Anti-CEA immunoconjugates and uses thereof

Abstract

The present invention provides immunoconjugates of formula (I) comprising an anti-CEA antibody linked by conjugation to one or more 8-phenyl-2-aminobenzazepine derivatives. The present invention also provides 8-phenyl-2-aminobenzazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions are suitable substrates for the formation of immunoconjugates via linkers or linking moieties. The present invention further provides methods for the treatment of cancer with the immunoconjugates described above.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 124,353, filed December 11, 2020, which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The said ASCII copy, created on December 6, 2021, is designated 17019 011WO1_SL.txt and is 55,248 bytes in size.

[0003] Technical Field The present invention relates generally to immunoconjugates comprising an anti-carcinoembryonic antigen (CEA) antibody conjugated to one or more 8-phenyl-2-aminobenzazepine molecules. [Background technology]

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

[0005] The present invention is generally directed to immunoconjugates comprising an anti-CEA antibody linked by conjugation to one or more 8-phenyl-2-aminobenzazepine derivatives. The present invention further relates to 8-phenyl-2-aminobenzazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions comprise an 8-phenyl-2-aminobenzazepine derivative intermediate composition in which the antibody is linked by a linker L to the formula: [ka] (In the formula, R 1 , R 2 , R 3, and R 4 One of the bonds is bonded to L, and the other is bonded to R 1-4 and X 1-4 The substituents are defined herein. is a suitable substrate for the formation of an immunoconjugate that can be covalently attached to an 8-Phe-2-aminobenzazepine (PhBz) moiety having the formula:

[0006] The present invention is further directed to the use of such immunoconjugates in the treatment of disease, particularly cancer.

[0007] One aspect of the invention is an immunoconjugate comprising an anti-CEA antibody covalently attached to a linker that is covalently attached to one or more 8-Phe-2-aminobenzazepine moieties.

[0008] Another aspect of the invention is the 8-phenyl-2-aminobenzazepine-linker compound.

[0009] Another aspect of the invention is a method of treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an anti-CEA antibody linked by conjugation to one or more 8-Phe-2-aminobenzazepine moieties.

[0010] Another aspect of the invention is the use of an immunoconjugate comprising an anti-CEA antibody linked by conjugation to one or more 8-Phe-2-aminobenzazepine moieties in the treatment of cancer.

[0011] Another aspect of the invention is a method for preparing an immunoconjugate by conjugation of one or more 8-Phe-2-aminobenzazepine moieties with an anti-CEA antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Reference will now be made in detail to certain specific 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 invention as defined by the appended 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 it is not intended that the present invention be limited in any way to the methods and materials described.

[0014] definition The term "immunoconjugate" or "immunostimulatory antibody conjugate" refers to an antibody construct covalently attached via a linker to an adjuvant moiety. The term "adjuvant" refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0015] 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 following administration of the immunoconjugate to a subject.

[0016] An "adjuvant" refers to a substance that can elicit an immune response in a subject exposed to the adjuvant.

[0017] The terms "Toll-like receptor" and "TLR" refer to any member of a family of highly conserved mammalian proteins that recognize pathogen-associated molecular patterns and act as 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.

[0018] The terms "Toll-like receptor 7" and "TLR7" refer to a nucleic acid or polypeptide that shares at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a published TLR7 sequence, e.g., GenBank Accession No. AAZ99026 for human TLR7 polypeptide, or GenBank Accession No. AAK62676 for mouse TLR7 polypeptide.

[0019] The terms "Toll-like receptor 8" and "TLR8" refer to a nucleic acid or polypeptide that shares at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a published TLR7 sequence, e.g., GenBank Accession No. AAZ95441 for human TLR8 polypeptide, or GenBank Accession No. AAK62677 for mouse TLR8 polypeptide.

[0020] 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 may indicate that an agonist stimulates or activates a TLR. Differences in signaling may be manifested, for example, as a change in expression of a target gene, a change in phosphorylation of a signaling component, a change in the subcellular localization of downstream elements such as nuclear factor kappa B (NF-kappa B), a change in the association of certain components (such as IL-1 receptor-associated kinase (IRAK)) with other proteins or subcellular structures, or a change in the biochemical activity of a component, such as a kinase (such as mitogen-activated protein kinase (MAPK)).

[0021] "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 (about 25 kDa) and one "heavy" chain (about 50-70 kDa) connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains are divided into different categories according to 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), and the N-terminus of each chain defines a variable region of about 100-110 or more amino acids, called the paratope, which is primarily responsible for antigen recognition, i.e., the antigen-binding domain. The light chains are classified as either κ or λ. The heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of about 150 kDa composed of four peptide chains. IgG antibodies contain two identical class γ heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus comprising a tetrameric quaternary structure. The two heavy chains are linked to each other and to the respective light chains by disulfide bonds. The resulting tetramer has two identical halves, which 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 in the specificity and affinity of binding to cancer cells.

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

[0023] 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 constitute 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 monovalent fragment consisting of a single group of antibodies. 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 linked by a synthetic linker that allows the synthesis of the two domains as a single polypeptide chain (i.e., V L and V H ) and single-chain Fv (scFv).

[0024] An antibody or antibody fragment may be part of a larger construct, e.g., a conjugate or fusion construct of the antibody fragment to an additional region. 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 a chimeric T cell receptor, e.g., by fusing to a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., hinge region)) and any intercellular signaling domains.

[0025] "Epitope" means 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.

[0026] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three major classes of Fc receptors: (1) 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).

[0027] 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 nucleotides or amino acid residues that are the same (i.e., identical) between the optimally aligned sequence of interest and the reference sequence, divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). 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 alignment of nucleic acid and amino acid sequences), 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 in, for example, 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, as follows: 100 x [(identical positions) / min(TG A ,T.G. B)], and 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 an alignment that minimizes (see, e.g., Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0028] 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 comprises three complementarity determining regions (CDR1, CDR2, and CDR3) connected by framework regions. The binding agent can be any of the various 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.

[0029] "Biosimilar" refers to an approved antibody construct that has similar activity characteristics as a CEA-targeting antibody, such as, for example, labetuzumab (CEA-CIDE™, MN-14, hMN14, Immunomedics) CAS Registry Number 219649-07-7.

[0030] "Biobetter" refers to an approved antibody construct that is an improvement of an already approved antibody construct, such as labetuzumab. A biobetter can have one or more modifications (e.g., an altered glycan profile or a unique epitope) relative to an already approved antibody construct. A biobetter is a recombinant protein drug that is similar to an existing biopharmaceutical, but is not identical and is superior to the original drug. A biobetter is not an entirely new drug, nor is it a generic version of a drug. Both biosimilars and biobetters are variations of biological drugs, the former being a very similar copy of the original drug, while the latter has improvements in efficacy, safety, tolerability, or dosing regimen.

[0031] "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 having the same molecular formula and intramolecular bonds, but differing 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.

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

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

[0034] 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 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 (i.e., carbon bound to hydrogen, carboxyl group, amino group) as a naturally occurring amino acid, but has a modified side 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 different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid.

[0035] "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.

[0036] "Linking moiety" 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 link 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.

[0037] "Divalent" refers to a chemical moiety that contains two points of attachment for linking two functional groups, and a polyvalent linking moiety can have additional points of attachment for linking further 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 that has 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.

[0038] Wavy line [ka] represents a point of attachment for the designated chemical moiety. When two wavy lines are present at a designated chemical moiety, it will be understood that the chemical moiety can be used bilaterally, i.e., read from left to right or right to left.

[0039] "Alkyl" refers to a straight-chain (linear) or branched-chain saturated aliphatic radical having the number of carbon atoms indicated. An alkyl can contain any number of carbons, for example, 1 to 12. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-3-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-4-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-5-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-6-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-7-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-8-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-1 ...2-propyl (t-Bu, t-butyl, -CH(CH3)CH Butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH 2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH Examples of "substituted alkyl" groups include, but are not limited to, 2,3-dimethyl-2-butyl (-C(CH)CH(CH), 3,3-dimethyl-2-butyl (-CH(CH)C(CH), 1-heptyl, 1-octyl, and the like. An alkyl group can be substituted or unsubstituted. A "substituted alkyl" group can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0040] The term "alkyldiyl" refers to a divalent alkyl radical. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and the like. Alkyldiyl groups are sometimes referred to as "alkylene" groups.

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

[0042] The terms "alkenylene" or "alkenyldiyl" refer to a straight-chain or branched divalent hydrocarbon radical. Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CHCH=CH-), and the like.

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

[0044] The terms "alkynylene" or "alkynyldiyl" refer to a divalent alkynyl radical.

[0045] "Carbocycle", "carbocyclyl", "carbocyclic" and "cycloalkyl" refer to saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic ring assemblies 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.

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

[0047] "Aryl" refers to an aromatic ring system having 6 to 20 carbon atoms (C6-C8) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. 20 ) refers to a monovalent aromatic hydrocarbon radical. 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.

[0048] The term "arylene" or "aryldiyl" refers to an aromatic ring system having 6 to 20 carbon atoms (C6-C7) derived by removing two hydrogen atoms from two carbon atoms of a parent aromatic ring system. 20 ) divalent aromatic hydrocarbon radical. Some aryldiyl groups are represented in the exemplary structures as "Ar". Aryldiyl includes bicyclic radicals that contain a saturated ring, a partially unsaturated ring, or an aromatic ring fused to an aromatic carbon ring. Common aryldiyl groups include, but are not limited to, benzene (phenyldiyl), substituted benzene, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Aryldiyl groups are also referred to as "arylenes", and are optionally substituted with one or more substituents as described herein.

[0049] The terms "heterocycle", "heterocyclyl" and "heterocycle" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms, in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally and independently substituted with one or more of the substituents described below. A heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S), or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), such as: bicyclo[4,5], [5,5], [5,6], or [6,6] systems. Heterocyclic rings are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially volumes 13, 14, 16, 19, and 28, and J. Am. Chem. Soc. (1960) 82:5566. "Heterocyclyl" also includes radicals in which the heterocyclic radical is fused to a saturated, partially unsaturated, or aromatic carbocyclic or heterocyclic ring.Examples of heterocycles include morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, homopiperazinyl, and azetidinyl. , oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl, and N-pyridyl urea. Spiroheterocyclyl moieties are also included within the scope of this definition. Examples of spiroheterocyclyl moieties include azaspiro[2.5]octanyl and azaspiro[2.4]heptanyl. Examples of heterocyclic groups in which two ring atoms are substituted with oxo (=O) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted, independently, with one or more substituents described herein.

[0050] The term "heterocyclyldiyl" refers to a divalent, saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic radical of three to about twenty ring atoms, in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are C, and one or more ring atoms are optionally and independently substituted with one or more substituents, as described. Examples of 5- and 6-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl.

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

[0052] The term "heteroaryldiyl" refers to a divalent aromatic radical of 5, 6, or 7 rings, including fused ring systems of 5 to 20 atoms, at least one of which is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of 5- and 6-membered heteroaryldiyls include pyridyldiyl, imidazolyldiyl, pyrimidyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyldiyl.

[0053] Heterocycle or heteroaryl groups may be carbon (carbon bond) or nitrogen (nitrogen bond) bonded, where possible. By way of example and not limitation, carbon bonded heterocycle or heteroaryl may be bonded at the 2-, 3-, 4-, 5-, or 6-position of pyridine, the 3-, 4-, 5-, or 6-position of pyridazine, the 2-, 4-, 5-, or 6-position of pyrimidine, the 2-, 3-, 5-, or 6-position of pyrazine, the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, the 2-, 4-, or 5-position of oxazole, imidazole or thiazole, the 3-, 4-, or 5-position of isoxazole, pyrazole or isothiazole, the 2- or 3-position of aziridine, the 2-, 3-, or 4-position of azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline.

[0054] By way of example, and without limitation, a nitrogen-linked heterocycle or heteroaryl is linked at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline.

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

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

[0057] 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).

[0058] The terms "treat," "treatment," and "treating" refer to an indication of success in treating or ameliorating an injury, medical condition, state (e.g., cancer), or symptom (e.g., cognitive impairment), including any objective or subjective parameter, such as relief, remission, alleviation of symptoms, or making the symptom, injury, medical condition, or condition more tolerable to the patient, slowing the rate of progression of the symptom, reducing the frequency or duration of the symptom or condition, or, in some circumstances, preventing the onset of the symptom. The treatment or amelioration of the symptom can be based on any objective or subjective parameter, including, for example, the results of a physical exam.

[0059] The terms "cancer," "neoplasm," and "tumor" are used herein to refer to cells that exhibit autonomous, unregulated growth, such that the cells exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Cells that are 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 the cancer burden refers to reducing the number of cancer cells or cancer cell volume in a subject. The term "cancer cell" as used herein refers to any cell that is a cancer cell (e.g., derived from any cancer that can treat an individual, e.g., isolated from an individual with cancer) or derived from a cancer cell, e.g., a clone of a cancer cell. For example, the 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, and the like. In some embodiments, the term can also refer to a portion of a cancer cell, such as an intracellular portion, a cell membrane portion, or a cell lysate of a cancer cell. Many types of cancer are known to those of skill in the art and include solid tumors, such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, as well as circulating cancers, such as leukemias.

[0060] As used herein, the term "cancer" includes any form of cancer, including, 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), carcinomas, soft tissue tumors, sarcomas, teratomas, melanomas, leukemias, lymphomas, and brain cancers, e.g., minimal residual disease, including both primary and metastatic tumors.

[0061] 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, neoplasms, premalignancies, malignant tumors, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0062] As used herein, the phrases "cancer recurrence" and "tumor recurrence" and their grammatical variations refer to 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, and 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.

[0063] 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 several steps 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.

[0064] 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 the skilled artisan 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 nd Edition, (Pharmaceutical Press, London, 2012). In the case of cancer, a therapeutically effective amount of the 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 of the symptoms associated with cancer. To the extent that 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 may be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0065] "Recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., human) for whom diagnosis, treatment, or therapy is desired. For purposes of treatment, "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.

