2-Amino-4-carboxamido-benzazepine immunoconjugates and uses thereof

Abstract

The present disclosure provides an immunoconjugate of formula I comprising an antibody linked by conjugation to one or more 2-amino-4-carboxamido-benzazepine derivatives. The present invention also provides 2-amino-4-carboxamido-benzazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions are suitable substrates for the formation of said immunoconjugates via linkers or linking sites. The present invention further provides methods of treating cancer using said immunoconjugates. [Formula 1] TIFF2024511088000082.tif56165

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 166,710, filed March 26, 2021, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on March 25, 2022, is named 17019_015WO1_SL.txt, and is 85,369 bytes in size.

[0003] The present invention relates generally to immunoconjugates comprising an antibody conjugated to one or more 2-amino-4-carboxamido-benzazepine molecules. [Background technology]

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

[0005] The present disclosure generally relates to immunoconjugates comprising an antibody linked by conjugation to one or more 2-amino-4-carboxamido-benzazepine derivatives. The invention further relates to 2-amino-4-carboxamido-benzazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions are suitable substrates for the formation of immunoconjugates, in which the antibody is linked by a linker to a compound of the formula: [ka] and can be covalently attached to the 4-position of a 2-amino-4-carboxamido-benzazepine moiety having the formula: R3 is attached to the linker L. The positions of the 3H-benzo[b]azepine structure are numbered according to the IUPAC rules. 2-3 and R 1-3 The substituents are defined herein.

[0006] The antibody binds to a target selected from the group consisting of PD-L1, HER2, TROP2 and CEA.

[0007] The present disclosure further relates to the use of such immunoconjugates in the treatment of disease, particularly cancer.

[0008] One aspect of the invention is an immunoconjugate comprising an antibody covalently bound to a linker that is covalently bound to one or more 2-amino-4-carboxamido-benzazepine moieties. Another aspect of the present invention is 2-amino-4-carboxamido-benzazepine linker compounds.

[0009] Another aspect of the invention is a method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an antibody linked by conjugation to one or more 2-amino-4-carboxamido-benzazepine moieties.

[0010] Another aspect of the present invention is the use of an immunoconjugate comprising an antibody linked by conjugation to one or more 2-amino-4-carboxamido-benzazepine moieties for the treatment of cancer.

[0011] Another aspect of the invention is a method of preparing an immunoconjugate by conjugating one or more 2-amino-4-carboxamido-benzazepines to an antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

[0014] definition The terms "Toll-like receptor" and "TLR" refer to members of a family of highly conserved mammalian proteins that recognize pathogen-associated molecular patterns and function 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 that is involved in TLR signaling.

[0015] 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 publicly available TLR7 sequence (e.g., GenBank Accession No. AAZ99026 for human TLR7 polypeptide or GenBank Accession No. AAK62676 for mouse TLR7 polypeptide).

[0016] 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 publicly published TLR7 sequence (e.g., GenBank Accession No. AAZ95441 for human TLR8 polypeptide or GenBank Accession No. AAK62677 for mouse TLR8 polypeptide).

[0017] A "TLR agonist" is a substance that directly or indirectly binds to a TLR (e.g., TLR7 and / or) and induces TLR signaling. A detectable difference in TLR signaling may indicate that the agonist stimulates or activates the TLR. Differences in signaling may be manifested, for example, as changes in expression of target genes, changes in phosphorylation of signaling components, changes in the subcellular localization of downstream elements such as nuclear factor-γB (NF-γB), changes in the association of certain components (e.g., IL-1 receptor-associated kinase (IRAK)) with other proteins or subcellular structures, or changes in the biochemical activity of components such as kinases (e.g., mitogen-activated protein kinases (MAPKs)).

[0018] "Antibody" refers to a polypeptide comprising an antigen-binding region (including complementarity determining regions (CDRs)) from an immunoglobulin gene or fragments 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 include, for example, the variable domains or regions of the light and heavy chains (V and V, respectively). L and V H ), light and heavy chain constant domains or regions (CL and C H ) and are classified into various categories according to size and function. 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. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, 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 gamma 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 a light chain by a disulfide bond, respectively. The resulting tetramer has two identical halves that together form a Y-shaped configuration. Both ends of the branch contain the same antigen-binding domain. In humans, there are four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), named in order of abundance in serum (i.e., IgG1 is the most abundant). The antigen-binding domain of an antibody is usually the most important for the specificity and affinity of binding to cancer cells.

[0019] A "bispecific" antibody (bsAb) is an antibody that binds two different epitopes to cancer (Suurs FV et al (2019) Pharmacology & Therapeutics 201:103-119). Bispecific antibodies can engage immune cells to destroy tumor cells, deliver payloads to tumors, and / or block tumor signaling pathways. Antibodies that target specific antigens include bispecific or multispecific antibodies that have at least one antigen-binding region that targets a specific antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody that has at least one antigen-binding region that targets tumor cells. Such antigens include, but are not limited to, mesothelin, prostate-specific membrane antigen (PSMA), HER2, TROP2, CEA, EGFR, 5T4, nectin 4, CD19, CD20, CD22, CD30, CD70, B7H3, B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), glypican 3, RG1, fucosyl-GMl, CTLA-4, and CD44 (WO2017 / 196598).

[0020] Antibodies that target specific antigens include bispecific or multispecific antibodies that have at least one antigen-binding region that targets a specific antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody that has at least one antigen-binding region that targets tumor cells. Such antigens include CD47, SIRPα, Dectin-2, PD-1, and PD-L1.

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

[0022] The term "immunoconjugate" refers to an antibody construct covalently attached to an adjuvant moiety via a linker. The immunoconjugate allows for targeted delivery of the active adjuvant moiety while the target antigen is bound.

[0023] "Adjuvant" refers to a substance that can induce an immune response in a subject exposed to the adjuvant. The term "adjuvant moiety" refers to an adjuvant that is covalently attached to an antibody construct, for example, via a linker, as described herein. The adjuvant moiety can induce an immune response while attached to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immune complex to a subject.

[0024] In some embodiments, the binding agent is an antigen-binding antibody "fragment," which is a construct that includes at least the antigen-binding region of an antibody, either alone or together with other components that together form an antigen-binding construct. Many different types of antibody "fragments" are known in the art, including, for example: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by disulfide bridges at the hinge region, and (iii) a V fragment consisting of a single arm of an antibody. L and V H (iv) F(ab') fragments, which result from cleavage of the disulfide bridges of the F(ab') fragment under mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (v) two domains of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized as one polypeptide chain (i.e., V L and V H ) and single-chain Fv (scFv).

[0025] An antibody or antibody fragment may be part of a larger construct, e.g., a conjugate or fusion construct to additional regions of the antibody fragment. 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 fusion to a transmembrane domain (optionally with an intervening linker or "stalk" (e.g., hinge region)) and optional intercellular signaling domain. For example, an antibody fragment may be fused to the gamma and / or delta chains of a t cell receptor to provide a T cell receptor-like construct that binds PD-L1. In yet another embodiment, an antibody fragment is part of a bispecific T cell inducer (BiTE) that includes a CD1 or CD3 binding domain and a linker.

[0026] A "cysteine ​​mutant antibody" is an antibody in which one or more amino acid residues of the antibody are replaced with a cysteine ​​residue. Cysteine ​​mutant antibodies can be prepared from parent antibodies by antibody engineering (Junutula, et al., (2008b) Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; U.S. Patent No. 7521541; U.S. Patent No. 7723485; U.S. Patent No. 2012 / 0121615; WO2009 / 052249). The cysteine ​​residue provides site-specific conjugation of adjuvants such as TLR agonists to the antibody through reactive cysteine ​​thiol groups at the engineered cysteine ​​site, but does not interfere with immunoglobulin folding and assembly or alter antigen binding and effector function. Cysteine ​​mutant antibodies can be conjugated to TLR agonist linker compounds with uniform stoichiometry of the immune complex (e.g., up to two TLR agonist moieties per antibody in antibodies with a single engineered mutant cysteine ​​site). The TLR agonist-linker compounds have a reactive electrophilic group that reacts specifically with the free cysteine ​​thiol group of the cysteine ​​mutant antibody.

