of the groups is —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 The immunoconjugate of any one of claims 1 to 7, wherein the nucleotide sequence is -L.

29. C １ -C ２０ Heteroaryldiyl is pyridinediyl, ２ -C ２０ 29. The immunoconjugate of claim 28, wherein the heterocyclyldiyl is piperidiyl.

30. Formula II: 【Chemistry 11】 Aminobenzazepine-linker compounds of (In the formula, Z is H, —O(C １ -C ８ alkyl), and N(X ２ R ２ ) (X ３ R ３ ) selected from; R １ , R ２ , R ３ , and R ４ is H, C １ -C １２ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryl, C ２ -C ９ Heterocyclyl, and C １ -C ２０ heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently selected from the group consisting of: -(C １ -C １２ alkyldiyl)-N(R ５ )-*; -(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C ３ -C １２ carbocyclyl); -(C ３ -C １２ carbocyclyl)-*; -(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)-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 alkyldiyl)-NR ５ - *; -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ ; -(C ２ -C ９ Heterocyclyl)-NR ５ -C(=NR ５ａ ) NR ５ - *; -(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(=O)-*; -C(=O)-(C ２ -C ２０ Heterocyclyldiyl)-*; -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 )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 )C(=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 ２ -C ５ heteroaryl); -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 heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-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 heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-OH; or R ２ and R ３ taken together form a 5- or 6-membered heterocyclyl ring; X １ , X ２ , X ３ , and X ４ represents a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N (R ５ ) independently selected from the group consisting of: R ５ is H, C ６ -C ２０ Aryl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups taken together form a 5- or 6-membered heterocyclyl ring; R ５ａ is C ６ -C ２０ Aryl and C １ -C ２０ selected from the group consisting of heteroaryl; where the asterisk * indicates the binding site of L, and where R １ , R ２ , R ３ and R ４ is attached to L; L is, 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 alkyldiyl)-; Q-C(=O)-(PEG)-C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-; 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 alkyldiyl)-; 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 )-(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 alkyldiyl)-OC(═O)-; Q-C(=O)-(PEG)-SS-(C １ -C １２ alkyldiyl)-C(═O)-; Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-(PEP)-; Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-(PEP)-N(R ５ )-(C １ -C １２ alkyldiyl)-; Q-C(=O)-(C １ -C １２ alkyldiyl)-C(═O)-(PEP)-N(R ５ )-(C １ -C １２ alkyldiyl)-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 the linker is selected from the group consisting of: Here, PEG is a group represented by 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 has the formula: 【Chemistry 12】 wherein AA １ and A.A. ２ are independently selected from amino acid side chains, or AA １ Or AA ２ and the adjacent nitrogen atom form a five-membered ring proline amino acid, the wavy line indicating the point of attachment; R ６ is C ６ -C ２０ Aryldiyl and C １ -C ２０ heteroaryldiyl, —CH ２ O—C(═O)— and optionally: 【Chemistry 13】 is substituted with; and MCgluc is a group: 【Chemistry 14】 wherein q is 1 to 8 and AA is an amino acid side chain; Q is F, Cl, NO ２ and SO ３ － is selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from Here, alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are selected from the group consisting of 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 P. ２ 、-NH ２ 、-NHCH ３ 、-N(CH ３ ) ２ -NHCOCH ３ 、-N(CH ３ )COCH ３ 、-NHS(O) ２ CH ３ 、-N(CH ３ )C(CH ３ ) ２ P. 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 SO ３ 、-OCH ２ CH ２ H 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, -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H).

31. PEP has the formula: 【Chemistry 15】 31. The aminobenzazepine-linker compound of claim 30, having the formula:

32. PEP is a group: 【Chemistry 16】 31. The aminobenzazepine-linker compound of claim 30 selected from: wherein n is 1 or greater and AA is an amino acid side chain.

33. A.A. １ and A.A. ２ 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, wherein are independently selected from the side chains of naturally occurring amino acids.

34. A.A. １ and A.A. ２ is 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 NHC (NH) NH ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ SO ３ H, and -CH ２ CH ２ CH ２ NHC(O)NH ２ 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, independently selected from:

35. A.A. １ -CH(CH ３ ) ２ and AA ２ Ga-CH ２ CH ２ CH ２ NHC(O)NH ２ 35. The aminobenzazepine-linker compound of claim 34, wherein:

36. A.A. １ and A.A. ２ GlcNAc aspartic acid, -CH ２ SO ３ H, and -CH ２ OPO ３ 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, wherein the aminobenzazepine-linker compound is independently selected from:

37. Formula IIa-d: 【Chemistry 17】 The aminobenzazepine-linker compound according to any one of claims 30 to 32, selected from

38. Formulae IIe and IIf: 【Chemistry 18】 wherein R of formula If is selected from ５ａ is selected from the group consisting of phenyl and pyridyl, and is selected from the group consisting of F, Cl, Br, I, —CN, —NO ２ and -OCH ３ 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, optionally substituted with one or more groups selected from:

39. 39. The aminobenzazepine-linker compound of claim 38, wherein L is Q-C(=O)-(PEG)- or Q-C(=O)-(PEG)-C(=O)-.

40. Formulae IIg and IIh: 【Chemistry 19】 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, selected from:

41. 41. The aminobenzazepine-linker compound of claim 40, wherein L is -C(=O)-(PEG)-C(=O)-(PEP)-.

42. R ２ and R ３ However, each １ -C ８ 42. The aminobenzazepine-linker compound of any one of claims 30 to 41, which is alkyl.