[0066] The phrase "synergistic adjuvant" or "synergistic combination" in the context of the present invention includes a combination of two immune modulators, such as receptor agonists, cytokines, and adjuvant polypeptides, which in combination induce a synergistic effect on immunity compared to either administered alone. In particular, the immunoconjugates disclosed herein include a synergistic combination of the claimed adjuvants and antibody constructs. These synergistic combinations upon administration induce a greater effect on immunity compared to, for example, when the antibody construct or the adjuvant is administered in the absence of the other moiety. Furthermore, a reduced amount of the immunoconjugate (measured by the total number of antibody constructs or the total number of adjuvants administered as part of the immunoconjugate) can be administered compared to when either the antibody construct or the adjuvant is administered alone.

[0067] 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.

[0068] The terms "about" and "approximately" used herein to modify a numerical value indicate a range of approximations surrounding the numerical value. Thus, when "X" is a value, "about X" or "approximately X" indicates a value between 0.9X and 1.1X, such as 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, such as "0.98X."

[0069] CEA antibody The immunoconjugates of the present invention include antibodies that target, bind to, or recognize carcinoembryonic antigen (CEA, CD66e, CEACAM5). Included within the scope of the present embodiments are functional variants of the antibody constructs or antigen-binding domains described herein. The term "functional variant" as used herein refers to an antibody construct having an antigen-binding domain that has substantial or significant sequence identity or similarity with a parent antibody construct or antigen-binding domain, where the functional variant retains the biological activity of the antibody construct or antigen-binding domain of which it is a variant. Functional variants encompass 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 CEA to a similar, comparable, or higher extent than the parent antibody construct or antigen-binding domain.

[0070] 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.

[0071] A functional variant may, for example, comprise an 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 may comprise an 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 may enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen-binding domain.

[0072] 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). , GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328, and A330. Additional Fc region modifications for modulating Fc receptor binding are described, for example, in U.S. Patent Application Publication No. 2016 / 0145350 and U.S. Patents Nos. 7,416,726 and 5,624,821, which are incorporated by reference in their entireties.

[0073] Antibodies comprising the immunoconjugates of the invention include glycan variants, such as afucosylation, in 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.

[0074] The amino acid substitutions in the antibody construct or antigen-binding domain of the invention are preferably conservative amino acid substitutions, which are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be a substitution of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), a substitution of an amino acid having a nonpolar side chain with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a substitution of a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), a substitution 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.), a substitution of an amino acid having a beta-branched side chain with an amino acid having another beta-branched side chain (e.g., Ile, Thr, and Val), a substitution of an amino acid having an aromatic side chain with an amino acid having another aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0075] An antibody construct or antigen-binding domain may 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.

[0076] 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.

[0077] In some embodiments, an antibody in an immunoconjugate (e.g., an antibody conjugated to at least two adjuvant moieties) comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region that modulate (e.g., increase or decrease) binding to one or more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), and / or FcγRIIIB (CD16b)) compared to a native antibody that does not have a mutation in the Fc region. In some embodiments, an antibody in an immunoconjugate comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region that decrease binding of the Fc region of the antibody to FcγRIIB. In some embodiments, the antibody in the immunoconjugate comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that reduce binding of the antibody to FcγRIIB while maintaining the same binding or increasing binding to FcγRI (CD64), FcγRIIA (CD32A), and / or FcRγIIIA (CD16a) compared to a native antibody that does not have a mutation in the Fc region. In some embodiments, the antibody in the immunoconjugate comprises one or more modifications in the Fc region that increase binding of the antibody's Fc region to FcγRIIB.

[0078] In some embodiments, the modulated binding is provided by mutations in the Fc region of the antibody compared to the native Fc region of the antibody. The mutations can be in the CH2 domain, the CH3 domain, or a combination thereof. A "native Fc region" is synonymous with a "wild-type Fc region" and comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature or that is identical to the amino acid sequence of an Fc region found in a native antibody (e.g., cetuximab). Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fcs include the various allotypes of Fcs (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0079] In some embodiments, mutations in the Fc region that result in modulation of 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 ... 2E), GASDALIE(G236A / S239D / A330L / I332E), V9(G237D / P238D / P271G / A330R), and V11(G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E233, G237, P238, H268, P271, L328, and A330. Further Fc region modifications for modulating Fc receptor binding are described, for example, in US2016 / 0145350, as well as US7416726 and US5624821, which are hereby incorporated by reference in their entirety.

[0080] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to alter the glycosylation pattern of the Fc region as compared to the native, unmodified Fc region.

[0081] Human immunoglobulins are glycosylated at the Asn297 residue in the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of a core heptasaccharide, N-acetylglucosamine4mannose3 (GlcNAc4Man3). Removal of the heptasaccharide by endoglycosidases or PNGaseF is known to cause conformational changes in the antibody Fc region that can significantly reduce antibody binding affinity to activating FcγRs and reduce effector function. The core heptasaccharide is often modified with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially affect Fc binding to activating and inhibitory FcγRs. Furthermore, it has been shown that α2,6-sialylation enhances anti-inflammatory activity in vivo, while defucosylation leads to improved FcγRIIIa binding and a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Thus, specific glycosylation patterns can be used to control inflammatory effector functions.

[0082] In some embodiments, the modification to alter the glycosylation pattern is a mutation, e.g., a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of controlling immune responses using antibodies that modulate FcγR regulatory signaling are described, for example, in U.S. Patent No. 7,416,726 and U.S. Patent Publications 2007 / 0014795 and 2008 / 0286819, which are hereby incorporated by reference in their entireties.

[0083] In some embodiments, the antibody of the immunoconjugate is modified to contain an engineered Fab region with a non-native glycosylation pattern. For example, hybridomas can be engineered to secrete defucosylated mAbs, desialylated mAbs, or deglycosylated Fc with specific mutations that can increase FcRγIIIa binding and effector function. In some embodiments, the antibody of the immunoconjugate is engineered to be defucosylated.

[0084] In some embodiments, the entire Fc region of the antibody in the immunoconjugate is replaced with a different Fc region, so that the Fab region of the antibody is bound to a non-natural Fc region. For example, the Fab region of cetuximab, which normally contains an IgG1 Fc region, can be bound to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which normally contains an IgG4 Fc region, can be bound to IgG1, IgG2, IgG3, IgA1, or IgG2. In some embodiments, the Fc-modified antibody with a non-natural Fc domain also contains one or more amino acid modifications, such as the S228P mutation in IgG4 Fc, that modulate the stability of the Fc domain as described. In some embodiments, the Fc-modified antibody with a non-natural Fc domain also contains one or more amino acid modifications, as described herein, that modulate Fc binding to FcR.

[0085] In some embodiments, the modifications that modulate binding of the Fc region to an FcR do not alter binding of the Fab region of the antibody to its antigen when compared to the native, unmodified antibody, while in other embodiments, the modifications that modulate binding of the Fc region to an FcR also increase binding of the Fab region of the antibody to its antigen when compared to the native, unmodified antibody.

[0086] In an exemplary embodiment, an immunoconjugate of the invention comprises an antibody construct that comprises an antigen-binding domain that specifically recognizes and binds CEA.

[0087] Increased expression of carcinoembryonic antigen (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of tumorigenesis, particularly tumor cell adhesion, metastasis, blocking cellular immune mechanisms, and having anti-apoptotic effects. CEA is a cell surface antigen and 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 analogue (labetuzumab govitecan, IMMU-130) targets CEA and is being investigated in patients with recurrent or refractory metastatic colorectal cancer (Sharkey, R. et al (2018), Molecula rCancer Therapeutics 17(1):196-203; Dotan, E. et al (2017), Journal of Clinical Oncology 35(9):3338-3346). 131 Labetuzumab conjugated to I is being evaluated in clinical trials for the treatment of colon cancer and other solid malignancies (Sharkey, R. et al (1995), Cancer Research (Suppl.) 55(23):5935s-5945s; Liersch, T. et al (2005), Journal of Clinical Oncology 23(27):6763-6770; Sahlmann, C.-O. et al (2017), Cancer 123(4):638-649).

[0088] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMN-14 / labetuzumab (SEQ ID NO: 1) disclosed in US6676924, which is incorporated herein by reference for this purpose. DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK SEQ ID NO: 1

[0089] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) sequence or light chain framework (LFR) sequence of hMN-14 / labetuzumab (US6676924) (SEQ ID NOs: 2-8). [Table 1]

[0090] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of hMN-14 / labetuzumab (SEQ ID NO: 9) disclosed in US6676924, which is incorporated herein by reference for this purpose. EVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPGKGLEWVAEIHPDSSTINYAPSLKDRFTISRDNSKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS SEQ ID NO: 9

[0091] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) sequence or heavy chain framework (HFR) sequence of hMN-14 / labetuzumab (US6676924) (SEQ ID NOs: 10-16). [Table 2]

[0092] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hPR1A3 (SEQ ID NO: 17) disclosed in US8642742, which is incorporated herein by reference for this purpose. DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIK SEQ ID NO: 17

[0093] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) sequence or light chain framework (LFR) sequence of hPR1A3 (US8642742) (SEQ ID NOs: 18-24). [Table 3]

[0094] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) sequence or heavy chain framework (HFR) sequence of hPR1A3 (US8642742) (SEQ ID NOs: 25-31). [Table 4]

[0095] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMFE-23 (SEQ ID NO: 32) as disclosed in US7232888, which is hereby incorporated by reference for this purpose. ENVLTQSPSSMSASVGDRVNIACSASSSVSYMHWFQQKPGKSPKLWIYSTSNLASGVPSRFSGSGSGTDYSLTISSMQPEDAATYYCQQRSSYPLTFGGGTKLEIK SEQ ID NO:32

[0096] In one 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 (US7232888) (SEQ ID NOs: 33-40). This embodiment includes two variants of LFR1, SEQ ID NO: 33 and SEQ ID NO: 34. [Table 5]

[0097] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of hMFE-23 (US7232888) (SEQ ID NO: 41). QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS SEQ ID NO: 41

[0098] In one 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 (US7232888) (SEQ ID NOs: 42-49). This embodiment includes two variants of HFR1, SEQ ID NO: 42 and SEQ ID NO: 43. [Table 6]

[0099] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VL kappa) of SM3E (US7232888) (SEQ ID NO:50). ENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK SEQ ID NO:50

[0100] In one 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 (US7232888) (SEQ ID NOs: 51-56 and 38-39). This embodiment includes two variants of LFR1, SEQ ID NO: 51 and SEQ ID NO: 52. [Table 7]

[0101] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain of NP-4 / arcitumomab (SEQ ID NO:57). QTVLSQSPAILSASPGEKVTMTCRASSSVTYIHWYQQKPGSSPKSWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQHWSSKPPTFGGGTKLEIK SEQ ID NO:57

[0102] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) sequence or light chain framework (LFR) sequence of NP-4 / arcitumomab (SEQ ID NOs: 58-64). [Table 8]

[0103] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of NP-4 / arcitumomab (SEQ ID NO: 65). EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKALEWLGFIGNKANGYTTEYSASVKGRFTISRDKSQSILYLQMNTLRAEDSATYYCTRDRGLRFYFDYWGQGTTLTVSS SEQ ID NO: 65

[0104] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) sequence or heavy chain framework (HFR) sequence of NP-4 (SEQ ID NOs: 66 to 72). [Table 9]

[0105] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of M5A / hT84.66 (SEQ ID NO: 73) disclosed in US7776330, which is incorporated herein by reference for this purpose. DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIK SEQ ID NO: 73

[0106] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) sequence or light chain framework (LFR) sequence of M5A / hT84.66 (US7776330) (SEQ ID NOs: 74-80). [Table 10]

[0107] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of M5A / hT84.66 (US7776330) (SEQ ID NO: 81). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSS SEQ ID NO: 81

[0108] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) sequence or heavy chain framework (HFR) sequence of M5A / hT84.66 (US7776330) (SEQ ID NOs: 82-88). [Table 11]

[0109] In one embodiment of the invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hAb2-3 (SEQ ID NO: 89) disclosed in US9617345, which is incorporated herein by reference for this purpose. DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 89

[0110] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) sequence or light chain framework (LFR) sequence of hAb2-3 (US9617345) (SEQ ID NOs: 90-96). [Table 12]

[0111] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises a variable heavy chain (VH) of SEQ ID NO: 97 (US9617345). EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS SEQ ID NO: 97

[0112] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) sequence or heavy chain framework (HFR) sequence of hAb2-3 (SEQ ID NOs: 98-104). [Table 13]

[0113] In one 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 (SEQ ID NO: 105) disclosed in US9982063, which is incorporated herein by reference for this purpose. QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL SEQ ID NO: 105

[0114] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the light chain CDR (complementarity determining region) sequence or light chain framework (LFR) sequence of A240VL-B9VH / AMG-211 (US9982063) (SEQ ID NOs: 106-112). [Table 14]

[0115] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of B9VH (US9982063) (SEQ ID NO: 113). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS SEQ ID NO: 113

[0116] In one 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 (US9982063) of SEQ ID NOs: 114 to 121. The embodiment includes two variants of CDR-H2, SEQ ID NO: 117 and SEQ ID NO: 118. [Table 15]

[0117] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of E12VH (US9982063) (SEQ ID NO: 122). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFILNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS SEQ ID NO: 122

[0118] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises a heavy chain CDR (complementarity determining region) sequence or a heavy chain framework (HFR) sequence of SEQ ID NOs: 123-129 (US9982063). [Table 16]

[0119] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of PR1A3 VH (US8642742) (SEQ ID NO: 130). QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSS SEQ ID NO: 130

[0120] 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 a variable (V) domain of an antibody heavy chain linked to a variable (V) domain of an antibody light chain via a synthetic peptide, and can be generated 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 an antigen-binding domain of an anti-CEA antibody, each variable region comprising CDR1, CDR2, and CDR3.

[0121] 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.

[0122] In some embodiments, the Fc region is modified by including a TGFβ1 receptor or a fragment thereof capable of binding transforming growth factor beta 1 (TGFβ1). For example, the receptor may be a 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 (incorporated herein). An "Fc linker" can be used to attach the IgG to the TGFβRII extracellular domain. The Fc linker can be a short and flexible peptide that allows for proper three-dimensional folding of the molecule while maintaining binding specificity to the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to the Fc of the antibody construct (with or without an Fc linker). In some embodiments, the C-terminus of the antibody construct heavy chain is fused to the TGFβ receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the antibody construct heavy chain is mutated to an alanine.