[0027] By "epitope" is meant any antigenic or epitopic determinant of an antigen to which an antigen-binding domain binds (i.e., in 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.

[0028] 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, including 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, IgG4).

[0029] As used herein, the phrase "immune checkpoint inhibitor" refers to any modulator that inhibits the activity of an immune checkpoint molecule. Immune checkpoint inhibitors may include, but are not limited to, immune checkpoint molecule binding proteins, small molecule inhibitors, antibodies (including bispecific and multispecific antibodies having at least one antigen-binding region that targets an immune checkpoint protein, e.g., bispecific or multispecific antibodies that do not target only an immune checkpoint protein, as well as dual immunomodulators (that target two immunomodulatory targets simultaneously) (resulting in blockade of inhibitory targets, depletion of suppressor cells, and / or activation of effector cells); tumor-targeted immunomodulators (that induce potent costimulation in tumor-infiltrating immune cells by targeting tumor antigens and costimulatory molecules such as CD40 or 4-1BB); NK cell redirecting agents (that redirect NK cells to malignant cells by targeting tumor antigens and CD16A); or T cell redirecting agents (that redirect T cells to malignant cells by targeting tumor antigens and CD3)), antibody derivatives (including Fc fusions, Fab fragments, and scFv), antibody drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides, and peptidomimetics.

[0030] The "identity" of a nucleic acid or amino acid sequence referred to herein may be determined by comparing a subject nucleic acid or amino acid sequence 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 subject sequence and the reference sequence divided by the length of the longest sequence (i.e., the length of either the subject sequence 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-Coffe, and ALIGN (for nucleic acid and amino acid sequence alignment), BLAST programs (e.g., BLAST2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SRESEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalism Models of Proteins and Nucleic Acids, Cambridge, UK (2009), Soding, Bioinformation, 21(7):951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17):3389-3402 1997) and Gusfield, Algorithms on Strings, Tree and Sequences, Cambridge University Press, Cambridge UK (1997)).Percent sequence identity (%) can also be calculated, for example, as 100×1 [(identical positions) / min(TGA,TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions of peptide sequences A and B in the alignment that minimizes TGA and TGB. See, e.g., Russell et al., J. Mol Biol., 244:332-350 (1994).

[0031] 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) linked 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.

[0032] "Biosimilar" refers to an approved antibody construct that has similar activity profiles as previously approved PD-L1-targeting antibody constructs, such as atezolizumab (TECENTRIQ™, Genentech, Inc.), durvalumab (IMFINZI™, AstraZeneca), and avelumab (BAVENCIO™, EMDSerono, Pfizer); previously approved HER2-targeting antibody constructs, such as trastuzumab (HERCEPTIN™, Genentech, Inc.) and pertuzumab (PERJETA™, Genentech, Inc.); or a CEA-targeting antibody, such as labetuzumab (CEA-CIDE™, MN-14, hMN14, Immunomedics) CAS Registry Number 219649-07-7).

[0033] "Biobetter" refers to an approved antibody construct that is an improvement of a previously approved antibody construct such as atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and labetuzumab. A biobetter can have one or more modifications (e.g., an altered glycan profile, or a unique epitope) relative to the previously approved antibody construct.

[0034] "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, and non-natural (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to an isomer that has the same molecular formula and intramolecular bond but differs in the three-dimensional arrangement of bonds and atoms (e.g., an l-amino acid and the corresponding d-amino acid). Amino acids can be glycosylated (e.g., N-linked glycan, O-linked glycan, phosphoglycan, C-linked glycan, or glypication) or deglycosylated. Amino acids may be represented herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0035] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0036] Naturally occurring amino acids include those formed in proteins by post-translational modifications, such as citrulline (Cit).

[0037] Non-natural (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 the L- or D-configuration that function similarly to natural amino acids. An "amino acid analog" can be a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to a hydrogen, a carboxyl group, and an amino group), but has a modified side group or peptide backbone, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions similarly to a natural amino acid.

[0038] "Linking site" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linking site can serve to covalently bond an adjuvant moiety to an antibody in an immunoconjugate.

[0039] "Linking site" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linking site can serve to covalently attach an adjuvant moiety to an antibody in an immunoconjugate. Useful bonds for connecting linking sites to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0040] "Divalent" refers to a chemical moiety that includes two points of attachment for linking two functional groups. 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.

[0041] Wavy line [ka] represents the point of attachment of a particular chemical moiety. [ka] When present, it is understood that the chemical moiety can be used in both ways, i.e., read from left to right or right to left. In some embodiments, the two wavy lines present [ka] A particular site having a suffix suffix is ​​considered to be used as read from left to right.

[0042] "Alkyl" refers to a straight chain (linear) or branched saturated aliphatic radical having the number of carbon atoms indicated. An alkyl can contain any number of carbons, for example, from 1 to 12. Examples of alkyl groups include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -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-9-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-1-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH3), 2-methyl-1-propyl (t-Bu, t-butyl, -CH(CH3)CH2CH 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 (-CH2CH2 CH2CH2CH2CH3), 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(C(CH3)2CH2CH2CH3), Examples of alkyl groups include, but are not limited to, 2,3-dimethyl-2-butyl (-C(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.

[0043] The term "alkyldiyl" refers to a divalent alkyl group. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and the like. Alkyldiyl groups are sometimes also referred to as "alkylene" groups.

[0044] "Alkenyl" refers to a straight chain (linear) or branched unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon double bond, sp. Alkenyl can contain from 2 to about 12 or more carbon atoms. Alkenyl groups are radicals having "cis" and "trans" orientations, alternatively "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 unsubstituted or substituted. "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.

[0045] The term "alkenylene" or "alkenyldiyl" refers to a straight-chain or branched divalent hydrocarbon radical. Examples include, but are not limited to, ethylenylene or vinylene (-CH=CH-), allyl (-CHCH=CH-).

[0046] "Alkynyl" refers to a straight chain (linear) or branched 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.

[0047] The terms "alkynylene" or "alkynyldiyl" refer to a divalent alkynyl radical.

[0048] The terms "carbocycle", "carbocyclyl", "carbocycle" and "cycloalkyl" refer to saturated or partially unsaturated monocyclic, fused bicyclic or bridged polycyclic assemblies containing 3 to 12 ring atoms or the number of atoms indicated. Saturated monocyclic carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocycles include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. Carbocyclic groups are partially unsaturated and may 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.

[0049] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0050] "Aryl" means an aromatic ring system having 6 to 20 carbon atoms (C6-C8) derived by the removal of one hydrogen atom from a carbon atom of a parent aromatic ring system. 20 ) 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 have 6 to 12 ring members, such as phenyl, naphthalene, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

[0051] "Arylene" or "aryldiyl" refers to an aryl group having 6 to 20 carbon atoms (C6-C8) derived by the removal of two hydrogen atoms from two carbon atoms of a parent aromatic ring system. 20 ) divalent aromatic hydrocarbon radical. Some aryldiyl groups are represented by "Ar" in the exemplary structures. Aryldiyl includes bicyclic radicals that include an aromatic ring fused to a saturated ring, a partially unsaturated ring, or an aromatic carbocyclic ring. Typical aryldiyl groups include, but are not limited to, radicals derived from benzene (phenylene), 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 described herein.