43. R ２ and R ３ are -CH ２ CH ２ CH ３ 43. The aminobenzazepine-linker compound of claim 42, wherein:

44. X ２ and X ３ are bonds, and R ２ or R ３ But -O-(C １ -C １２ 44. The aminobenzazepine-linker compound of claim 43, which is alkyl.

45. R ２ or R ３ But, -OCH ２ CH ３ 45. The aminobenzazepine-linker compound of claim 44, wherein:

46. R １ and R ４ One of the two is -(C ６ -C ２０ Aryldiyl)-S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ and -(C ６ -C ２０ Aryldiyl)-S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, selected from: (alkyldiyl)-OH.

47. C ６ -C ２０ Aryldiyl is phenyldiyl, ２ -C ２０ 47. The aminobenzazepine-linker compound of claim 46, wherein the heterocyclyldiyl is azetidinediyl.

48. R １ and R ４ one of which is of the formula: 【Chemistry 20】 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, selected from:

49. R １ and R ４ One of the groups is —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 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, wherein:

50. C １ -C ２０ Heteroaryldiyl is pyridinediyl, ２ -C ２０ 50. The aminobenzazepine-linker compound of claim 49, wherein the heterocyclyldiyl is piperidiyl.

51. Q is, 【Chemical 21】 33. The aminobenzazepine-linker compound of any one of claims 30 to 32, selected from:

52. 31. The aminobenzazepine-linker compound of claim 30 selected from Table 2a.

53. 31. The aminobenzazepine-linker compound of claim 30 selected from Table 2b.

54. 31. The aminobenzazepine-linker compound of claim 30 selected from Table 2c.

55. An immunoconjugate prepared by conjugating an antibody with the aminobenzazepine-linker compound of any one of claims 52-54.

56. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 29 and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

57. 57. A method of treating cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 56 to a subject in need thereof.

58. 58. The method of claim 57, wherein the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8 agonism.

59. 58. The method of claim 57, wherein the cancer is a PD-L1-expressing cancer.

60. 58. The method of claim 57, wherein the cancer is a HER2-expressing cancer.

61. 58. The method of claim 57, wherein the cancer is a CEA-expressing cancer.

62. 62. The method of any one of claims 57 to 61, 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.

63. 63. The method of claim 62, wherein the breast cancer is triple-negative breast cancer.

64. 63. The method of claim 62, wherein the Merkel cell carcinoma is metastatic Merkel cell carcinoma.

65. 63. The method of claim 62, wherein the gastric cancer is a HER2-overexpressing gastric cancer.

66. 63. The method of claim 62, wherein the cancer is gastroesophageal junction adenocarcinoma.

67. 30. Use of the immunoconjugate of any one of claims 1 to 29 for treating cancer.

68. 31. A method for preparing an immunoconjugate of formula I according to claim 1, wherein the aminobenzazepine-linker compound of formula II according to claim 30 is conjugated to an antibody.

69. the antibody construct is a type A PD-L1 antibody and comprises an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide, the immunoglobulin heavy chain variable region polypeptide comprises a type A complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 1-23, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 24-57, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 58-95; or the immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 96-128, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 129-151, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 152-155; 3. The immunoconjugate of claim 2, wherein the sequence is from Figures 1-4.

70. the antibody construct is a type A PD-L1 antibody and comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 223-264, or at least the CDRs thereof, and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 265-306, or at least the CDRs thereof; 3. The immunoconjugate of claim 2, wherein the sequence is from Figures 1-4.

71. the antibody construct is a type A PD-L1 antibody and comprises an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 223-264, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 265-306; 3. The immunoconjugate of claim 2, wherein the sequence is from Figures 1-4.

72. 3. The immunoconjugate of claim 2, wherein the antibody construct is a type A PD-L1 antibody and comprises the heavy and light chain immunoglobulin polypeptides of the PD-L1-binding agent of Figures 1A-D, or at least the CDRs thereof.

73. the antibody construct is a type B PD-L1 antibody and comprises an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide, wherein: the immunoglobulin heavy chain variable region polypeptide comprises a complementarity determining region 1 (HCDR1) comprising any one of SEQ ID NOs: 308-321, a complementarity determining region 2 (HCDR2) comprising any one of SEQ ID NOs: 322-338, and a complementarity determining region 3 (HCDR3) comprising any one of SEQ ID NOs: 339-359; or the immunoglobulin light chain variable region polypeptide comprises a complementarity determining region 1 (LCDR1) comprising any one of SEQ ID NOs: 360-374, a complementarity determining region 2 (LCDR2) comprising any one of SEQ ID NOs: 131 and 375-386, and a complementarity determining region 3 (LCDR3) comprising any one of SEQ ID NOs: 387-398; 3. The immunoconjugate of claim 2, wherein the sequence is from Figures 5-8.

74. the antibody construct is a type B PD-L1 antibody and comprises an immunoglobulin heavy chain variable region of any one of SEQ ID NOs: 430-450, or at least the CDRs thereof, and an immunoglobulin light chain variable region of any one of SEQ ID NOs: 451-471, or at least the CDRs thereof; 3. The immunoconjugate of claim 2, wherein the sequence is from Figures 5-8.

75. the antibody construct is a type B PD-L1 antibody and comprises an immunoglobulin heavy chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 430-450, and an immunoglobulin light chain variable region polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 451-471; 3. The immunoconjugate of claim 2, wherein the sequence is from Figures 5-8.

76. 3. The immunoconjugate of claim 2, wherein the antibody construct is a type B PD-L1 antibody and comprises the heavy and light chain immunoglobulin polypeptides of the PD-L1-binding agent of Figures 5A-B, or at least the CDRs thereof.

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