[0123] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0124] In some embodiments, the antibody in the immunoconjugate is a cysteine ​​engineered antibody that provides for site-specific conjugation of adjuvants, labels, or drug moieties to the antibody through cysteine ​​substitution at sites where the engineered cysteines are available for conjugation, but does not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; US2012 / 0121615; WO2009 / 052249). A "cysteine ​​engineered antibody" or "cysteine ​​engineered antibody variant" is an antibody in which one or more residues of an antibody have been replaced with a cysteine ​​residue. Cysteine ​​engineered antibodies can be conjugated to 8-Phe-2-aminobenzazepine adjuvant moieties as 8-phenyl-2-aminobenzazepine-linker compounds with uniform stoichiometry (e.g., for antibodies with a single engineered cysteine ​​site, up to two 8-Phe-2-aminobenzazepine moieties per antibody).

[0125] In some embodiments, the cysteine ​​engineered antibody used to prepare the immunoconjugates of Table 3 has a cysteine ​​residue introduced at the 149-lysine site of the light chain (LC K149C). In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced at the 118-alanine site (EU numbering) of the heavy chain (HC A118C). This site is numbered 121 in the sequential numbering system and 114 in the Kabat numbering system. In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced in the light chain at G64C or R142C according to the Kabat numbering, or in the heavy chain at D101C, V184C or T205C according to the Kabat numbering system.

[0126] 8-Phenyl-2-aminobenzazepine adjuvant compounds The immunoconjugates of the present invention include an 8-Phe-2-aminobenzazepine adjuvant moiety. The adjuvant moieties described herein are compounds that induce an immune response (i.e., immunostimulants). In general, 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 a variety of pathogen-associated molecular patterns from bacteria, viruses, and fungi and serve as a first line of defense against invading pathogens. TLRs induce overlapping but 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) through 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 recruitment of TNF receptor-associated factor 6 (TRAF6), which in turn 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 control of TLR signaling include TIR domain-containing adaptor-inducing interferon-β (TRIF)-dependent induction of TNF receptor-associated factor 6 (TRAF6) and activation of MyD88-independent pathways via TRIF and TRAF3, which leads to 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.

[0127] Generally, the adjuvant moieties described herein are TLR7 and / or TLR8 agonists. Both TLR7 and TLR8 are expressed on monocytes and dendritic cells. In humans, TLR7 is also expressed on plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is expressed primarily on cells of bone marrow origin, namely 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 TLR8 agonists can result in the production of high levels of IL-12, IFN-γ, IL-1, TNF-α, IL-6, and other inflammatory cytokines. Similarly, stimulation of TLR7 expressing cells, such as pDCs, with TLR7 agonists can result in the production of high levels of IFN-α and other inflammatory 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.

[0128] Exemplary 8-phenyl-2-aminobenzazepine compounds (PhBz) of the invention are shown in Table 1. Each compound was synthesized, purified, characterized by mass spectrometry, and found to have the mass indicated. Further experimental procedures are described in the Examples. Activity against human embryonic kidney (HEK) 293 NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. The 8-phenyl-2-aminobenzazepine compounds of Table 1 demonstrate surprising and unexpected properties of TLR8 agonist selectivity that may be predictive of therapeutic activity useful for treating cancer and other disorders. [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6]

[0129] 8-Phenyl-2-aminobenzazepine-Linker Compounds The immunoconjugates of the present invention are prepared by conjugating an anti-CEA antibody with an 8-phenyl-2-aminobenzazepine-linker compound, PhBzL. The 8-phenyl-2-aminobenzazepine-linker compound comprises an 8-Phe-2-aminobenzazepine (PhBz) moiety covalently attached to a linker unit. The linker unit comprises functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. The linker unit comprises a reactive functional group that reacts, i.e., conjugates, with a reactive functional group of an antibody. For example, a nucleophilic group, such as a lysine side chain amino of an antibody, reacts with an electrophilic reactive functional group of an Hx-linker compound to form an immunoconjugate. Also, for example, a cysteine ​​thiol of an antibody reacts with a maleimide or bromoacetamide group of an Hx-linker compound to form an immunoconjugate.

[0130] Suitable electrophilic reactive functional groups for Hx-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.

[0131] The present invention provides solutions to limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers are unstable in the bloodstream, which can result in unacceptable amounts of adjuvant / drug released prior to internalization in target cells (Khot, A. et al (2015) Bioanalysis 7 (13): 1633-1648). Other linkers may provide stability in the bloodstream, but the efficacy of intracellular release may be adversely affected. Linkers that provide the 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.

[0132] An exemplary embodiment is a compound of formula II: [ka] (In the formula, R 1 , R 2 , R 3 , and R 4 are independently 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 and optionally selected from the group consisting of: -(C1-C 12Alkyldiyl)-N(R 5 )- * , -(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C1-C 12 Alkyldiyl)-OR 5 , -(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)-(C2-C 20 Heterocyclyldiyl)- * , -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R5 )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)-C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 - * , -(C2-C9 heterocyclyl)-NR 5 -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -(C2-C9 heterocyclyl)-(C6-C 20 Aryldiyl)- * , -(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 )- * , -(C1-C 20 Heteroaryl)-N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -C(=O)- * , -C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -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 )CO2R5 , -NR 5 C(=NR 5a )N(R 5 )2, -NR 5 C(=NR 5a )N(R 5 )- * , -NR 5 C(=NR 5a )R 5 , -N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -N(R 5 )-(C2-C5 heteroaryl), -N(R 5 )-S(=O)2-(C1-C 12 alkyl), -O-(C1-C 12 alkyl), -O-(C1-C 12 Alkyldiyl)-N(R 5 )2, -O-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -OC(=O)N(R 5 )2, -OC(=O)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-C20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-OH substituted with one or more groups selected from or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring, X 1 , X 2 , X 3 , and X 4 are independently a bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, and S(O)2N(R 5 ) selected from the group consisting of R 5 are independently H, C6-C 20 Aryl, C3-C 12 Carbocyclyl, C6-C 20 Aryldiyl, C1-C 12 Alkyl, and C1-C 12 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 Heteroaryl; However, the asterisk * indicates the binding site of L, and R 1 , R 2 , R 3 and R 4 One of the is bonded to L, L is QC(=O)-PEG-, QC(=O)-PEG-C(=O)N(R 6 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, QC(=O)-PEG-O-, QC(=O)-PEG-OC(=O)-, QC(=O)-PEG-C(=O)-, QC(=O)-PEG-C(=O)-PEP-, QC(=O)-PEG-N(R 6 )-, QC(=O)-PEG-N(R 6 )-C(=O)-, QC(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-, QC(=O)-PEG-N + (R 6 )2-PEG-C(=O)-PEP-, QC(=O)-PEG-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-, QC(=O)-PEG-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, 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 6 )-(C1-C 12 Alkyldiyl)-, QC(=O)-(C1-C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-N(R 5 )-C(=O), QC(=O)-(C1-C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-N(R 6)C(=O)-(C2-C5 monoheterocyclyldiyl)-, Q-(CH2) m -C(=O)N(R 6 )-PEG-, Q-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, Q-(CH2) m -C(=O)N(R 6 )-PEG-O-, Q-(CH2) m -C(=O)N(R 6 )-PEG-OC(=O)-, Q-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)-, Q-(CH2) m -C(=O)N(R 6 )-PEG-N(R 5 )-, Q-(CH2) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-, Q-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-, Q-(CH2) m -C(=O)N(R 6 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-, Q-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-, Q-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-, and Q-(CH2) m-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)- is a linker selected from the group consisting of R 6 are independently H or C1-C6 alkyl; 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; Gluc has the formula: [ka] having PEP has the formula: [ka] having wherein AA is independently selected from a natural or unnatural amino acid side chain, or one or more AA and adjacent nitrogen atoms form a five-membered proline amino acid ring; The wavy lines indicate the attachment points. Cyc optionally comprises F, Cl, NO2, -OH, -OCH3, and: [ka] C6-C substituted with one or more groups selected from glucuronic acid having the structure 20 Aryldiyl and C1-C 20 heteroaryldiyl; R 7 is -CH(R 8 )O-, -CH2-, -CH2N(R 8 )-, and -CH(R 8 )OC(═O)—, R 8 is H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and -C(=O)N(R 9 )2, and R 9 are independently H, C1-C 12Alkyl, and -(CH2CH2O) n -(CH2) m -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R 9 the groups taken together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1, Q is independently F, Cl, NO2, and SO3 - N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, each of which is substituted with one or more groups selected from provided that alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH3, -C H2CH2CH3, -CH(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)2C N, -CH2CN, -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 , -NHS(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, OCH2F, OCHF2, OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H). The compound includes the 8-phenyl-2-aminobenzazepine-linker compound of the formula:

[0133] Exemplary embodiments of the 8-phenyl-2-aminobenzazepine-linker compound of formula II include those in which Q is [ka] The linker compound is selected from the above.

[0134] Exemplary embodiments of the 8-phenyl-2-aminobenzazepine-linker compounds of formula II include the above linker compounds where Q is phenoxy substituted with one or more F.

[0135] Exemplary embodiments of the 8-phenyl-2-aminobenzazepine-linker compounds of formula II include those linker compounds where Q is 2,3,5,6-tetrafluorophenoxy.

[0136] Exemplary embodiments of 8-phenyl-2-aminobenzazepine-linker (PhBzL) compounds are selected from Table 2. Each compound was synthesized, purified, characterized by mass spectrometry, and found to have the mass indicated. Further experimental procedures are described in the Examples. The 8-phenyl-2-aminobenzazepine-linker compounds in Table 2 exhibit surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. The 8-phenyl-2-aminobenzazepine-linker intermediate compound of formula II in Table 2 is used for conjugation with antibodies by the method of Example 201 to form the immunoconjugates of Tables 3a and 3b. [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6] [Table 18-7] [Table 18-8] [Table 18-9] [Table 18-10] [Table 18-11] [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4]

[0137] CEA immunoconjugates Immunostimulatory antibody conjugates, or immunoconjugates, deliver TLR7 / 8 agonists into tumors to activate tumor-infiltrating myeloid cells and initiate broad innate and adaptive anti-tumor immune responses (Ackerman, et al., (2021) Nature Cancer 2:18-33).

[0138] CEA (CEACAM5) is a well-validated cell surface antigen that is highly expressed in a variety of solid tumors. The favorable properties of CEA, including robust cell surface expression, low internalization rate, and limited expression in normal tissues, suggest that this antigen may be a suitable target for immunoconjugates in a multifunctional approach to treat CEA-expressing cancers.

[0139] An exemplary embodiment of the immunoconjugate comprises one or more 8-Phe-2-aminobenzazepine (PhBz) moieties covalently attached by a linker and having formula I: [ka] or a pharma- ceutically acceptable salt thereof, Ab is an antibody construct having an antigen-binding domain that binds to CEA; p is an integer from 1 to 8; PhBz has the formula: [ka] (R 1 , R 2 , R 3 , and R 4 are independently 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 and optionally selected from the group consisting of: -(C1-C 12 Alkyldiyl)-N(R 5 )- * , -(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C1-C 12 Alkyldiyl)-OR5 , -(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)-(C2-C 20 Heterocyclyldiyl)- * , -(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)-C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 - * , -(C2-C9 heterocyclyl)-NR 5 -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -(C2-C9 heterocyclyl)-(C6-C 20 Aryldiyl)- * , -(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 )- * , -(C1-C 20 Heteroaryl)-N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -C(=O)- * , -C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -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 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -N(R 5 )-(C2-C5 heteroaryl), -N(R 5 )-S(=O)2-(C1-C 12 alkyl), -O-(C1-C 12 alkyl), -O-(C1-C 12 Alkyldiyl)-N(R 5 )2, -O-(C1-C 12 Alkyldiyl)-N(R 5 )- * , -OC(=O)N(R 5 )2, -OC(=O)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 substituted with one or more groups selected from or R 2 and R 3together form a 5- or 6-membered heterocyclyl ring, X 1 , X 2 , X 3 , and X 4 are independently a bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, and S(O)2N(R 5 ) selected from the group consisting of R 5 are independently H, C6-C 20 Aryl, C3-C 12 Carbocyclyl, C6-C 20 Aryldiyl, C1-C 12 Alkyl, and C1-C 12 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 Heteroaryl; However, the above asterisk * indicates the binding site of L, and R 1 , R 2 , R 3 and R 4 is bonded to L) wherein the 8-phenyl-2-aminobenzazepine moiety has the formula: L is -C(=O)-PEG-, -C(=O)-PEG-C(=O)N(R 6 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, -C(=O)-PEG-O-, -C(=O)-PEG-OC(=O)-, -C(=O)-PEG-C(=O)-, -C(=O)-PEG-C(=O)-PEP-, -C(=O)-PEG-N(R 6 )-, -C(=O)-PEG-N(R6 )-C(=O)-, -C(=O)-PEG-N(R 6 )-PEG-C(=O)-PEP-, -C(=O)-PEG-N + (R 6 )2-PEG-C(=O)-PEP-, -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-, -C(=O)-PEG-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, -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 6 )-(C1-C 12 Alkyldiyl)-, -C(=O)-(C1-C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-N(R 5 )-C(=O), -C(=O)-(C1-C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C1-C 12 alkyldiyl)-C(=O)-Gluc-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-O-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-OC(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-N(R 5 )-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-N(R 5 )-C(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-SS-(C1-C 12 alkyldiyl)-OC(=O)-, -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)-, -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 6 )-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-, and -Succinimidyl-(CH2) m -C(=O)-PEP-N(R 6)-(C1-C 12 Alkyldiyl)N(R 6 )C(=O)-(C2-C5 monoheterocyclyldiyl)- is a linker selected from the group consisting of R 6 are independently H or C1-C6 alkyl; 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; Gluc has the formula: [ka] having PEP has the formula: [ka] having wherein AA is independently selected from a natural or unnatural amino acid side chain, or one or more AA and adjacent nitrogen atoms form a five-membered proline amino acid ring; The wavy lines indicate the attachment points. Cyc optionally comprises F, Cl, NO2, -OH, -OCH3, and [ka] C6-C substituted with one or more groups selected from glucuronic acid having the structure 20 Aryldiyl and C1-C 20 heteroaryldiyl; R 7 is -CH(R 8 )O-, -CH2-, -CH2N(R 8 )-, and -CH(R 8 )OC(═O)—, R 8 is H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and -C(=O)N(R 9 )2, and R 9 are independently H, C1-C 12 Alkyl, and -(CH2CH2O)n -(CH2) m -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R 9 the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1, provided that alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally, independently selected from F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C≡CH, -C≡CCH 3, -CH2CH2CH3, -CH(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, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -CO CH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH 3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH 2, -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) nSubstituted with one or more groups selected from H, OCH2F, OCHF2, OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0140] Exemplary embodiments of the immunoconjugate of Formula I include those wherein the antibody is selected from labetuzumab and arcitumomab, or a biosimilar or biobetter thereof.