[0052] The terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in 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 substituted independently with one or more substituents described below. The heterocycle may be a monocyclic ring of 3 to 7 members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicyclic ring of 7 to 10 members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), such as a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocyclic rings are described in Paquette, Leo A., "Principles of Modern Heterocyclic Chemistry" (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-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 the heterocyclic ring 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, piperazinyl, homopiperazinyl, azopyranyl, tetra ... Examples of heterocyclic ... 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.

[0053] The term "heterocyclyldiyl" refers to a divalent saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic radical of 3 to about 20 ring atoms, where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, and the remaining ring atoms are C, and where one or more ring atoms are optionally substituted independently with one or more of the substituents described. Examples of 5- and 6-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl.

[0054] The term "heteroaryl" refers to a monovalent 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 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.

[0055] 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, pyrimidinyldiyl, pyrazolyldiyl, triazolyldiyl, pyrazinyldiyl, tetrazolyldiyl, furyldiyl, thienyldiyl, isoxazolyldiyl, thiazolyldiyl, oxadiazolyldiyl, oxazolyldiyl, isothiazolyldiyl and pyrrolyldiyl.

[0056] The heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked, where possible. By way of example and not limitation, a carbon-linked heterocycle or heteroaryl is linked at the 2-, 3-, 4-, 5-, or 6-position of a pyridine, the 3-, 4-, 5-, or 6-position of a pyridazine, the 2-, 4-, 5-, or 6-position of a pyrimidine, the 2-, 3-, 5-, or 6-position of a pyrazine, the 2-, 3-, 4-, or 5-position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of an oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of an isoxazole, pyrazole, or isothiazole, the 2-, or 3-position of an aziridine, the 2-, 3-, or 4-position of an azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of a quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of an isoquinoline.

[0057] By way of example, and without limitation, a nitrogen-linked heterocycle or heteroaryl may be bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or β-carboline.

[0058] The terms "halo" or "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0059] 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 an oxygen and bonded to two other groups in the carbonyl-containing moiety.

[0060] 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).

[0061] The terms "treat," "treatment," and "treating" refer to any indicia of success in treating or ameliorating an injury, condition, state (e.g., cancer) or symptom (e.g., cognitive impairment), and include any objective or subjective parameter, such as remission, remission, relief of symptoms, or making the symptom, injury, condition, or condition more tolerable to the patient, reducing the rate of progression of a symptom, reducing the frequency or duration of a symptom or condition, or, in some circumstances, preventing the onset of a symptom. Treatment or amelioration of a symptom can be based on any objective or subjective parameter, including the results of a physical examination.

[0062] The terms "cancer," "neoplasm," and "tumor" are used herein to refer to cells that exhibit autonomous, uncontrolled proliferation, such as those that 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, pre-metastatic 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 or is derived from a cancer cell (e.g., isolated from any cancer that can treat an individual, e.g., from an individual with cancer) (e.g., a clone of a cancer cell). For example, the cancer cell may be from an established cancer cell line, may be a primary cell isolated from an individual with cancer, may be a progeny cell from a primary cell isolated from an individual with cancer, etc. In some embodiments, the term can also refer to a portion of a cancer cell, such as an intracellular portion of the cancer cell, a cell membrane portion, or a cell lysate. Many types of cancer are known to those of skill in the art, including solid tumors, such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myelomas, as well as circulating cancers, such as leukemias.

[0063] 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, including minimal residual disease, and including both primary and metastatic tumors.

[0064] "PD-L1 expression" refers to cells that have PD-L1 receptors on their cell surface. As used herein, "PD-L1 overexpression" refers to cells that have more PD-L1 receptors compared to corresponding non-cancerous cells.

[0065] "HER2" refers to the protein human epidermal growth factor receptor 2.

[0066] "HER2 expression" refers to a cell that has a HER2 receptor on the cell surface. For example, a cell may have about 20,000 to about 50,000 HER2 receptors on the surface of the cell. As used herein, "HER2 overexpression" refers to a cell that has more than about 50,000 HER2 receptors. For example, the cell has 2, 5, 10, 100, 1,000, 10,000, 100,000, or 1,000,000 times the number of HER2 receptors compared to a corresponding non-cancer cell (e.g., about 1 or 2 million HER2 receptors). HER2 is estimated to be overexpressed in breast cancer by about 25% to about 30%.

[0067] "TROP2 expression" refers to cells that have TROP2 receptors on the cell surface. As used herein, "TROP2 overexpression" refers to cells that have more TROP2 receptors compared to corresponding normal non-cancerous cells. TROP2 is estimated to be overexpressed in approximately 74% of breast cancers, 72% of colorectal cancers, and 64% of lung cancers, as well as other organ cancer types.

[0068] The "pathology" of cancer includes any phenomenon that compromises the well-being of a patient, including, but not limited to, abnormal or uncontrolled cell proliferation, metastasis, inhibition of normal function of neighboring cells, release of abnormal levels of cytokines or other secretions, suppressed or exacerbated inflammatory or immune responses, neoplasms, premalignant or malignant tumors, and invasion of surrounding or distant tissues or organs, such as lymph nodes.

[0069] As used herein, the phrases "cancer recurrence" and "tumor recurrence" and their grammatical variations refer to further growth of tumors or cancer cells after a cancer diagnosis. In particular, recurrence can occur when further cancer cell growth occurs in cancer tissue. Similarly, "tumor spread" occurs when tumor cells are dispersed to local or distant tissues or organs, and thus includes tumor metastasis. "Tumor invasion" occurs when tumor growth spreads locally and impairs the function of the involved tissue by compressing, destroying or preventing normal organ function.

[0070] As used herein, the term "metastasis" refers to the growth of a cancer tumor in an organ or body part that is not directly connected to the organ in which the cancer tumor resides. Metastasis will be understood to include micrometastasis, which is the presence of undetectable amounts of cancer cells in an organ or body part that is not directly connected to the organ in which the cancer tumor resides. Metastasis can also be defined as a several-step process, including the departure of cancer cells from the original tumor site, the migration and / or infiltration of cancer cells to other parts of the body.

[0071] The phrases "effective amount" and "therapeutically effective amount" refer to the dosage or amount of a substance, such as an immunoconjugate, that produces the therapeutic effect for which it is administered. The actual amount administered will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22nd 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 inhibit the growth of and / or kill existing cancer cells, it 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).

[0072] The terms "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 therapy, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, zoo, sport, and pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, and the like. In certain embodiments, the mammal is a human.

[0073] 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 when administered alone. In particular, the immunoconjugates disclosed herein include synergistic combinations of the claimed adjuvants and antibody constructs. These synergistic combinations upon administration induce a greater effect on immunity compared to when, for example, the antibody construct or the adjuvant is administered in the absence of the other moiety. Furthermore, a reduced amount of the immunoconjugate may be administered (measured by the total number of antibody constructs or the total number of adjuvants administered as part of the immunoconjugate) compared to when administered alone with either the antibody construct or the adjuvant.

[0074] As used herein, the term "administering" means parenterally, intravenously, intraperitoneally, intramuscularly, intratumorally, intralesionally, intranasally or subcutaneously, orally, as a suppository, topically, intrathecally, or by implantation of a sustained release device, e.g., a mini-osmotic pump, into the subject.

[0075] The terms "about" and "approximately" used herein to modify a numerical value indicate a close range surrounding that 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 present specification for a claim limitation of, for example, "0.98X."

[0076] antibody The immune complexes of the present invention comprise antibodies. Included within the scope of the embodiments of the present invention are functional variants of the antibody constructs or antigen-binding domains described herein. As used herein, the term "functional variant" refers to an antibody construct having an antigen-binding domain with substantial or significant sequence identity or similarity with a parent antibody construct or antigen-binding domain, and this functional variant retains the biological activity of the antibody construct or antigen-binding domain of which it is a variant. Functional variants include variants of the antibody constructs or antigen-binding domains described herein (parent antibody constructs or antigen-binding domains) that retain, for example, a similar, equal or greater ability to recognize target cells expressing PD-L1, HER2, CEA or TROP2 than the parent antibody construct or antigen-binding domain.

[0077] With respect to an antibody construct or antigen-binding domain, a functional variant is one that is, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the antibody construct or antigen-binding domain.

[0078] 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, such that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen-binding domain.

[0079] 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 that replace one amino acid with particular physical and / or chemical properties with another amino acid with the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid (e.g., Asp or Glu) substituted for another acidic / negatively charged polar amino acid, a positively charged amino acid substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituted for another amino acid with a beta-branched side chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0080] An antibody construct or antigen-binding domain may consist essentially of a particular amino acid sequence(s) as 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.

[0081] In some embodiments, the antibody in the immune complex comprises a modified Fc region, where the modification modulates binding of the Fc region to one or more Fc receptors.

[0082] In some embodiments, an antibody in an immune complex (e.g., an antibody conjugated to at least two adjuvant moieties) comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in an Fc region that result in modulated binding (e.g., increased or decreased 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 lacking the Fc region mutations. In some embodiments, an antibody in an immune complex comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in an Fc region that reduce binding of the Fc region of the antibody to FcγRIIB. In some embodiments, the antibody in the immune complex comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that decrease binding of the antibody to FcγRIIB while maintaining the same or increased binding to FcγRI (CD64), FcγRIIA (CD32A) and / or FcγRIIIA (CD16a) compared to a native antibody lacking the Fc region mutation. In some embodiments, the antibody in the immune complex comprises one or more modifications in the Fc region of the antibody that increase binding of the Fc region of the antibody to FcγRIIB.

[0083] 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 identical to the amino acid sequence of an Fc region found in nature or 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 Fc includes the various allotypes of Fc (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0084] In some embodiments, the mutations in the Fc region that result in modulated binding to one or more Fc receptors are the following mutations: SD(S239D) (SDIE(S239D / I332E), SE(S267E), SELF(S267E / L328F), SDIE(S239D / I332E), SDIEAL(S239D / I332E / A330L), GA(G236A), ALIE(A330L / I332E), GA(G236A), ALIE(A330L / I332E), GA(G236B), ALIE(A330L / I332E), GA(G236C), ALIE(A330L / I332E), GA(G236D ... 32E), 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 to modulate Fc receptor binding are described, for example, in U.S. Patent Nos. 2016 / 0145350 and 7,416,726 and 5,624,821, which are incorporated by reference in their entireties.

[0085] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0086] Human immunoglobulins are glycosylated at the Asn297 residue in the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of the core heptasaccharide N-acetylglucosamine4mannose3 (GlcNAc4Man3). Removal of the heptasaccharide by endoglycosidases or PNGaseF is known to cause conformational changes in the antibody Fc region, which can significantly reduce antibody binding affinity for activating FcγRs and reduce effector functions. 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 demonstrated that α2,6-sialylation enhances anti-inflammatory activity in vivo, and defucosylation improves FcγRIIIa binding, as well as 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.

[0087] In some embodiments, the modification to change the glycosylation pattern is a mutation, such as a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of regulating 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 Application Publication Nos. 2007 / 0014795 and 2008 / 0286819, which are incorporated herein by reference in their entirety.

[0088] In some embodiments, the antibody of the immunoconjugate is modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern. For example, hybridomas can be engineered to secrete defucosylated mAbs, desialylated mAbs or deglycosylated Fc with specific mutations that allow for increased FcRγIIIa binding and effector function. In some embodiments, the antibody of the immunoconjugate is engineered to be defucosylated.

[0089] In some embodiments, the entire Fc region of the antibody in the immune complex is replaced with a different Fc region, so that the Fab region of the antibody is conjugated to a non-natural Fc region. For example, the Fab region of cetuximab, which normally comprises an IgG1 Fc region, can be conjugated to IgG2, IgG3, IgG4 or IgA, or the Fab region of nivolumab, which normally comprises an IgG4 Fc region, can be conjugated to IgG1, IgG2, IgG3, IgA1 or IgG2. In some embodiments, the Fc-modified antibody with a non-natural Fc domain also comprises one or more amino acid modifications, such as the S228P mutation in IgG4 Fc, that modulate the stability of the described Fc domain. In some embodiments, the Fc-modified antibody with a non-natural Fc domain also comprises one or more amino acid modifications, as described herein, that modulate Fc binding to FcR.

[0090] In some embodiments, 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, hi other embodiments, 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.

[0091] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct that comprises an antigen-binding domain that specifically recognizes and binds to PD-L1.

[0092] Programmed cell death ligand 1 (PD-L1, cluster of differentiation 274, CD274, B7-homolog 1, or B7-H1) belongs to the B7 protein superfamily and is a ligand for programmed cell death protein 1 (PD-1, PDCD1, cluster of differentiation 279, or CD279). PD-L1 can also interact with B7.1 (CD80), and such interaction is believed to inhibit T cell priming. The PD-L1 / PD-1 axis plays a major role in suppressing adaptive immune responses. More specifically, binding of PD-L1 to its receptor, PD-1, is believed to result in signals that inhibit T cell activation and proliferation. Agents that bind to PD-L1 and prevent the ligand from binding to the PD-1 receptor can prevent this immune suppression and thus enhance the immune response, as needed, for example, for the treatment of cancer or infectious diseases. The PD-L1 / PD-1 pathway also contributes to the prevention of autoimmunity, therefore agonistic agents against PD-L1, or agents that deliver an immunoinhibitory payload, may be useful in the treatment of autoimmune disorders.

[0093] Several antibodies targeting PD-L1, such as atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™), have been developed for the treatment of cancer. Nevertheless, there remains a need for new PD-L1 binding agents, including agents that bind with high affinity to PD-L1 and effectively inhibit PD-L1 / PD-1 signaling, and agents that can deliver therapeutic payloads to PD-L1-expressing cells. Additionally, there is a need for novel PD-L1 binding agents for the treatment of autoimmune disorders and infections.

[0094] Provided is a method of delivering a TLR agonist 2-amino-4-carboxamido-benzazepine payload to a cell expressing PD-L1, the method comprising administering to the cell or a mammal comprising the cell an immunoconjugate comprising an anti-PD-L1 antibody covalently attached to a linker that is covalently attached to one or more 2-amino-4-carboxamido-benzazepine moieties.

[0095] The invention also provides methods for enhancing or reducing or inhibiting an immune response in a mammal responsive to PD-L1 inhibition, and methods of treating a disease, disorder or condition in a mammal, said methods comprising administering to the mammal a PD-L1 immunoconjugate.

[0096] The present invention provides PD-L1 antibodies comprising an immunoglobulin heavy chain variable region polypeptide and an immunoglobulin light chain variable region polypeptide. The PD-L1 antibodies specifically bind to PD-L1. The binding specificity of the antibodies allows for targeting of cells expressing PD-L1, for example, to deliver a therapeutic payload to such cells. In some embodiments, the PD-L1 antibodies bind to human PD-L1. However, antibodies that bind to any PD-L1 fragment, homolog, or paralog are also encompassed.