[0141] An exemplary embodiment of the immunoconjugate of formula I is wherein the antibody construct comprises: a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; b) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; c) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 55, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; e) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 59, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 61, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 69, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 71; f) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 75, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; g) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 91, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 93, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 95, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 99, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 103; h) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120, or i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 126, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128 The immunoconjugate comprises the above immunoconjugate.

[0142] Exemplary embodiments of the immunoconjugate of Formula I include the above immunoconjugate, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105, and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130.

[0143] Exemplary embodiments of the immunoconjugate of Formula I include the above immunoconjugates, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105, and a variable heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130.

[0144] Exemplary embodiments of the immunoconjugate of Formula I include the above immunoconjugate, wherein the antibody construct comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain CDR (complementarity determining region) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 118.

[0145] Exemplary embodiments of the immunoconjugate of Formula I include the immunoconjugate described above, wherein the antibody construct comprises a variable light chain comprising the amino acid sequence of SEQ ID NO:105, and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:113.

[0146] An exemplary embodiment of the immunoconjugate of formula I is 2 is a bond and R 2 is C1-C8 alkyl.

[0147] An exemplary embodiment of the immunoconjugate of formula I is 2 and X 3 are bonds, and R 2 and R 3 are independently C1-C8 alkyl, -O-(C1-C 12Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 , -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 , -(C1-C 12 alkyl)-OC(O)N(R 5 )2, -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 alkyl)-OC(O)N(R 5 ) 2.

[0148] An exemplary embodiment of the immunoconjugate of formula I is 2 is C1-C8 alkyl, R 3 -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 The immunoconjugate includes the above immunoconjugate,

[0149] An exemplary embodiment of the immunoconjugate of formula I is 2 is -CH2CH2CH3, and R 3 is selected from -CH2CH2CH2NHCO2(t-Bu), -OCH2CH2NHCO2(cyclobutyl), and -CH2CH2CH2NHCO2(cyclobutyl).

[0150] An exemplary embodiment of the immunoconjugate of formula I is 2 and R 3 are each independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH, and -CH2CH2CH2OH.

[0151] An exemplary embodiment of the immunoconjugate of formula I is 2 and R 3 is each -CH2CH2CH3.

[0152] An exemplary embodiment of the immunoconjugate of formula I is 2 is -CH2CH2CH3, and R 3 is -OCH2CH3.

[0153] An exemplary embodiment of the immunoconjugate of formula I is 3 -R 3 but [ka] The immunoconjugate is selected from the group consisting of:

[0154] An exemplary embodiment of the immunoconjugate of formula I is 4 is a bond and R 4 is H.

[0155] An exemplary embodiment of the immunoconjugate of formula I is 1 is linked to L.

[0156] An exemplary embodiment of the immunoconjugate of formula I is 2 or R 3 is linked to L.

[0157] An exemplary embodiment of the immunoconjugate of formula I is 3 -R 3 -L, [ka] (wherein the wavy line indicates the point of attachment to N) The immunoconjugate is selected from the group consisting of:

[0158] An exemplary embodiment of the immunoconjugate of formula I is 4 C1-C 12 is alkyl.

[0159] An exemplary embodiment of the immunoconjugate of formula I is 4 Ga-(C1-C 12 Alkyldiyl)-N(R 5 )- * and an asterisk * indicates a binding site for L.

[0160] Exemplary embodiments of immunoconjugates of Formula I include the above immunoconjugates wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0161] Exemplary embodiments of the immunoconjugates of Formula I include those immunoconjugates described above where L is attached to a cysteine ​​thiol of the antibody.

[0162] Exemplary embodiments of the immunoconjugates of Formula I include those immunoconjugates described above wherein, for PEG, m is 1 or 2 and n is an integer from 2 to 10.

[0163] Exemplary embodiments of the immunoconjugates of Formula I include the above immunoconjugates wherein n is 10.

[0164] An exemplary embodiment of the immunoconjugate of formula I is one in which L comprises PEP, wherein PEP is a dipeptide and has the formula: [ka] The immunoconjugate comprises the above immunoconjugate having the formula:

[0165] Exemplary embodiments of the immunoconjugate of Formula I include the above immunoconjugates, wherein AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2, or AA1 and AA2 form a five-membered ring of proline amino acid.

[0166] Exemplary embodiments of the immunoconjugate of Formula I include the above immunoconjugates wherein AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0167] Exemplary embodiments of the immunoconjugates of Formula I include those above wherein AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H.

[0168] An exemplary embodiment of the immunoconjugate of Formula I is an immunoconjugate wherein the PEP has the formula: [ka] wherein AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids. The immunoconjugate comprises the above immunoconjugate having the formula:

[0169] An exemplary embodiment of the immunoconjugate of formula I is one in which L comprises PEP, where PEP is a tripeptide and has the formula: [ka] The immunoconjugate comprises the above immunoconjugate having the formula:

[0170] An exemplary embodiment of the immunoconjugate of Formula I is one in which L comprises PEP, wherein PEP is a tetrapeptide and has the formula: [ka] The immunoconjugate comprises the above immunoconjugate having the above structure.

[0171] An exemplary embodiment of the immunoconjugate of formula I is AA1 is selected from the group consisting of Abu, Ala, and Val; AA2 is selected from the group consisting of Nle(O-Bzl), Oic, and Pro; AA3 is selected from the group consisting of Ala and Met(O)2; The immunoconjugates described above include those wherein AA4 is selected from the group consisting of Oic, Arg(NO2), Bpa, and Nle(O-Bzl).

[0172] Exemplary embodiments of immunoconjugates of Formula I include those wherein L comprises PEP, wherein PEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala-Ala-Pro-Ala (SEQ ID NO: 131), Ala-Ala-Pro-Val (SEQ ID NO: 132), and Ala-Ala-Pro-Nva (SEQ ID NO: 133).

[0173] An exemplary embodiment of the immunoconjugate of formula I is where L comprises PEP, wherein PEP has the structure: [ka] The immunoconjugate includes the above immunoconjugate, wherein the immunoconjugate is selected from the group consisting of

[0174] An exemplary embodiment of the immunoconjugate of formula I is wherein L has the structure: [ka] (In the formula, the wavy line is R 5 (indicating a bond to The immunoconjugate includes the above immunoconjugate, wherein the immunoconjugate is selected from the group consisting of

[0175] An exemplary embodiment of the immunoconjugate of Formula I is represented by formula Ia: [ka] The immunoconjugate comprises the above immunoconjugate having the formula:

[0176] An exemplary embodiment of the immunoconjugate of formula Ia is 4 is a bond and R 4 is H.

[0177] An exemplary embodiment of the immunoconjugate of formula Ia is 2 and X 3 are bonds, and R 2 and R 3 are independently C1-C8 alkyl, -O-(C1-C 12 Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 , -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 , -(C1-C 12 alkyl)-OC(O)N(R 5 )2, -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 alkyl)-OC(O)N(R 5 ) 2.

[0178] Exemplary embodiments of the immunoconjugates of formula Ia are represented by formulas Ib through If: [ka] [ka] is selected from.

[0179] An exemplary embodiment of the immunoconjugate of formula Ia is 2 and X 3 are bonds, and R 2 and R 3 are independently C1-C8 alkyl, -O-(C1-C 12Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 , -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 The immunoconjugate includes the above immunoconjugate, wherein the immunoconjugate is selected from the group consisting of

[0180] An exemplary embodiment of the immunoconjugate of formula Ia is 2 and X 3 are bonds, and R 2 is C1-C8 alkyl, R 3 -O-(C1-C 12 Alkyl) and -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 The immunoconjugate includes the above immunoconjugate, wherein the immunoconjugate is selected from the group consisting of

[0181] The present invention includes all reasonable combinations of the embodiments of Formula I and permutations of features.

[0182] In certain embodiments, the immunoconjugate compounds of the present invention include those with immune stimulatory activity. The antibody-drug conjugates of the present invention selectively deliver effective doses of 8-phenyl-2-aminobenzazepine drugs to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated 8-phenyl-2-aminobenzazepine.

[0183] Drug loading is the number of PhBz moieties per antibody in the immunoconjugate of formula I, represented by p. Drug (PhBz) loading can range from 1 to 8 drug moieties (D) per antibody. The immunoconjugate of formula I includes a mixture or collection 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, for example, 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 ranges therebetween). Exemplary immunoconjugates 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 without the use of engineering to form intrachain disulfide bonds. In that case, 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.

[0184] For some anti-immunoconjugates, p may be limited by the number of binding sites on the antibody. For example, as in certain exemplary embodiments described herein, when the linkage is a cysteine ​​thiol, 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 the Hx-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.

[0185] 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 Hx-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.

[0186] It should be understood that if more than one nucleophilic group of an antibody reacts with a drug, the resulting product is a mixture of immunoconjugate compounds with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody may be calculated from the mixture by a double ELISA antibody assay that is specific for the antibody and specific for the drug. Individual immunoconjugate molecules may be identified in the mixture by mass spectrometry and separated by HPLC, 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., Proceedings of the AACR, Volume 45, March 2004, No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004.) In certain embodiments, homogenous immunoconjugates having a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.

[0187] Exemplary embodiments of immunoconjugates of Formula I are selected from the anti-CEA, PhBz immunoconjugates of Tables 3a and 3b. Evaluation of immunoconjugate activity in vivo was performed according to the method of Example 203. [Table 20] [Table 21]

[0188] Composition of Immunoconjugates The invention provides compositions, e.g., pharma- ceutical or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates described herein and, optionally, a carrier, e.g., a pharma- ceutical or pharmacologically acceptable carrier, for the immunoconjugates. The immunoconjugates may be identical or different in composition, i.e., the compositions may include immunoconjugates having the same number of adjuvants linked to the same position on the antibody construct, and / or the same number of Hx 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.

[0189] In an exemplary embodiment, the composition comprising the immunoconjugate compounds comprises a mixture of the immunoconjugate compounds, and the average drug (Hx) loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5.

[0190] The immunoconjugate compositions of the invention can have an average adjuvant-to-antibody construct ratio (DAR) of about 0.4 to about 10. One of skill in the art will recognize that the number of 8-phenyl-2-aminobenzazepine adjuvants conjugated to an antibody construct may vary among different 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 may be 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.

[0191] The average number of adjuvant moieties per antibody (DAR) in immunoconjugate preparations from conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the immunoconjugate in the composition in terms of p can also be measured. In some cases, separation, purification, and characterization of homogenous immunoconjugates with a particular value of p from immunoconjugates with other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0192] In some embodiments, the composition further comprises one or more pharma- ceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the present invention can be formulated for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. Alternatively, the immunoconjugates can be injected into a tumor. A composition for injection generally comprises a solution of the immunoconjugate dissolved in a pharma- ceutically acceptable carrier. Among the acceptable vehicles and solvents that can be used are isotonic solutions of water and one or more salts, such as sodium chloride, e.g., Ringer's solution. In addition, sterile, fixed oils can be conventionally used as a solvent or suspending medium. 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 be used in the preparation of injectables as well. These compositions are desirably sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharma- ceutically 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.

[0193] 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, weight, etc., according to the particular mode of administration selected and the needs of the patient. In certain embodiments, the concentration of immunoconjugate in an injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0194] Methods of Treating Cancer with Immunoconjugates The present invention provides a method of treating cancer. The method comprises administering to a subject in need thereof, e.g., a subject having cancer and in need of cancer treatment, a therapeutically effective amount of an immunoconjugate described herein (e.g., as a composition described herein). The method comprises administering a therapeutically effective amount of an immunoconjugate (IC) selected from Tables 3a and 3b.

[0195] 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.

[0196] In another aspect, the immunoconjugate is provided for use as a medicament.In certain embodiments, the present invention provides an immunoconjugate for use in a method for treating an individual, comprising administering to the individual an effective amount of the immunoconjugate.In one such embodiment, the method further comprises, for example, administering to the individual an effective amount of at least one additional therapeutic agent as described herein.

[0197] In a further aspect, the present invention provides the use of the 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 having cancer.In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, for example as described herein, to the individual.

[0198] Carcinomas are malignant tumors originating from epithelial tissue. Epithelial cells line the exterior of the body, line internal cavities, and line glandular tissue. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer that arises in glandular (secretory) cells, such as breast, pancreatic, lung, prostate, stomach, gastroesophageal junction, and colon cancer), adrenocortical carcinoma, hepatocellular carcinoma, renal cell carcinoma, ovarian carcinoma in situ, ductal carcinoma, breast carcinoma, basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, colon carcinoma, nasopharyngeal carcinoma, multilocular cystic renal cell carcinoma, oat cell carcinoma, large cell lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, and the like. 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 CEA (e.g., labetuzumab, or a biosimilar or biobetter thereof).

[0199] 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, hemangiomatoid fibrous histiocytoma, chondromyxoid fibroma, and sarcoma. 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, chondrotic 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, mesothelioma, neuroblastoma, osteocarcinoma These include, but are not limited to, osteoma, osteosarcoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, benign or malignant schwannoma, synovial sarcoma, Evans' tumor, nodular fasciitis, desmoid-type 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.

[0200] Sarcoma is a rare type of cancer that begins in cells of mesenchymal origin, such as bone or soft tissues of the body, including 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 bone, liposarcoma begins in fat, and rhabdomyosarcoma begins in 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 protuberans (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).

[0201] 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 derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm).Teratomas occur most frequently in the ovaries in women, the testes in men, and the coccyx in children.

[0202] Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma may begin in a mole (cutaneous melanoma) but can also begin in other pigmented tissues, such as the eye or intestine.