[0097] In some embodiments, the PD-L1 antibody binds to PD-L1 without substantially inhibiting or preventing PD-L1 from binding to its receptor, PD-1. However, in other embodiments, the PD-L1 antibody can completely or partially block (inhibit or prevent) the binding of PD-L1 to its receptor, PD-1, and the antibody can be used to inhibit PD-L1 / PD-1 signaling (e.g., for therapeutic purposes). The antibody or antigen-binding antibody fragment can be monospecific for PD-L1, or can be bispecific or multispecific. For example, in a bivalent or multivalent antibody or antibody fragment, the binding domains can be different, targeting different epitopes of the same antigen, or targeting different antigens. Methods for constructing multivalent binding constructs are known in the art. Bispecific and multispecific antibodies are known in the art. Additionally, diabodies, triabodies, or tetrabodies, which are dimers, trimers, or tetramers of polypeptide chains, respectively, can be provided, each comprising a V-terminal domain and a V-terminal domain on the same polypeptide chain. H and V L V due to a peptide linker that is too short to allow pairing between L V connected toH , which allows for different V H -V L It drives pairing between complementary domains on the polypeptide chains to generate multimeric molecules with two, three or four functional antigen binding sites. It is also possible to generate bis-scFv fragments, which are small scFv fragments with two different variable regions, to generate bispecific bis-scFv fragments capable of binding two different epitopes. Fab dimers (Fab2) and Fab trimers (Fab3) can be generated using genetic engineering methods to create multispecific constructs based on Fab fragments.

[0098] A PD-L1 antibody may be or be derived from a human, non-human, humanized or chimeric antibody, or the corresponding antibody fragment. A "chimeric" antibody is typically an antibody or fragment thereof that comprises a human constant region and a non-human variable region. A "humanized" antibody is typically a monoclonal antibody that comprises a human antibody scaffold but contains non-human derived amino acids or sequences in at least one CDR (e.g., one, two, three, four, five or all six CDRs).

[0099] The PD-L1 antibody can be internalizing as described in WO2021 / 150701, which is incorporated herein by reference, or the PD-L1 antibody can be non-internalizing as described in WO2021 / 150702, which is incorporated herein by reference.

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

[0101] Several anti-HER2 monoclonal antibodies have been approved and are in clinical development (Costa, RLB et al (2020) Breast Cancer 6 (10): 1-11).

[0102] In a particular embodiment, the immunoconjugate of the invention comprises an anti-HER2 antibody, such as that prepared by the method of Example 201. In one embodiment of the invention, the anti-HER2 antibody of the immunoconjugate of the invention comprises a humanized anti-HER2 antibody, such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8, as described in Table 3 of U.S. Pat. No. 5,821,337, which is specifically incorporated herein by reference. These antibodies include human framework regions with the complementarity determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also known as trastuzumab and is commercially available under the trade name HERCEPTIN™ (Genentech, Inc.).

[0103] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1κ, monoclonal antibody that is a humanized version of the murine anti-HER2 antibody (4D5) that selectively binds with high affinity (Kd=5 nM) to the extracellular domain of HER2 in cell-based assays (U.S. Pat. Nos. 5,677,171, 5,821,337, 6,054,297, 6,165,464, 6,339,142, 6,407,213, 6,639,055, 6,719,971, 6,800,738, 7,074,404; Coussens et al (1985) Science 230:1132-9; Slamon et al. (1986) al(1989)Science244:707-12, Slamon et al(2001)New Engl.J.Med.344:783-792).

[0104] In one embodiment of the invention, the antibody construct or antigen binding domain comprises the CDR regions of trastuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises the framework regions of trastuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0105] In another embodiment of the invention, the anti-HER2 antibody of the immunoconjugate of the invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US Patent No. 7,862,817. An exemplary humanized 2C4 antibody is pertuzumab (CAS Registry Number 380610-27-5), PERJETA™ (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI), which functions by inhibiting the ability of the antibody to form active heterodimers or homodimers with other HER receptors (e.g., EGFR / HER1, HER2, HER3 and HER4). See, e.g., Harari and Yarden Oncogene 19:6102-14 (2000), Yarden and Sliwkowski. Nat Rev Mol Cell Biol 2:127-37 (2001), Sliwkowski Nat Struct Biol 10:158-9 (2003), Cho et al. Nature 421:756-60 (2003), and Malik et al. Pro Am Soc Cancer Res 44:176-7 (2003). PERJETA™ is approved for the treatment of breast cancer.

[0106] In one embodiment of the invention, the antibody construct or antigen binding domain comprises the CDR regions of pertuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises the framework regions of pertuzumab. In one embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of pertuzumab.

[0107] Margetuximab (MGAH22, MARGENZA™, Macrogenics, Inc.), CAS Registry No. 1350624-75-7, is an FDA-approved anti-HER2 monoclonal antibody. The Fc region of margetuximab has been optimized to increase binding to activating FcγRs but decrease binding to inhibitory Fc.γ.Rs on immune effector cells (Nordstrom, JL, et al (2011) Breast Cancer Res. 13(6):R123; Rugo, HS, et al (2021) JAMA Oncol.; 7(4):573-584; Markham, A. (2021) Drugs 81:599-604). Margetuximab is approved by the FDA for the treatment of patients with recurrent or refractory advanced breast cancer whose tumors express HER2 at the 2+ level by immunohistochemistry and without evidence of HER2 gene amplification by FISH.

[0108] HT-19 is another anti-HER2 monoclonal antibody that binds to an epitope on human HER2 that is distinct from that of trastuzumab or pertuzumab. HT-19 has been shown to inhibit HER2 signaling comparable to trastuzumab and to enhance HER2 degradation in combination with trastuzumab and pertuzumab. XMT-1522 is an antibody-drug conjugate that contains the HT-19 antibody (Bergstrom DA et al., (2015) Cancer Res.; 75: LB-231).

[0109] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds CEA. Carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), also known as CD66e (cluster of differentiation 66e), is a member of the carcinoembryonic antigen (CEA) gene family.

[0110] Increased expression of carcinoembryonic antigen (CEA, CD66e, CEACAM5) is associated with various biological aspects of tumors, especially tumor cell adhesion, metastasis, blocking cellular immune mechanisms, and having anti-apoptotic functions. 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 has been studied in patients with recurrent or refractory metastatic colorectal cancer (Sharkey, R. et al (2018), Molecular Cancer Therapeutics 17 (1): 196-203; Dotan, E. et al (2017), Journal of Clinical Oncology 35 (9): 3338-3346). Also, 131 Labetuzumab conjugated to I has been evaluated in clinical trials for the treatment of colorectal 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).

[0111] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VLκ) of hMN-14 / labetuzumab SEQ ID NO:1, as disclosed in U.S. Pat. No. 6,676,924, which is incorporated herein by reference for this purpose. DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK SEQ ID NO: 1

[0112] 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 hMN-14 / labetuzumab SEQ ID NOs:2-8 (U.S. Pat. No. 6,676,924) (full length sequence disclosed as SEQ ID NO:1). [Table 1]

[0113] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable heavy chain (VH) of hMN-14 / labetuzumab SEQ ID NO:9, as disclosed in U.S. Pat. No. 6,676,924, which is incorporated herein by reference for this purpose. EVQLVESGGGVVQPGRSLRLSCSSSGFDFTTYWMSWVRQAPGKGLEWVAEIHPDSSTINYAPSLKDRFTISRDNSKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS SEQ ID NO: 9

[0114] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hMN-14 / labetuzumab SEQ ID NO: 10-16 (U.S. Patent No. 6,676,924) (full length sequence disclosed as SEQ ID NO: 9). [Table 2]

[0115] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VLκ) of hPR1A3 SEQ ID NO:17, as disclosed in U.S. Pat. No. 8,642,742, which is incorporated herein by reference for this purpose. DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIK SEQ ID NO: 17

[0116] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of hPR1A3 SEQ ID NO: 18-24 (U.S. Patent No. 8,642,742) (full length sequence disclosed as SEQ ID NO: 17). [Table 3]