[0203] 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 called cutaneous neuroendocrine carcinoma. In some embodiments, the method of treating Merkel cell carcinoma includes administering an immunoconjugate containing an antibody construct (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof) that can bind to CEA. In some embodiments, the Merkel cell carcinoma has metastasized when administration is performed.

[0204] Leukemia is a cancer that begins in blood-forming tissues, such as bone marrow, and produces large numbers of abnormal blood cells that enter the bloodstream. For example, leukemia can occur in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for how quickly the disease develops and progresses (e.g., acute vs. chronic) and the type of white blood cells 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).

[0205] Lymphoma is a cancer that begins in the 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, lymphocytopenic CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0206] 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 those 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 lymphoma, Burkitt-like lymphoma (small noncleaved 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.

[0207] Brain cancer includes any cancer of brain tissue. Examples of brain cancer include, but are not limited to, glioma (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningioma, pituitary adenoma, and vestibular schwannoma, primitive neuroectodermal tumor (medulloblastoma).

[0208] 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 included 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.

[0209] The immunoconjugates of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing depends in part on whether administration is brief or chronic, and may be by any suitable route, for example, injections such as intravenous or subcutaneous injections. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various times, bolus administration, and pulse infusion.

[0210] 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 labetuzumab, its biosimilars, and its biobetters. For example, the method can include administering the immunoconjugate to provide a dose of about 100 ng / kg to about 50 mg / kg to the subject. 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. Doses of immunoconjugates may fall outside these ranges depending on the particular 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 a month to about five times a week. In some embodiments, the immunoconjugate is administered once a week.

[0211] 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.

[0212] Some embodiments of the present invention provide a method of treating cancer as described above, where the cancer is breast cancer. Breast cancer can originate from 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, medullary, mucinous, papillary, or cribriform carcinoma of the breast), lobular carcinoma in situ, invasive lobular carcinoma, inflammatory breast cancer, and triple negative (tests negative for estrogen receptor, progesterone receptor, and excess HER2 protein) breast cancer. In some embodiments, the method of treating breast cancer includes administering an immunoconjugate containing an antibody construct (e.g., labetuzumab, its biosimilar, or biobetter) capable of binding to CEA or tumors that overexpress CEA.

[0213] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8.

[0214] In some embodiments, a therapeutically effective amount of the immunoconjugate is administered to a patient in need of treatment for cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, or breast cancer. The Merkel cell carcinoma can be metastatic Merkel cell carcinoma. The breast cancer can be triple negative breast cancer. The esophageal cancer can be gastroesophageal junction adenocarcinoma. EXAMPLES

[0215] Example L-7 Synthesis of 1-(1-((3-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)phenyl)sulfonyl)azetidin-3-yl)-3-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2-azahexatriacontan-36-oic acid 2,3,5,6-tetrafluorophenyl, PhBzL-7 [ka] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[2-amino-4]-[ethoxy[(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylazetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-7b To a solution of 2-amino-8-[3-[3-(aminomethyl)azetidin-1-yl]sulfonylphenyl]-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, a solution of PhBzL-7a (270 mg, 431 umol, 1 equiv, TFA) in DMF (2 mL) was added Et3N (131 mg, 1.29 mmol, 180 uL, 3 equiv) and 3-[2-[2- [2-[2-[2-[2-[2-[2-[2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (2,3,5,6-tetrafluorophenyl), TFP-PEG10-CO2H (329 mg, 431 umol, 1 eq.) was added and then stirred at 0° C. for 1 h. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 35%-57%, 8 min) to give PhBzL-7b (270 mg, 243 umol, 56.45% yield) as a colorless oil.

[0216] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[2-amino-4-[ethoxy][(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylazetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-7c To a solution of PhBzL-7b (270 mg, 243 umol, 1 equiv) in CH3CN (2 mL) and HO (2 mL) was added TFA (222 mg, 1.95 mmol, 144 uL, 8 equiv), then stirred at 80° C. for 1 h. The mixture was concentrated, the residue was diluted with water (10 mL), and the pH of the aqueous phase was adjusted to about 5 by slowly adding aqueous NaHCO3, extracted with DCM:i-PrOH=3:1 (10 mL×3), and the organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 75×30 mm×3 um; mobile phase: [water (0.2% FA)-ACN]; B%: 20%-50%, 8 min) to give PhBzL-7c (50 mg, 47.52 umol, 19.51% yield) as a colorless oil. 1 H NMR (400 MHz, MeOD) δ8.16-8.09 (m, 2H), 7.94-7.79 (m, 2H), 7.75 (s, 1H), 7.73-7.62 (m, 2H), 7.41 (s, 1H), 3.97 (q, J = 7.0 Hz, 2H), 3.86 (t, J = 8.2 Hz, 2H), 3.79-3.69 (m, 4H), 3.66-3.49 (m, 40H), 3.32 (s, 2H), 3.18 (d, J = 6.4 Hz, 2H), 2.71-2.61 (m, 1H), 2.48 (t, J = 6.5 Hz, 2H), 2.30 (t, J = 6.0 Hz, 2H), 1.78 (sxt, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.2 Hz, 3H).

[0217] Preparation of PhBzL-7 To a solution of PhBzL-7c (50 mg, 72 umol, 1 equiv, TFA) in DCM (2 mL) and DMA (0.1 mL) was added 2,3,5,6-tetrafluorophenol (95 mg, 503 umol, 8 equiv) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI (140 mg, 700 umol, 10 equiv), then the mixture was stirred at 25° C. for 0.5 h. The reaction mixture was diluted with water and purified by HPLC to give PhBzL-7 (0.046 g, 0.038 mmol, 53%). LC / MS [M+H] 1200.50 (calculated); LC / MS [M+H] 1200.80 (observed).

[0218] Example L-11 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4]-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluorobenzenesulfonic acid, PhBzL-11 [ka] Preparation of methyl 4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]benzoate, PhBz-4 A mixture of 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide, PhBz-4a (0.2 g, 546 umol, 1 equiv.), (4-methoxycarbonylphenyl)boronic acid (98.3 mg, 546 umol, 1 equiv.), K2CO3 (151 mg, 1.09 mmol, 2 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, Pd(dppf)Cl2 (40.0 mg, 54.6 umol, 0.1 equiv.) in dioxane (50 mL) and HO (5 mL) was degassed and purged with N2 three times, then stirred under N2 atmosphere at 90° C. for 2 h. The mixture was diluted with HO (10 mL) and extracted with EtOAc (30 mL×3). The combined organic layer was washed with saturated brine (50 mL×2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-45%, 8 min) to give PhBz-4 (0.25 g, crude) as a white solid. 1 H NMR (MeOD, 400 MHz) δ 8.15 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.79-7.75 (m, 1H), 7.71-7.67 (m, 2H), 7.45 (s, 1H), 4.01-3.96 (m, 2H), 3.95 (s, 3H), 3.76 (t, J = 7.2 Hz, 2H), 3.43 (s, 2H), 1.80-1.75 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.6Hz, 3H). HPLC: 98.776% (220 nm), 99.813% (254 nm). LC / MS [M+H] 422.2 (calculated); LC / MS [M+H] 422.1 (observed).

[0219] Preparation of 4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoic acid, PhBzL-11a To a solution of PhBz-4 (0.2 g, 474 umol, 1 equiv) in MeOH (20 mL) and HO (10 mL) was added LiOH·HO (119 mg, 2.85 mmol, 6 equiv) and then stirred for 12 h at 20° C. The pH of this mixture was adjusted to approximately 7 with HCl (4 M) and then concentrated under reduced pressure to give PhBzL-11a (0.16 g, 393 umol, 82.75% yield) as a brown solid. 1 H NMR (DMSO-d6, 400 MHz) δ 8.06 (br d, J = 8.4 Hz, 2H), 7.83 (br d, J = 8.4 Hz, 2H), 7.78-7.63 (m, 3H), 7.32-7.24 (m, 1H), 4.02-3.77 (m, 2H), 3.63 (t, J = 7.2 Hz, 2H), 3.37 (s, 2H), 1.74-1.58 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 7.6 Hz, 3H).

[0220] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PhBz-11b To a solution of PhBz-11a (0.11 g, 270 umol, 1 equiv.) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (190 mg, 324 umol, 1.2 equiv.) in DMF (2 mL) was added azabenzotriazole tetramethyluronium hexafluorophosphate, HATU (113 mg, 297 umol, 1.1 equiv.) and DIEA (174 mg, 1.35 mmol, 235 uL, 5 equiv.), then stirred at 20° C. for 12 h. The reaction mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna C18 100×30 mm×5 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-40%, 10 min) to give PhBz-11b (0.09 g, 92.29 umol, 34.19% yield) as a white solid.

[0221] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-11c To a solution of PhBzL-11b (0.09 g, 92.3 umol, 1 equiv) in MeCN (1 mL) and HO (2 mL) was added HCl (12 M, 153 uL, 20 equiv) and then stirred for 1 h at 80° C. The reaction mixture was concentrated under reduced pressure to give PhBzL-11c (0.06 g, 65.3 umol, 70.74% yield) as a white solid.

[0222] Preparation of PhBzL-11 To a solution of PhBzL-11c (0.06 g, 65.3 umol, 1 equiv.) and (2,3,5,6-tetrafluoro-4-hydroxyphenyl)sulfonyloxysodium (87.5 mg, 326 umol, 5 equiv.) in DCM (2 mL) and DMA (0.2 mL), EDCI (62.6 mg, 326 umol, 5 equiv.) was added and then stirred at 20° C. for 1 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 10 min) to give PhBzL-11 (0.005 g, 4.36 umol, 6.68% yield) as a yellow oil. 1 H NMR (MeOH, 400 MHz) δ 7.98 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H), 7.69-7.65 (m, 1H), 7.45 (s, 1H), 3.98 (q, J = 7.2 Hz, 2H), 3.85 (t, J = 5.6 Hz, 2H), 3.76 (t, J = 7.2 Hz, 2H), 3.71-3.53 (m, 38H), 3.44 (s, 2H), 2.96 (t, J = 5.6 Hz, 2H), 1.88-1.71 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.6 Hz, 3H). HPLC: 95.471 % (220 nm), 94.988 % (254 nm). LC / MS [M+H] 1147.4 (calculated value); LC / MS [M+H] 1147.4 (measured value).

[0223] Example L-17 Synthesis of (3-(2-amino-8-(3-((3-(15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl))-3,13-dioxo-6,9-dioxa-2,12-diazapentadecyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)tert-butyl carbamate, PhBzL-17 [ka] To a solution of tert-butyl (3-(2-amino-8-(3-((3-(aminomethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, PhBzL-17a (50 mg, 0.08 mmol, 1 equiv.) and 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethoxy)ethoxy)propanoate (34 mg, 0.08 mmol, 1 equiv.) in 2:1 ACN:DMF (3 ml), was added 2,4,6-collidine (21 μl, 0.16 mmol, 2 equiv.). The reaction mixture was stirred at room temperature for 2 h, then diluted with water and purified by preparative HPLC to give PhBzL-17 (39 mg, 0.041 mmol, 52%) as a white solid after lyophilization. LC / MS [M+H] 935.4 (calculated); LC / MS [M+H] 935.8 (observed).

[0224] Example L-18 Synthesis of (3-(2-amino-8-(3-((3-(39-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl))-3,37-dioxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2,36-diazanonatriacontyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)tert-butyl carbamate, PhBzL-18 [ka] (3-(2-amino-8-(3-((3-(aminomethyl)azetidin-1-yl)sulfonyl)phenyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)tert-butyl carbamate, PhBzL-17a (50 mg, 0.08 mmol, 1 equiv.) and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) To a solution of 3-oxo-7,10,13,16,19,22,25,28,31,34-decaoxa-4-azaheptatriacontan-37-oic acid (52.8 mg, 0.078 mmol, 0.97 equiv.) in DMF (1 ml) was added DIPEA (28 μl, 0.16 mmol, 2 equiv.), followed by HATU (36.5 mg, 0.096 mmol, 1.2 equiv.). The reaction mixture was stirred at room temperature for 2 h, then concentrated and purified by preparative HPLC to give PhBzL-18 (28.9 mg, 0.022 mmol, 28%). LC / MS [M+H] 1287.6 (calculated); LC / MS [M+H] 1288.1 (observed).

[0225] Example L-19 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[2-amino-4-[2-(cyclobutoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylazetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluorobenzenesulfonic acid, PhBzL-19 [ka] Preparation of cyclobutyl N-[2-[[2-amino-8-[3-[3-[(tert-butoxycarbonylamino)methyl]azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, PhBz-12b To a mixture of cyclobutyl N-[2-(propylaminooxy)ethyl]carbamate (288 mg, 1.14 mmol, 1.5 equiv, HCl) and 2-amino-8-[3-[3-[(tert-butoxycarbonylamino)methyl]azetidin-1-yl]sulfonylphenyl]-3H-1-benzoazepine-4-carboxylic acid, PhBz-12a (400 mg, 760 umol, 1.0 equiv) in DCM (10 mL) and DMA (3 mL) at 25 °C under N2, EDCI (582 mg, 3.04 mmol, 4.0 equiv) was added in one portion, followed by stirring at 25 °C for 2 h. DCM (10 mL) was removed in vacuum, water (15 mL) was added, the aqueous phase was extracted with ethyl acetate (10 mL × 4), and the combined organic phase was washed with saturated brine (20 mL × 2), dried over anhydrous Na2SO4, filtered, and concentrated in 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 = 10 / 1, 0 / 1) to give PhBz-12b (340 mg, 469 umol, 61.7% yield) as a brown solid. 1 H NMR (400 MHz, MeOD) δ8.12-8.05 (m, 2H), 7.90-7.83 (m, 1H), 7.82-7.76 (m, 1H), 7.58-7.50 (m, 2H), 7.49-7.42 (m, 1H), 7.33 (s, 1H), 4.76-4.67 (m, 1H), 3.96 (t, J = 5.2 Hz, 2H), 3.85 (t, J = 8.0 Hz, 2H), 3.75 (t, J = 7.2 Hz, 2H), 3.61-3.53 (m, 2H), 3.05 (d, J = 6.8 Hz, 2H), 2.63-2.54 (m, 1H), 2.19 (d, J = 8.9 Hz, 2H), 1.95-1.85 (m, 2H), 1.83-1.75 (m, 2H), 1.66 (d, J = 10.0 Hz, 1H), 1.60-1.48 (m, 1H), 1.39 (s, 9H), 1.00 (t, J = 7.2 Hz, 3H).

[0226] Preparation of cyclobutyl N-[2-[[2-amino-8-[3-[3-(aminomethyl)azetidin-1-yl]sulfonylphenyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, PhBz-12 To a solution of PhBz-12b (290 mg, 400 umol, 1.0 equiv) in MeCN (5 mL) and HO (5 mL) under N2 at 25 °C, TFA (456 mg, 4.00 mmol, 296 uL, 10 equiv) was added in one portion and the mixture was stirred at 80 °C for 1 h. MeCN (5 mL) was removed in vacuo and the aqueous phase was extracted with methyl tert-butyl ether (5 mL × 3) to remove excess TFA, then the aqueous phase was lyophilized to give PhBz-12 (280 mg, 379 umol, 94.7% yield, TFA) as a yellow solid.

[0227] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[1-[3-[2-amino-4-[2-(cyclobutoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylazetidin-3-yl]methylamino]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-19a A mixture of PhBz-12 (100 mg, 160 umol, 1.0 equiv.) and EtN (48.6 mg, 480 umol, 66.8 uL, 3.0 equiv.) in THF (2 mL) was treated with 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, TFP-PEG, ethyl ether, and ethyl ether were added at 0 °C under N2. 10-CO2H (113 mg, 160 umol, 1.0 equiv) was added in one portion and the mixture was stirred at 0° C. for 30 min, then heated to 25° C. and stirred for an additional 0.5 h. The reaction mixture was quenched with TFA at 0° C. until pH was 6, then water (5 mL) was added, the aqueous phase was extracted with ethyl acetate (3 mL), the ethyl acetate phase was discarded, then the aqueous phase was further extracted with DCM:i-PrOH / 3:1 (5 mL×3), and the combined organic phase was concentrated in vacuo to give PhBzL-19a (160 mg, 137 umol, 85.7% yield) as a yellow oil.

[0228] Preparation of PhBzL-19 To a mixture of PhBzL-19a (80.0 mg, 68.6 umol, 1.0 equiv) and (2,3,5,6-tetrafluoro-4-hydroxyphenyl)sulfonyloxysodium (92.0 mg, 343 umol, 5.0 equiv) in DCM (1 mL) and DMA (0.2 mL) at 25 °C under N2, EDCI (65.8 mg, 343 umol, 5.0 equiv) was added in one portion and the mixture was stirred at 25 °C for 1 h. The reaction mixture was filtered and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-60%, 8 min) to give PhBzL-19 (45.0 mg, 25.2 umol, 36.6% yield, 78.0% purity) as a yellow oil, and the crude product was further purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-50%, 8 min) to give PhBzL-19 (13.8 mg, 9.37 umol, 29.0% yield, 94.6% purity) as a yellow oil. 1H NMR (400 MHz, MeOD) δ8.16-8.08 (m, 2H), 7.93 (d, J = 7.6 Hz, 1H), 7.89-7.80 (m, 3H), 7.79 (s, 1H), 7.53 (s, 1H), 4.69-4.66 (m, 1H), 3.99 (t, J = 4.8 Hz, 2H), 3.93-3.84 (m, 4H), 3.81-3.74 (m, 2H), 3.72-3.50 (m, 40H), 3.46 (s, 2H), 3.18 (d, J = 6.4 Hz, 2H), 2.99 (t, J = 5.6 Hz, 2H), 2.74-2.64 (m, 1H), 2.30 (t, J = 6.0 Hz, 2H), 2.24-2.15 (m, 2H), 1.94-1.84 (m, 2H), 1.79 (br dd, J = 7.2, 14.4 Hz, 2H), 1.71-1.62 (m, 1H), 1.59-1.49 (m, 1H), 1.02 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1393.5 (calculated value); LC / MS [M+H] 1393.2 (actual value).

[0229] Example L-21 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluorobenzenesulfonic acid, PhBzL-21 [ka] Preparation of (2R)-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]pyrrolidine-2-carboxylate, PhBz-16b To a solution of (2R)-methyl pyrrolidine-2-carboxylate (334 mg, 2.02 mmol, 1 equiv, HCl) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid, PhBz-16a (0.5 g, 2.02 mmol, 1 equiv) in DMF (5 mL) was added HATU (766 mg, 2.02 mmol, 1 equiv) and DIEA (781 mg, 6.05 mmol, 1.05 mL, 3 equiv), then stirred at 20° C. for 2 h. The reaction mixture was quenched by addition of HO (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layer was washed with 20 mL of saturated brine, dried over NaSO, filtered, and concentrated under reduced pressure to give PhBz-16b (1.5 g, crude) as a yellow oil.

[0230] Preparation of (2R)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carboxylate, PhBz-16 A mixture of PhBz-16b (0.7 g, 1.95 mmol, 1 equiv), 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide (714 mg, 1.95 mmol, 1 equiv), K2CO3 (539 mg, 3.90 mmol, 2 equiv), Pd(dppf)Cl2 (143 mg, 195 umol, 0.1 equiv) in dioxane (20 mL) and H2O (2 mL) was degassed and purged with N2 three times, then stirred at 90 °C under N2 atmosphere for 2 h. The reaction mixture was extracted with EtOAc (30 mL x 3). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-50%, 8 min) to give PhBz-16 (0.5 g, 964 umol, 49.48% yield) as a white solid. 1H NMR (MeOH, 400 MHz) δ 7.86-7.64 (m, 7H), 7.45 (s, 1H), 4.64 (dd, J = 5.2, 8.4 Hz, 1H), 3.98 (q, J = 7.2 Hz, 2H), 3.83-3.73 (m, 5H), 3.72-3.58 (m, 2H), 3.48 (s, 2H), 2.50-2.33 (m, 1H), 2.14-1.91 (m, 3H), 1.78 (t, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H), 1.01 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 519.3 (calculated); LC / MS [M+H] 519.2 (observed).

[0231] Preparation of (R)-1-(4-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)benzoyl)pyrrolidine-2-carboxylic acid, PhBzL-21a To a solution of PhBz-16 (0.5 g, 964 umol, 1 equiv) in MeOH (20 mL) was added a solution of LiOH·HO (121 mg, 2.89 mmol, 3 equiv) in HO (2 mL) and then stirred for 2 h at 20 °C. The pH of the reaction mixture was adjusted to about 5 with HCl (4 M) and then filtered to give PhBzL-21a (0.2 g, 396 umol, 41.11% yield) as a brown solid.

[0232] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-21b To a solution of PhBzL-21a (0.2 g, 396 umol, 1 equiv.) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (279 mg, 476 umol, 1.2 equiv.) in DMF (2 mL) was added DIEA (256 mg, 1.98 mmol, 345 uL, 5 equiv.) and HATU (166 mg, 436 umol, 1.1 equiv.), then stirred at 20° C. for 2 h. The reaction mixture was filtered and purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-50%, 8 min) to give PhBzL-21b (0.15 g, 139.89 umol, 35.29% yield) as a yellow oil.

[0233] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2R)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-21c To a solution of PhBzL-21b (0.15 g, 140 umol, 1 equiv) in MeCN (2 mL) and HO (1 mL) was added HCl (12 M, 233 uL, 20 equiv) and then stirred for 1 h at 80° C. The reaction mixture was concentrated under reduced pressure to give PhBzL-21c (0.11 g, 108 umol, 77.38% yield) as a yellow oil.

[0234] Preparation of PhBzL-21 To a solution of PhBzL-21c (0.11 g, 108 umol, 1 equiv.) and (2,3,5,6-tetrafluoro-4-hydroxyphenyl)sulfonyloxysodium (116 mg, 433 umol, 4 equiv.) in DCM (2 mL) and DMA (0.1 mL), EDCI (83.0 mg, 433 umol, 4 equiv.) was added and then stirred at 20° C. for 1 h. The reaction mixture was filtered and concentrated in vacuo to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 8 min) to give PhBzL-21 (53.8 mg, 43.24 umol, 39.94% yield) as a yellow oil. 1 H NMR (MeOH, 400 MHz) δ7.85-7.64 (m, 6H), 7.56 (br d, J = 8.0 Hz, 1H), 7.45 (s, 1H), 4.62-4.39 (m, 1H), 3.98 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 5.6 Hz, 2H), 3.82-3.70 (m, 4H), 3.69-3.49 (m, 36H), 3.49-3.35 (m, 5H), 3.24-3.05 (m, 1H), 2.96 (t, J = 6.0 Hz, 2H), 2.49-2.26 (m, 1H), 2.12-1.87 (m, 3H), 1.84-1.71 (m, 2H), 1.28-1.15 (m, 3H), 1.01 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 1244.5 (calculated value); LC / MS [M+H] 1244.4 (actual value).

[0235] Example L-22 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluorobenzenesulfonic acid, PhBzL-22 [ka] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(3-bromophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-22b To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (500 mg, 854 umol, 1 eq.) and 3-bromobenzenesulfonyl chloride, PhBzL-22a (218 mg, 854 umol, 123 uL, 1 eq.) in DCM (5 mL), Et3N (173 mg, 1.71 mmol, 23 uL, 2 eq.) was added and then stirred at 25° C. for 0.5 h. The reaction mixture was diluted with water (10 mL) and extracted with DCM (20 mL×3). The combined organic layers were washed with saturated brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to ethyl acetate:MeOH = 10:1) to give PhBzL-22b (400 mg, 497 umol, 58.2% yield) as a yellow oil.

[0236] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-22c To a solution of PhBzL-22b (200 mg, 249 umol, 1 equiv.) and 2-amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-benzo[b]azepine-4-carboxamide (103 mg, 249 umol, 1 equiv.) in dioxane (2 mL) was added a solution of K2CO3 (68.7 mg, 497 umol, 2 equiv.) in water (0.5 mL) and Pd(dppf)Cl2 (9.09 mg, 12.4 umol, 0.05 equiv.) under N2, and the mixture was stirred at 90° C. for 5 hours. The mixture was filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex luna C18 100×40 mm×5 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-53%, 8 min) to give PhBzL-22c (50 mg, 49.5 umol, 19.90% yield) as a yellow oil. 1 H NMR (400MHz, MeOD) δ 8.22 (s, 1H), 7.98 (dd, J = 8.0, 16.6 Hz, 2H), 7.83-7.72 (m, 4H), 7.49 (s, 1H), 4.01 (q, J = 7.2 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 3.69 (t, J = 6.4 Hz, 2H), 3.66-3.52 (m, 34H), 3.51-3.46 (m, 6H), 3.15 (t, J = 5.2 Hz, 2H), 2.47 (t, J = 6.4 Hz, 2H), 1.84-1.77 (m, 2H), 1.72-1.65 (m, 1H), 1.46 (s, 9H), 1.24 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H).

[0237] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-22d To a solution of PhBzL-22c (50 mg, 49.5 umol, 1 equiv) in MeCN (0.2 mL) and water (2 mL) was added HCl (12 M, 61.8 uL, 15 equiv) and then stirred for 2 h at 80° C. The mixture was concentrated under reduced pressure to give PhBzL-22d (45 mg, 45.4 umol, 91.8% yield, HCl) as a yellow oil.

[0238] Preparation of PhBzL-22 To a solution of PhBzL-22d (45 mg, 45.4 umol, 1 equiv, HCl) and sodium 2,3,5,6-tetrafluoro-4-hydroxybenzenesulfonate (48.7 mg, 182 umol, 4 equiv) in DCM (0.3 mL) and DMA (0.3 mL), EDCI (34.8 mg, 182 umol, 4 equiv) was added and stirred at 25° C. for 0.5 h. The mixture was filtered, concentrated under reduced pressure, and purified by preparative HPLC (TFA condition; column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-50%, 8 min) to give PhBzL-22 (22 mg, 18.6 umol, 40.97% yield) as a yellow solid. 1 H NMR (400MHz, MeOD) δ 8.22 (s, 1H), 7.97 (dd, J = 8.4, 16.8 Hz, 2H), 7.83-7.68 (m, 4H), 7.48 (s, 1H), 4.00 (q, J = 6.8 Hz, 2H), 3.87 (t, J = 6.0 Hz, 2H), 3.78 (t, J = 7.2 Hz, 2H), 3.66-3.46 (m, 42H), 3.15 (t, J = 5.2 Hz, 2H), 2.98 (t, J = 6.0 Hz, 2H), 1.85-1.74 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 1183.4 (calculated value); LC / MS [M+H] 1183.6 (actual value).

[0239] Example L-26 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluorobenzenesulfonic acid, PhBzL-26 [ka] Preparation of (2S)-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoyl]pyrrolidine-2-carboxylate, PhBz-11b To a solution of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (500 mg, 2.02 mmol, 1.0 equiv) and (S)-methyl pyrrolidine-2-carboxylate, PhBz-11a (367 mg, 2.22 mmol, 1.1 equiv, HCl) in DMF (3 mL) under N at 25 °C, DIEA (1.04 g, 8.06 mmol, 1.40 mL, 4.0 equiv) and HATU (766 mg, 2.02 mmol, 1.0 equiv) were added in one portion and the mixture was stirred at 25 °C for 1.5 h. Water (10 mL) was added, the aqueous phase was extracted with ethyl acetate (10 mL×3), and the combined organic phase was washed with saturated brine (10 mL×2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give PhBz-11b (700 mg, crude) as a colorless oil.

[0240] Preparation of methyl 1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carboxylate, PhBz-11 Intermediate PhBz-11b (650 mg, 1.81 mmol, 1.0 equiv), 2-amino-8-bromo-N-ethoxy-N-propyl-3H-1-benzazepine-4-carboxamide (663 mg, 1.81 mmol, 1.0 equiv), Pd(dppf)Cl2 (132 mg, 181 umol, 0.1 equiv), and K2CO3 (500 mg, 3.62 mmol, 2.0 equiv) in dioxane (8 mL) and HO (2 mL) were degassed and then heated at 95 °C under N2 for 2 h. Dioxane was removed in vacuum, then water (10 mL) was added, the aqueous phase was extracted with ethyl acetate (10 mL x 3), and the combined organic phase was washed with saturated brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated in 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 = 10 / 1, 0 / 1) to give PhBz-11 (700 mg, 1.35 mmol, yield 74.6%) as a yellow solid.