[0117] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence of hPR1A3 SEQ ID NO:25-31 (U.S. Patent No. 8,642,742) (full length sequence disclosed as SEQ ID NO:130). [Table 4]

[0118] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of hMFE-23 SEQ ID NO: 32, as disclosed in U.S. Pat. No. 7,232,888, which is incorporated herein by reference for this purpose. ENVLTQSPSSMSASVGDRVNIACSASSSVSYMHWFQQKPGKSPKLWIYSTSNLASGVPSRFSGSGSGTDYSLTISSMQPEDAATYYCQQRSSYPLTFGGGTKLEIK SEQ ID NO:32

[0119] In one embodiment of the invention, a 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 SEQ ID NOs: 33-40 (US Pat. No. 7,232,888). Said embodiment includes two variants of LFR1, SEQ ID NO: 33 and SEQ ID NO: 34 (full length sequences disclosed as SEQ ID NO: 32 and SEQ ID NO: 172, respectively, in apparent order). [Table 5]

[0120] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of hMFE-23 SEQ ID NO:41 (US Pat. No. 7,232,888). QVKLEQSGAEVVKPGASVKLSCKASGFNIKDSYMHWLRQGPGQRLEWIGWIDPENGDTEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYYCNEGTPTGPYYFDYWGQGTLVTVSS SEQ ID NO: 41

[0121] In one embodiment of the invention, a CEA-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of hMFE-23 SEQ ID NOs: 42-49 (US Pat. No. 7,232,888). Said embodiment includes two variants of HFR1, SEQ ID NO: 42 and SEQ ID NO: 43 (full-length sequences disclosed as SEQ ID NO: 41 and SEQ ID NO: 173, respectively, in apparent order). [Table 6]

[0122] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of SM3E SEQ ID NO:50 (US Pat. No. 7,232,888). ENVLTQSPSSMSVSVGDRVTIACSASSSVPYMHWLQQKPGKSPKLLIYLTSNLASGVPSRFSGSGSGTDYSLTISSVQPEDAATYYCQQRSSYPLTFGGGTKLEIK SEQ ID NO:50

[0123] In one embodiment of the invention, a CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequences of SM3E SEQ ID NOs: 51-56 and 38-39 (US Pat. No. 7,232,888). Said embodiment includes two variants of LFR1, SEQ ID NO: 51 and SEQ ID NO: 52 (full length sequences disclosed as SEQ ID NO: 50 and SEQ ID NO: 174, respectively, in apparent order). [Table 7]

[0124] 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

[0125] 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 NP-4 / arcitumomab SEQ ID NO:58-64 (full length sequence disclosed as SEQ ID NO:57). [Table 8]

[0126] 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

[0127] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of NP-4 SEQ ID NO: 66-72 (full length sequence disclosed as SEQ ID NO: 65). [Table 9]

[0128] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the variable light chain (VLκ) of M5A / hT84.66 SEQ ID NO:73, as disclosed in U.S. Pat. No. 7,776,330, which is incorporated herein by reference for this purpose. DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIK SEQ ID NO: 73

[0129] 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) sequence of M5A / hT84.66 SEQ ID NO:74-80 (U.S. Patent No. 7,776,330) (full length sequence disclosed as SEQ ID NO:73). [Table 10]

[0130] 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 SEQ ID NO:81 (US Pat. No. 7,776,330). EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSS SEQ ID NO: 81

[0131] 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) sequence of M5A / hT84.66 SEQ ID NO: 82-88 (U.S. Patent No. 7,776,330) (full length sequence disclosed as SEQ ID NO: 81). [Table 11]

[0132] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VLκ) of hAb2-3 SEQ ID NO:89, as disclosed in U.S. Pat. No. 9,617,345, which is incorporated herein by reference for this purpose. DIQMTQSPASLSASVGDRVTITCRASENIFSYLAWYQQKPGKSPKLLVYNTRTLAEGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQHHYGTPFTFGSGTKLEIK SEQ ID NO: 89

[0133] 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) sequence of hAb2-3 SEQ ID NO:90-96 (U.S. Pat. No. 9,617,345) (full length sequence disclosed as SEQ ID NO:89). [Table 12]

[0134] 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 (US Pat. No. 9,617,345). EVQLQESGPGLVKPGGSLSLSCAASGFVFSSYDMSWVRQTPERGLEWVAYISSGGGITYAPSTVKGRFTVSRDNAKNTLYLQMNSLTSEDTAVYYCAAHYFGSSGPFAYWGQGTLVTVSS SEQ ID NO: 97

[0135] 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 hAb2-3 SEQ ID NO: 98-104 (full length sequence disclosed as SEQ ID NO: 97). [Table 13]

[0136] In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises a variable light chain (VLκ) of A240VL-B9VH / AMG-211 SEQ ID NO: 105, as disclosed in U.S. Pat. No. 9,982,063, which is incorporated herein by reference for this purpose. QAVLTQPASLSASPGASASLTCTLRRGINVGAYSIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVSSRFSASKDASANAGILLISGLQSEDEADYYCMIWHSGASAVFGGGTKLTVL SEQ ID NO: 105

[0137] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) or light chain framework (LFR) sequence of A240VL-B9VH / AMG-211 SEQ ID NO:106-112 (U.S. Pat. No. 9,982,063) (full length sequence disclosed as SEQ ID NO:105). [Table 14]

[0138] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of B9VH SEQ ID NO:113 (US Pat. No. 9,982,063). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFIRNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS SEQ ID NO: 113

[0139] 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 SEQ ID NOs: 114-121 (US Pat. No. 9,982,063). Said embodiment includes CDR-H2, two variants of SEQ ID NO: 117 and SEQ ID NO: 118 (full length sequences disclosed as SEQ ID NO: 113 and SEQ ID NO: 175, respectively, in apparent order). [Table 15]

[0140] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of E12VH SEQ ID NO: 122 (US Pat. No. 9,982,063). EVQLVESGGGLVQPGRSLRLSCAASGFTVSSYWMHWVRQAPGKGLEWVGFILNKANGGTTEYAASVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGLRFYFDYWGQGTTVTVSS SEQ ID NO: 122

[0141] In one embodiment of the present invention, the CEA targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequences of SEQ ID NOs: 123-129 (U.S. Pat. No. 9,982,063) (full length sequence disclosed as SEQ ID NO: 122). [Table 16]

[0142] In one embodiment of the invention, the CEA targeting antibody construct or antigen binding domain comprises the variable heavy chain (VH) of PR1A3 VH SEQ ID NO: 130 (US Pat. No. 8,642,742). QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSS SEQ ID NO: 130

[0143] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct that comprises an antigen-binding domain that specifically recognizes and binds TROP2.

[0144] Tumor-associated calcium signaling factor 2 (TROP2) is a transmembrane glycoprotein encoded by the TACSTD2 gene (Linnenbach AJ, et al. (1993) Mol Cell Biol. 13(3): 1507-15; Calabrese G, et al (2001) Cytogenet Cell Genet. 92(1-2): 164-5). It is an intracellular calcium signaling factor that is differentially expressed in many cancers. It signals cells for self-renewal, proliferation, invasion, and survival. It has properties similar to stem cells. TROP2 is expressed in many normal tissues, but in contrast, it is overexpressed in many cancers (Ohmachi T,et al.,(2006)Clin.Cancer Res.,12(10),3057-3063;Muhlmann G,et al.,(2009)J.Clin.Pathol.,62(2),152-158;Fong D,et al.,(2008)Br. J.Cancer,99(8),1290-1295;Fong D,et al.,(2008)Mod. Pathol.,21(2),186-191;Ning S,et al.,(2013)Neurol. Sci.,34(10),1745-1750). TROP2 overexpression has prognostic significance. Several ligands that interact with TROP2 have been proposed. TROP2 signals to cells through different pathways, which are transcriptionally regulated by a complex network of several transcription factors.