[0241] Preparation of 1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carboxylic acid, PhBzL-26a To a solution of PhBz-11 (300 mg, 578 umol, 1.0 equiv) in MeOH (5 mL) and HO (5 mL) under N2 at 25 °C, LiOH·HO (97.1 mg, 2.31 mmol, 4.0 equiv) was added in one portion and then stirred at 25 °C for 10 h. The reaction mixture was quenched with HCl (4 M) until pH = 7, MeOH (5 mL) was removed in vacuo, and the aqueous phase was then extracted with DCM / iPr-OH = 3 / 1 (5 mL × 3), and the combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give PhBzL-26a (280 mg, 555 umol, 95.9% yield) as a brown oil.

[0242] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-26b To a mixture of PhBzL-26a (200 mg, 396 umol, 1.0 equiv), tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (348 mg, 594 umol, 1.5 equiv), and DIEA (102 mg, 793 umol, 138 uL, 2 equiv) in DMF (3 mL) was added HATU (151 mg, 396 umol, 1.0 equiv) in one portion at 0° C. under N2, which was stirred at 0° C. for 30 min, then heated to 25° C. and stirred for an additional 0.5 h. The reaction mixture was filtered and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-55%, 8 min) to give PhBzL-26b (250 mg, 233 umol, 58.8% yield) as a yellow oil.

[0243] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[4-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]benzoyl]pyrrolidine-2-carbonyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-26c To a solution of PhBzL-26b (120 mg, 112 umol, 1.0 equiv) in MeCN (1 mL) and HO (2 mL) under N2 at 25 °C was added HCl (12 M, 280 uL, 30 equiv) in one portion and then stirred at 80 °C for 1 h. The reaction mixture was concentrated in vacuo to give PhBzL-26c (110 mg, 108 umol, 96.7% yield) as a yellow oil.

[0244] Preparation of PhBzL-26 To a mixture of PhBzL-26c (110 mg, 108 umol, 1.0 equiv.) and (2,3,5,6-tetrafluoro-4-hydroxyphenyl)sulfonyloxysodium (145 mg, 541 umol, 5.0 equiv.) in DCM (2 mL) and DMA (0.3 mL) was added EDCI (103 mg, 541 umol, 5.0 equiv.) in one portion under N2 at 25° C., which was stirred for 1 h at 25° C. The reaction mixture was filtered and the filtrate was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-45%, 8 min) to give PhBzL-26 (26.3 mg, 20.1 umol, 18.5% yield, 94.9% purity) as a light tan solid. 1H NMR (400 MHz, MeOD) δ7.86-7.74 (m, 5H), 7.71-7.66 (m, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 4.63-4.42 (m, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.88 (t, J = 6.0 Hz, 2H), 3.78 (t, J = 7.2 Hz, 4H), 3.71-3.55 (m, 38H), 3.49-3.40 (m, 5H), 2.99 (t, J = 6.0 Hz, 2H), 2.43-2.31 (m, 1H), 2.11-1.99 (m, 2H), 1.97-1.87 (m, 1H), 1.80 (d, J = 7.2 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1144.5 (calculated value); LC / MS [M+H] 1144.3 (actual value).

[0245] Example L-27 Synthesis of 4-((3-(2-(2-(2-((3-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-)yl)phenyl)sulfonamido)ethoxy)ethoxy)ethoxy)propanoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonic acid, PhBzL-27 [ka] Preparation of tert-butyl 3-[2-[2-[2-[(3-bromophenyl)sulfonylamino]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-27c To a mixture of tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoate, PhBzL-27b (0.47 g, 1.69 mmol, 1 equiv.) in DCM (5 mL) was added Et3N (514 mg, 5.08 mmol, 708 uL, 3 equiv.) and 3-bromobenzenesulfonyl chloride, PhBzL-27a (433 mg, 1.69 mmol, 245 uL, 1 equiv.) at 0° C., then stirred for 3 h at 20° C. The mixture was washed with water (5 ml), then the organic phase was dried over Na2SO4 and concentrated to give PhBzL-27c (0.8 g, 1.61 mmol, 95.1% yield) as a colorless oil. 1 H NMR (400MHz, MeOD) δ8.10 (d, J = 1.6 Hz, 1H), 7.96-7.83 (m, 2H), 7.59 (t, J = 7.8 Hz, 1H), 3.79 (t, J = 6.4 Hz, 2H), 3.72-3.64 (m, 6H), 3.60-3.52 (m, 4H), 3.18-3.14 (m, 2H), 2.57 (t, J = 6.4 Hz, 2H), 1.54 (s, 9H).

[0246] Preparation of tert-butyl 3-[2-[2-[2-[[3-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]propanoate, PhBzL-27e To a mixture of PhBzL-27c (300 mg, 605 umol, 1 equiv.) and 2-amino-N-ethoxy-N-propyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3H-1-benzazepine-4-carboxamide, PhBzL-27d (250 mg, 605 umol, 1 equiv.) in dioxane (10 mL) and HO (1 mL), Pd(dppf)Cl (22.1 mg, 30.2 umol, 0.05 equiv.) and KCO (209 mg, 1.51 mmol, 2.5 equiv.) were added and stirred at 100° C. for 1 h under N. The mixture was filtered through Celite and concentrated to give a residue. The residue was diluted with EtOAc (20 mL) and water (10 ml). The organic layer was separated, dried over Na2SO4, and concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-55%, 8 min) to give PhBzL-27e (0.2g, 285umol, 47.0% yield) as a colorless oil. LC / MS [M+H] 703.3 (calculated); LC / MS [M+H] 703.2 (observed).

[0247] Preparation of 3-[2-[2-[2-[[3-[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzoazepin-8-yl]phenyl]sulfonylamino]ethoxy]ethoxy]ethoxy]propanoic acid, PhBzL-27f To a mixture of PhBzL-27e (240 mg, 341 umol, 1 equiv) in water (10 mL) was added HCl (12 M, 569 uL, 20 equiv) and then stirred at 80° C. for 0.5 h. The mixture was concentrated to give PhBzL-27f (0.2 g, 309 umol, 90.6% yield) as a yellow oil. LC / MS [M+H] 647.3 (calculated); LC / MS [M+H] 647.3 (observed).

[0248] Preparation of PhBzL-27 To a mixture of PhBzL-27f (0.2 g, 309 umol, 1 equiv) and sodium 2,3,5,6-tetrafluoro-4-hydroxybenzenesulfonate (415 mg, 1.55 mmol, 5 equiv) in DMA (0.3 mL) and DCM (3 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDCI, CAS Registry Number 1892-57-5 (296 mg, 1.55 mmol, 5 equiv) and then stirred at 20° C. for 0.5 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150×25×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 8 min) to give PhBzL-27 (80.7 mg, 87.4 umol, yield 28.3%, purity 94.70%) as a white solid. 1 H NMR (400MHz, MeOD) δ8.18 (d, J = 1.6 Hz, 1H), 8.02-7.83 (m, 2H), 7.81-7.63 (m, 4H), 7.46 (s, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.90-3.71 (m, 4H), 3.69-3.42 (m, 12H), 3.16-3.10 (m, 2H), 2.96 (t, J = 5.6Hz, 2H), 1.88-1.69 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.2Hz, 3H). LC / MS [M+H] 875.2 (calculated); LC / MS [M+H] 875.3 (observed).

[0249] Example 201 Preparation of Immunoconjugates (IC) To prepare lysine-conjugated immunoconjugates, the antibody is buffer exchanged into conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3 using G-25 SEPHADEX™ desalting columns (Sigma-Aldrich, St. Louis, MO) or Zeba™ spin desalting columns (Thermo Fisher Scientific). The eluent is then adjusted to a concentration of about 1-10 mg / ml each using buffer, then sterile filtered. The antibody is preheated to 20-30° C. and rapidly mixed with 2-20 (e.g., 7-10) molar equivalents of an 8-phenyl-2-aminobenzazepine-linker (PhBzL) compound of formula II dissolved in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) at a concentration of 5-20 mM. The reaction is allowed to proceed for approximately 16 hours at 30° C. and the immunoconjugate (IC) is separated from the reactants by passage through two successive G-25 desalting columns equilibrated in phosphate buffered saline (PBS) pH 7.2, a Zeba™ spin desalting column to yield the immunoconjugate (IC) in Table 3. The adjuvant-to-antibody ratio (DAR) is measured by liquid chromatography mass spectrometry using a C4 reversed-phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, Mass.) interfaced with a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0250] To prepare cysteine-conjugated immunoconjugates, antibodies are buffer-exchanged into conjugation buffer containing PBS, pH 7.2, with 2 mM EDTA using Zeba™ spin desalting columns (Thermo Fisher Scientific). Interchain disulfides are reduced using a 2-4 fold molar excess of tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT) for 30 min to 2 h at 37°C. Excess TCEP or DTT was removed using Zeba™ spin desalting columns pre-equilibrated with the conjugation buffer described above. The concentration of the buffer-exchanged antibodies was adjusted to approximately 5-20 mg / ml using the conjugation buffer described above and sterile filtered. Maleimide-PhBzL compounds are dissolved in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) at a concentration of 5-20 mM. For conjugation, the antibody is mixed with 10-20 molar equivalents of maleimide-PhBzL. Optionally, up to 20% (v / v) additional DMA or DMSO was added to improve the solubility of maleimide-PhBzL in the conjugation buffer. The reaction is allowed to proceed for approximately 30 min to 4 h at 20 °C. The resulting conjugate is purified from unreacted maleimide-PhBzL using two consecutive Zeba™ spin desalting columns. The columns are pre-equilibrated with phosphate buffered saline (PBS), pH 7.2. The adjuvant-to-antibody ratio (DAR) is estimated by liquid chromatography-mass spectrometry using a C4 reversed-phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0251] For conjugation, the antibody may be dissolved in an aqueous buffer system known in the art that does not adversely affect the antibody stability or antigen binding specificity. Phosphate buffered saline can be used. The PhBzL compound is dissolved in a solvent system that includes at least one polar aprotic solvent as described elsewhere herein. In some such embodiments, PhBzL is dissolved in a pH 8 Tris buffer (e.g., 50 mM Tris) to a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM, and ranges thereof, such as about 5 mM to about 50 mM or about 10 mM to about 30 mM. In some embodiments, the 8-phenyl-2-aminobenzazepine-linker intermediate is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide), or acetonitrile, or another suitable dipolar aprotic solvent.

[0252] Alternatively, in the conjugation reaction, an equivalent excess of the 8-phenyl-2-aminobenzazepine-linker (PhBzL) intermediate solution may be diluted and mixed with the antibody solution. The 8-phenyl-2-aminobenzazepine-linker intermediate solution may be suitably diluted with at least one polar aprotic solvent and at least one polar protic solvent, examples of which include water, methanol, ethanol, n-propanol, and acetic acid. The molar equivalent of 8-phenyl-2-aminobenzazepine-linker intermediate to antibody can be about 1.5:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1, and within these ranges, for example, about 1.5:1 to about 20:1, about 1.5:1 to about 15:1, about 1.5:1 to about 10:1, about 3:1 to about 15:1, about 3:1 to about 10:1, about 5:1 to about 15:1, or about 5:1 to about 10:1. The reaction can be suitably monitored for completion by methods known in the art, such as LC-MS. The conjugation reaction is generally complete in a range of about 1 hour to about 16 hours. After the reaction is complete, a reagent can be added to the reaction mixture to quench the reaction. If the thiol groups of the antibody have been reacted with a thiol-reactive group, such as the maleimide of the 8-phenyl-2-aminobenzazepine-linker intermediate, the unreacted antibody thiol groups can be reacted with a capping reagent. One example of a suitable capping reagent is ethylmaleimide.

[0253] After conjugation, the immunoconjugate can be purified and separated from unconjugated reactants and / or conjugate aggregates by purification methods known in the art, such as, but not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof. For example, prior to purification, the immunoconjugate can be diluted with 20 mM sodium succinate, pH 5, or the like. The diluted solution is applied to a cation exchange column, followed by washing with, for example, at least 10 column volumes of 20 mM sodium succinate, pH 5. The conjugate can be suitably eluted with a buffer such as PBS.

[0254] Example 202 HEK reporter assay Human embryonic kidney (HEK293) reporter cells expressing human TLR7 or human TLR8 (Invivogen, San Diego CA) were used for cell growth and experiments according to the supplier's protocol. Briefly, cells were grown to 80-85% confluence in DMEM supplemented with 10% FBS, ZEOCIN™, and blasticidin at 5% CO2. Cells were then cultured at 4 × 10 ng / ml with HEK detection medium and substrate containing immune stimulatory molecules. 4 Cells / well were seeded into 96-well flat-bottom plates. Activity was measured at wavelengths of 620-655 nm using a plate reader.

[0255] Example 203 Evaluation of Immunoconjugate Activity In Vitro This example demonstrates that the immunoconjugates of the invention are effective in inducing immune activation and are therefore useful in the treatment of cancer. a) Isolation of human antigen-presenting cells: Human myeloid antigen-presenting cells (APCs) were negatively selected from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation using ROSETTESEP™ Human Monocyte Enrichment Coctail (Stem Cell Technologies, Vancouver, Canada) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR. Immature APCs were subsequently purified to greater than 90% purity by negative selection using the EASYSEP™ Human Monocyte Enrichment Kit (Stem Cell Technologies) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR, but without CD16 depletion. b) Myeloid APC activation assay: 2×10 5 APCs are incubated in 96-well plates (Corning, Corning, NY) in Iscove's Modified Dulbecco's Medium (IMDM) (Lonza) supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL (micrograms per milliliter) streptomycin, 2 mM L-glutamine, sodium pyruvate, non-essential amino acids, and, where indicated, various concentrations of unconjugated (naked) antibodies and immunoconjugates of the invention (prepared according to the examples above). Cell-free supernatants are analyzed after 18 hours by ELISA to measure TNFα secretion as a readout for the proinflammatory response. c) PBMC activation assay: Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California). PBMCs were incubated in co-culture with CEA-expressing tumor cells (e.g., MKN-45, HPAF-II) in 96-well plates (Corning, Corning, NY) at an effector to target cell ratio of 10:1. Cells were stimulated with various concentrations of unconjugated (naked) antibodies and immunoconjugates of the invention (prepared according to the above examples). Cell-free supernatants were analyzed by cytokine bead array using the LegendPlex™ kit according to the manufacturer's guidelines (BioLegend®, San Diego, CA). d) Isolation of human conventional dendritic cells: Human conventional dendritic cells (cDCs) were negatively selected from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. Briefly, cells were first enriched by using ROSETTESEP™ Human CD3 Depletion Cocktail (Stem Cell Technologies, Vancouver, Canada) to remove T cells from the cell preparation. cDCs were then further enriched by negative selection using EASYSEP™ Human Myeloid DC Enrichment Kit (Stem Cell Technologies). e) cDC activation assay: 8×10 4APCs were co-cultured with tumor cells expressing ISAC target antigens at an effector (cDC)-target (tumor cell) ratio of 10:1. Cells were incubated in 96-well plates (Corning, Corning, NY) containing RPMI-1640 medium supplemented with 10% FBS and, where indicated, various concentrations of the indicated immunoconjugates of the present invention (prepared according to the above examples). After approximately 18 hours of overnight incubation, cell-free supernatants were collected and analyzed for cytokine (including TNFα) secretion using BioLegend LEGENDPLEX cytokine bead arrays.