[0145] Human TROP2 (TACSTD2: tumor-associated calcium signaling factor 2, GA733-1, EGP-1, M1S1; hereafter referred to as hTROP2) is a single-pass transmembrane type 1 cell membrane protein consisting of 323 amino acid residues. On the other hand, the existence of a cell membrane protein involved in immune resistance (common to human trophoblasts and cancer cells) was previously suggested (Faulk WP, et al. (1978), Proc. Natl. Acad. Sci. 75 (4): 1947-1951), and an antigen molecule recognized by a monoclonal antibody against a cell membrane protein in a human choriocarcinoma cell line was identified and named TROP2 as one of the molecules expressed in human trophoblasts (Lipinski M, et al. (1981), Proc. Natl. Acad. Sci. 78 (8), 5147-5150). This molecule is also called tumor antigen GA733-1, recognized by mouse monoclonal antibody GA733 (Linnenbach AJ, et al., (1989) Proc. Natl. Acad. Sci. 86(1), 27-31), obtained by immunization with gastric cancer cell lines, or epithelial glycoprotein (EGP-1; Basu A, et al., Int. J. Cancer, 62(4), 472-479(1995)), recognized by mouse monoclonal antibody RS7-3G11, obtained by immunization with non-small cell lung cancer cells. However, in 1995, the cloning of the TROP2 gene confirmed that all these molecules are the same molecule (Fornaro M, et al., (1995) Int. J. Cancer, 62(5), 610-618). The DNA and amino acid sequences of hTROP2 are published in public databases and can be found, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0146] In response to such information suggesting a relationship with cancer, several anti-hTROP2 antibodies have been established and their antitumor effects have been investigated. Among these antibodies, for example, non-conjugated antibodies that show antitumor activity by themselves in nude mouse xenograft models (WO2008 / 144891; WO2011 / 145744; WO2011 / 155579; WO2013 / 077458) and antibodies that show antitumor activity as ADCs with cytotoxic drugs (WO2003 / 074566; WO2011 / 068845; WO2013 / 068946; U.S. Patent No. 7,999,083) are disclosed. However, the strength or scope of their activity is still insufficient, and medical needs for therapeutic targeting of hTROP2 are not met.

[0147] TROP2 expression in cancer cells correlates with drug resistance. Several strategies have targeted TROP2 on cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, etc. In vitro and preclinical studies using these various therapeutic treatments significantly inhibited tumor cell growth both in vitro and in vivo in mice. Clinical studies have explored the application of Trop2 as a prognostic biomarker and as a potential therapeutic target for reverse resistance.

[0148] Sacituzumab govitecan (TRODELVY®, Immunomedics, IMMU-132), an antibody-drug conjugate comprising a TROP2-directed antibody linked to a topoisomerase inhibitor, is indicated for the treatment of metastatic triple-negative breast cancer (mTNBC) in adult patients who have received at least two prior therapies. The TROP2 antibody in sacituzumab govitecan is conjugated to SN-38, an active metabolite of irinotecan (US 2016 / 0297890; WO 2015 / 098099).

[0149] In one embodiment of the invention, the TROP2 targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) sequence of hRS7 (humanized RS7), SEQ ID NOs: 131-133 (U.S. Patent No. 7,238,785; U.S. Patent No. 7,420,040, incorporated herein by reference). [Table 17]

[0150] In one embodiment of the present invention, the TROP2-targeting antibody construct or antigen-binding domain comprises the heavy chain CDR (complementarity determining region) sequences of hRS7 (humanized RS7), SEQ ID NOs: 134-136 (U.S. Pat. Nos. 7,238,785; 9,797,907; 9,382,329; WO2020 / 142659, each of which is incorporated herein by reference). [Table 18]

[0151] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) sequences of AR47A6.4.2, SEQ ID NOs: 134, 137, 138 (U.S. Patent No. 7,420,040, incorporated herein by reference). [Table 19]

[0152] In one embodiment of the invention, the TROP2 targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) sequences of humanized KM4097, SEQ ID NOs: 139-141 (US 2012 / 0237518, incorporated herein by reference). [Table 20]

[0153] In one embodiment of the invention, the TROP2 targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) sequences of humanized KM4097, SEQ ID NOs: 142-144 (US 2012 / 0237518, incorporated herein by reference). [Table 21]

[0154] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) sequence of hTINA1-H1L1, SEQ ID NOs: 132, 133, 145 (U.S. Pat. No. 10,227,417, incorporated herein by reference). [Table 22]

[0155] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) sequence of hTINA1-H1L1, SEQ ID NOs: 146-148 (U.S. Pat. No. 10,227,417, incorporated herein by reference). [Table 23]

[0156] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) sequence of hTINA1-H1L1, SEQ ID NOs: 149-151 (U.S. Patent No. 8,871,908, incorporated herein by reference). [Table 24]

[0157] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) sequence of hTINA1-H1L1, SEQ ID NOs: 152-157 (U.S. Patent No. 8,871,908, incorporated herein by reference). [Table 25]

[0158] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the light chain CDR (complementarity determining region) sequences of SEQ ID NOs: 150, 151, 158 of hTINA1-H1L1 (U.S. Pat. No. 8,871,908, incorporated herein by reference). [Table 26]

[0159] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the heavy chain CDR (complementarity determining region) sequences of SEQ ID NOs: 152-154, 157, 159, 160 of hTINA1-H1L1 (U.S. Patent No. 8,871,908, incorporated herein by reference). [Table 27]

[0160] In one embodiment of the present invention, the immune complex comprises a cysteine ​​mutated antibody having a light chain sequence selected from SEQ ID NOs: 161-163. [Table 28]

[0161] In one embodiment of the invention, the cysteine ​​mutated, TROP2 targeting antibody comprises a heavy chain (HC) of SEQ ID NO:164. QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 164

[0162] In one embodiment of the invention, the light chain (LC) of the TROP2 targeting antibody is selected from SEQ ID NOs: 165-167. [Table 29]

[0163] In one embodiment of the invention, the immunoconjugate comprises a cysteine ​​mutated antibody having the heavy chain sequence of SEQ ID NO:168. [Table 30]

[0164] In one embodiment of the invention, the light chain (LC) of the cysteine ​​mutated TROP2 targeting antibody has the sequence of SEQ ID NO:169. DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 169

[0165] In one embodiment of the invention, the heavy chain (HC) of the cysteine ​​mutated TROP2 targeting antibody has the sequence of SEQ ID NO: 170. QVQLVQSGAEVKKPGASVKVSCKASGDTFTNHYMHWVRQAPGQGLEWMGWINPNSGHTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAVAGPMDVWGQGT TVTVSSACTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 170

[0166] In some embodiments, the antibody construct further comprises an Fc domain. In certain embodiments, the antibody construct is an antibody. In certain embodiments, the antibody construct is a fusion protein. The antigen-binding domain may be a single chain variable fragment (scFv). A single chain variable fragment (scFv) is a truncated Fab fragment comprising the variable (V) domain of an antibody heavy chain linked to the 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 can comprise one or more variable regions (e.g., two variable regions) of an antigen-binding domain of an anti-PD-L1 antibody, an anti-HER2 antibody, or an anti-CEA antibody, each variable region comprising CDR1, CDR2, and CDR3. In some embodiments, the antibody in the immune complex comprises a modified Fc region, where the modification modulates binding of the Fc region to one or more Fc receptors.