[0256] In addition to the described assays utilizing different myeloid cell populations, various screening assays can be used to measure activation of myeloid cell types. These include: monocytes isolated from healthy donor blood, M-CSF-differentiated macrophages, GM-CSF-differentiated macrophages, GM-CSF+IL-4 monocyte-derived dendritic cells, conventional dendritic cells (cDCs) isolated from healthy donor blood, and myeloid cells polarized to an immunosuppressive state (also called myeloid-derived suppressor cells or MDSCs). Examples of MDSC-polarized cells include M2a MΦ (IL4 / IL13), M2c MΦ (IL10 / TGFb), GM-CSF / IL6 MDSCs, and monocytes differentiated to an immunosuppressive state, such as tumor-educated monocytes (TEMs). TEM differentiation can be performed using tumor-conditioned medium (e.g., 786.O, MDA-MB-231, HCC1954). Primary tumor-associated myeloid cells can also include primary cells present in dissociated tumor cell suspensions (Discovery Life Sciences).

[0257] Assessment of activation of the described myeloid cell populations may be performed as monocultures or as cocultures with cells expressing the antigen of interest to which the immunoconjugate may bind via the CDR region of the antibody. After 18-48 hours of incubation, activation may be assessed by upregulation of cell surface costimulatory molecules using flow cytometry or by measuring secreted inflammatory cytokines. For cytokine measurements, cell-free supernatants are collected and analyzed by cytokine bead arrays (e.g., LegendPlex from Biolegend) using flow cytometry.

[0258] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference into this specification to the same extent as if each reference was individually and specifically indicated to be incorporated by reference herein and was set forth in its entirety herein.

Claims

1. 4. The antibody covalently linked by a linker to one or more 8-phenyl-2-aminobenzazepine moieties, comprising formula I: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, Ab is an antibody construct having an antigen-binding domain that binds to CEA; p is an integer from 1 to 8; PhBz has the formula: 【Chemistry 2】 (R １ , R ２ , R ３ , and R ４ are independently 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 and optionally selected from the group consisting of: - (C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , - (C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C １ -C １２ Alkyldiyl)-OR ５ , - (C ３ -C １２ Carbocyclyl), - (C ３ -C １２ 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 ) N.R. ５ - ＊ , - (C ６ -C ２０ Aryl), - (C ６ -C ２０ Aryl)- ＊ , - (C ６ -C ２０ Aryldiyl)-N(R ５ ) - ＊ , - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ Alkyldiyl)-(C ２ -C ２０ Heterocyclyldiyl)- ＊ , - (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)-C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , - (C ２ -C ９ Heterocyclyl)-NR ５ -C(=NR ５ａ ) N.R. ５ - ＊ , - (C ２ -C ９ Heterocyclyl)-NR ５ - (C ６ -C ２０ Aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , - (C ２ -C ９ Heterocyclyl)-(C ６ -C ２０ Aryldiyl)- ＊ , - (C １ -C ２０ Heteroaryl), - (C １ -C ２０ Heteroaryl)- ＊ , - (C １ -C ２０ Heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , - (C １ -C ２０ Heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , - (C １ -C ２０ Heteroaryl)-NR ５ -C(=NR ５ａ ) N (R ５ ) - ＊ , - (C １ -C ２０ Heteroaryl)-N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , -C(=O)- ＊ 、 -C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , -C(=O)-(C ２ -C ２０ 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(=O)-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , -N(R ５ )-(C ２ -C ５ Heteroaryl), -N(R ５ ) -S(=O) ２ - (C １ -C １２ alkyl), -O-(C １ -C １２ alkyl), -O-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -O-(C １ -C １２ alkyldiyl)-N(R ５ ) - ＊ , -O-C(=O)N(R ５ ) ２ 、 -O-C(=O)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 and substituted with one or more groups selected from Or R ２ and R ３ together form a 5- or 6-membered heterocyclyl ring; X １ , X ２ , X ３ , and X ４ are independently a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N (R ５ ) selected from the group consisting of R ５ are independently H, C ６ -C ２０ Aryl, C ３ -C １２ Carbocyclyl, 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 Heteroaryl; However, the asterisk ＊ indicates the binding site of L, and R １ , R ２ , R ３ and R ４ is bonded to L. the 8-phenyl-2-aminobenzazepine moiety having the formula: L, -C(=O)-PEG-, -C(=O)-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(=O)-Gluc-, -C(=O)-PEG-O-, -C(=O)-PEG-O-C(=O)-, -C(=O)-PEG-C(=O)-, -C(=O)-PEG-C(=O)-PEP-, -C(=O)-PEG-N(R ６ )-、 -C(=O)-PEG-N(R ６ )-C(=O)-、 -C(=O)-PEG-N(R ６ )-PEG-C(=O)-PEP-、 -C(=O)-PEG-N ＋ (R ６ ) ２ 2019-02-20 10:00:00 AM -C(=O)-PEG-C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)-, -C(=O)-PEG-C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-, -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 ６ )-(C １ -C １２ Alkyldiyl)-, -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ alkyldiyl)-N(R ５ )-C(=O), -C(=O)-(C １ -C １２ alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ alkyldiyl)-N(R ６ ) C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(=O)-Gluc-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ ) -, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-PEP-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(═O)—, and -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)- is a linker selected from the group consisting of R ６ are independently H or C １ -C ６ is alkyl, PEG has the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -, m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc has the formula: 【Chemistry 3】 having PEP has the formula: 【Chemistry 4】 having wherein AA is independently selected from a natural or unnatural amino acid side chain, or one or more AA and adjacent nitrogen atoms form a 5-membered ring proline amino acid; The wavy lines indicate the attachment points. Cyc is optionally F, Cl, NO ２ , -OH, -OCH ３ , and: 【Chemistry 5】 C substituted with one or more groups selected from glucuronic acid having the structure ６ -C ２０ Aryldiyl and C １ -C ２０ heteroaryldiyl; R ７ is -CH(R ８ ) O-, -CH ２ --, --CH ２ N (R ８ )- and -CH(R ８ )O—C(═O)—; R ８ are independently H, C １ -C ６ Alkyl, C(=O)-C １ -C ６ Alkyl, and -C(=O)N(R ９ ) ２ is selected from R ９ is H, C １ -C １２ Alkyl, and -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R ９ the groups taken together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; provided that alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from 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 N.H. ２ , -CH ２ N.H.S.O. ２ CH ３ , -CH ２ N.H.C.H. 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, O.C.H. ２ F, O.C.H.F. ２ , O.C.F. ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H; The immunoconjugate or a pharma- ceutically acceptable salt thereof.

2. The immunoconjugate of claim 1 , wherein the antibody is selected from labetuzumab and arcitumomab.

3. The antibody construct comprises: a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:3, CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, CDR-L3 comprising the amino acid sequence of SEQ ID NO:7, CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, CDR-H2 comprising the amino acid sequence of SEQ ID NO:13, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:15; b) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 26, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; c) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:53, CDR-L2 comprising the amino acid sequence of SEQ ID NO:55, CDR-L3 comprising the amino acid sequence of SEQ ID NO:39, CDR-H1 comprising the amino acid sequence of SEQ ID NO:44, CDR-H2 comprising the amino acid sequence of SEQ ID NO:46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:48; e) CDR-L1 comprising the amino acid sequence of SEQ ID NO:59, CDR-L2 comprising the amino acid sequence of SEQ ID NO:61, CDR-L3 comprising the amino acid sequence of SEQ ID NO:63, CDR-H1 comprising the amino acid sequence of SEQ ID NO:67, CDR-H2 comprising the amino acid sequence of SEQ ID NO:69, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:71; f) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 75, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 77, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 83, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 87; g) CDR-L1 comprising the amino acid sequence of SEQ ID NO:91, CDR-L2 comprising the amino acid sequence of SEQ ID NO:93, CDR-L3 comprising the amino acid sequence of SEQ ID NO:95, CDR-H1 comprising the amino acid sequence of SEQ ID NO:99, CDR-H2 comprising the amino acid sequence of SEQ ID NO:101, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:103; h) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 117 or 118, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120, or i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 107, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 124, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 126, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:

128. The immunoconjugate of claim 1 , comprising:

4. 2. The immunoconjugate of claim 1, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105, and a variable heavy chain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130.

5. 2. The immunoconjugate of claim 1, wherein the antibody construct comprises a variable light chain comprising an amino acid sequence selected from SEQ ID NOs: 1, 17, 32, 50, 57, 73, 89, and 105, and a variable heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 9, 41, 65, 81, 97, 113, 122, and 130.

6. The immunoconjugate of claim 5, wherein the antibody construct comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 105, and a heavy chain CDR (complementarity determining region) CDR-H2 comprising the amino acid sequence of SEQ ID NO:

118.

7. The immunoconjugate of claim 6, wherein the antibody construct comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 105 and a variable heavy chain comprising the amino acid sequence of SEQ ID NO:

113.

8. X ２ is a bond, and R ２ C １ -C ８ The immunoconjugate of any one of claims 1 to 7, wherein the aryl group is alkyl.

9. X ２ and X ３ are bonds, and R ２ and R ３ Independently, C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ Alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , -(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ , -O-(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ The immunoconjugate of any one of claims 1 to 7, selected from:

10. R ２ C １ -C ８ alkyl, R ３ But -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ The immunoconjugate of claim 9 ,

11. R ２ Ga-CH ２ CH ２ CH ３ and R ３ But -CH ２ CH ２ CH ２ N.H.C.O. ２ (t-Bu), -OCH ２ CH ２ N.H.C.O. ２ (cyclobutyl), and -CH ２ CH ２ CH ２ N.H.C.O. ２ 11. The immunoconjugate of claim 10, wherein said aryl group is selected from (cyclobutyl).

12. R ２ and R ３ each independently represents -CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -OCH ２ CF ３ , -CH ２ CH ２ CF ３ , -OCH ２ CH ２ OH and -CH ２ CH ２ CH ２ 10. The immunoconjugate of claim 9, wherein said amino acid is selected from the group consisting of OH.

13. R ２ and R ３ are -CH ２ CH ２ CH ３ The immunoconjugate of claim 12,

14. R ２ CH ２ CH ２ CH ３ and R ３ OCH ２ CH ３ The immunoconjugate of claim 12,

15. X ３ -R ３ but, 【Chemistry 6】 The immunoconjugate of any one of claims 1 to 7, selected from the group consisting of:

16. X ４ is a bond, and R ４ The immunoconjugate of any one of claims 1 to 7, wherein

17. R １ The immunoconjugate of any one of claims 1 to 7, wherein:

18. R ２ Or R ３ The immunoconjugate of any one of claims 1 to 7, wherein:

19. X ３ -R ３ -L is, 【Chemistry 7】 where the wavy line indicates the point of attachment to N. The immunoconjugate of claim 18, selected from the group consisting of:

20. The immunoconjugate of any one of claims 1 to 7, wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

21. The immunoconjugate of any one of claims 1 to 7, wherein L is attached to a cysteine ​​thiol of the antibody.

22. The immunoconjugate of any one of claims 1 to 7, wherein, with respect to the PEG, m is 1 or 2 and n is an integer from 2 to 10.

23. 23. The immunoconjugate of claim 22, wherein n is 10.

24. L has the structure: 【Chemistry 13】 (In the formula, the wavy line represents R ５ (showing the bond to The immunoconjugate of any one of claims 1 to 7, selected from:

25. Formula Ia: 【Chemistry 14】 The immunoconjugate of any one of claims 1 to 7, having the formula:

26. X ４ is a bond and R ４ The immunoconjugate of claim 25, wherein

27. X ２ and X ３ are bonds, and R ２ and R ３ Independently, C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ Alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , -(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ , -O-(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ 26. The immunoconjugate of claim 25, selected from:

28. X ２ The immunoconjugate of claim 25 , wherein

29. Formulae Ib to If: 【Chemistry 15-1】 【Chemistry 15-2】 The immunoconjugate of any one of claims 1 to 7, selected from:

30. X ２ and X ３ are bonds, and R ２ and R ３ Independently, C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ Alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ 30. The immunoconjugate of claim 29, selected from:

31. X ２ and X ３ are bonds, and R ２ C １ -C ８ alkyl, R ３ -O-(C １ -C １２ alkyl) and -O-(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ 30. The immunoconjugate of claim 29, selected from:

32. An 8-phenyl-2-aminobenzazepine-linker compound selected from the group consisting of:

33. 33. An immunoconjugate prepared by conjugation of an anti-CEA antibody with the 8-phenyl-2-aminobenzazepine-linker compound of claim 32.

34. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 7, and one or more pharma- ceutically acceptable diluents, vehicles, carriers, or excipients.

35. 32. A medicament for treating cancer comprising the immunoconjugate of any one of claims 1 to 31, wherein the cancer is selected from cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, 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.

36. A method for preparing an immunoconjugate of formula I according to claim 1, comprising conjugating an 8-phenyl-2-amino-thienoazepine-linker compound according to claim 32 with said anti-CEA antibody.

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