[0167] In some embodiments, the Fc region is modified by including a transforming growth factor beta 1 (TGFβ1) receptor or a fragment thereof capable of binding to TGFβ1. For example, the receptor can be TGFβ receptor II (TGFβRII). In some embodiments, the TGFβ receptor is a human TGFβ receptor. In some embodiments, the IgG has a C-terminal fusion to the TGFβRII extracellular domain (ECD), as described in U.S. Pat. No. 9,676,863, which is incorporated herein. An "Fc linker" can be used to link the IgG to the TGFβRII extracellular domain, e.g., G4S4G Fc linker (SEQ ID NO: 171). The Fc linker can be a short, flexible peptide that allows for proper three-dimensional folding of the molecule while maintaining binding specificity to the target. In some embodiments, the N-terminus of the TGFβ receptor is fused to the Fc of the antibody construct (with or without an Fc linker). In some embodiments, the C-terminus of the antibody construct heavy chain is fused to the TGFβ receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the antibody construct heavy chain is mutated to alanine.

[0168] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0169] In some embodiments, the antibody of the immunoconjugate is a cysteine ​​engineered antibody that provides site-specific conjugation of adjuvants to the antibody through cysteine ​​substitution at sites where the engineered cysteine ​​is available for conjugation, but does not perturb immunoglobulin folding and assembly or alter antigen binding and effector function (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; U.S. Patent Nos. 7,521,541; 7,723,485; 2012 / 0121615; WO 2009 / 052249). A "cysteine ​​engineered antibody" or "cysteine ​​engineered antibody variant" is an antibody in which one or more residues of an antibody are replaced with a cysteine ​​residue. Cysteine ​​engineered antibodies can be conjugated to thiol-reactive electrophilic groups, such as maleimides, on a 2-amino-4-carboxamido-benzazepine linker compound (Formula II) with uniform stoichiometry (e.g., up to two 2-amino-4-carboxamido-benzazepine moieties per antibody in antibodies with a single engineered cysteine ​​site).

[0170] In some embodiments, cysteine ​​engineered antibodies are used to prepare immunoconjugates. The immunoconjugates may have a reactive cysteine ​​thiol residue introduced at a site on the light chain, such as the 149-lysine site (LC K149C), or at a site on the heavy chain, such as the 122-serine site (HC S122C), as numbered by Kabat numbering. 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 SEQ ID NO: system and 114 in the Kabat numbering. In other embodiments, the cysteine ​​engineered antibody has cysteine ​​residues introduced into (i) the light chain at G64C, R142C, K188C, L201C, T129C, S114C or E105C according to the Kabat numbering, (ii) the heavy chain at D101C, V184C, T205C or 122C according to the Kabat numbering, or (iii) other cysteine ​​mutated antibodies and as described in Bhakta, S. et al., (2013) "Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers", Laurent Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular Biology, vol. 1045, pages 189-203; WO 2011 / 156328; U.S. Patent No. 9,000,130.

[0171] 2-Amino-4-carboxamido-benzazepine adjuvant compounds The immunoconjugates of the present invention include a 2-amino-4-carboxamido-benzazepine adjuvant moiety. The adjuvant moieties described herein are compounds that induce an immune response (i.e., immune stimulants). Generally, the adjuvant moieties described herein are TLR agonists. TLRs are type I transmembrane proteins involved in the initiation of innate immune responses in vertebrates. TLRs recognize 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. When activated (e.g., by natural stimuli or synthetic TLR agonists), TLRs initiate a signaling cascade that leads to the activation of nuclear factor kappa-B (NF-κB) through the adaptor protein myeloid differentiation primary response gene 88 (MyD88), and the recruitment of IL-1 receptor-associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF receptor-associated factor 6 (TRAF6), which results in phosphorylation of the NF-κB inhibitor I-κB. As a result, NF-κB enters the cell nucleus and initiates the transcription of genes whose promoters contain NF-κB binding sites, such as cytokines. Additional regulatory modes of TLR signaling include TIR domain-containing adaptor-inducing interferon-β (TRIF)-dependent induction of TNF receptor-associated factor 6 (TRAF6), as well as activation of MyD88-independent pathways via TRIF and TRAF3, leading to phosphorylation of interferon response factor 3 (IRF3). Similarly, the MyD88-dependent pathway also activates several IRF family members, including IRF and IRF7, and the TRIF-dependent pathway also activates the NF-κB pathway.

[0172] Typically, 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. An exemplary compound of the invention (2Am4CBza) is shown in Table 1. Each compound was characterized by mass spectrometry and shown to have the mass indicated. Activity against HEK293NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 203. [Table 31]

[0173] 2-Amino-4-carboxamido-benzazepine linker compounds The immunoconjugates of the present invention are prepared by conjugation of an antibody with a 2-amino-4-carboxamido-benzazepine linker compound. The 2-amino-4-carboxamido-benzazepine linker compound comprises a 2-amino-4-carboxamido-benzazepine (2Am4CBza) moiety covalently attached to a linker unit L and a reactive electrophilic group Q. 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 a 2Am4CBza linker compound to form an immunoconjugate (IC). Also, for example, a cysteine ​​thiol of an antibody reacts with a maleimide or bromoacetamide group of a 2Am4CBza linker compound to form an immunoconjugate.

[0174] Suitable reactive electrophilic functional groups for the 2Am4CBza linker compounds (Q in formula II) 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) or sulfotetrafluorophenyl (SulfoTFP) esters (amine reactive); imide esters (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.

[0175] 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 may release unacceptable amounts of adjuvant / drug before 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 usually have poor stability in the bloodstream. Alternatively, bloodstream stability and intracellular release are usually 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 correlated. For example, aggregate formation is generally positively correlated to 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 the immunoconjugate and make the process difficult to scale up.

[0176] An exemplary embodiment of a 2-amino-4-carboxamido-benzazepine linker compound has formula II: [ka] Including, X 2 and X 3 are each 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 1a , R 1b and R 2 H, C1~C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6~C 20Aryl, C2-C9 heterocyclyl, and C1-C 20 Heteroaryl; R 3 teeth, -(C1~C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-C(=O)*; -(C1~C 12 Alkyldiyl)-N(R 5 )-C(=O)O-(C3~C 12 Carbocyclyldiyl)-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-(C1~C 20 Heteroaryldiyl)-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-(C1~C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-S(O2)-*; -(C1~C 12 Alkyldiyl)-OC(=O)-(C2-C9 heterocyclyldiyl)-*; -(C1~C 12 Alkyldiyl)-O-*; -(C1~C 12 Alkyldiyl)-(C3~C 12 Carbocyclyldiyl)-*; -(C1~C 12 Alkyldiyl)-(C6~C 20 Alkyldiyl)-*; -(C1~C 12 Alkyldiyl)-(C6~C 20 Aryl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C6~C20 Aryl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C2-C9 heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C3~C 12 Carbocyclyldiyl)-*; -(C3~C 12 Carbocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C3~C 12 Carbocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C3~C 12 Carbocyclyldiyl)-NR 5 -C(=NR 5a )-N(R 5 )-*; -(C6~C 20 Aryldiyl)-*; -(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 )-C(=NR 5a )-N(R 5 )-*; -(C2~C 20 Heterocyclyldiyl)-*; -(C2-C9 heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C2-C9 heterocyclyldiyl)-N(R 5 )-C(=NR 5a )-N(R 5 )-*; -(C1~C 20 Heteroaryldiyl)-*; -(C1~C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 20 Heteroaryldiyl)-(C1-C 12 alkyldiyl)-O-*; and -(C1~C 20 Heteroaryldiyl)-N(R 5 )-C(=NR 5a )-N(R 5 )-*, The asterisk * indicates the attachment site of the linker L. R 5 H, C6~C 20 Aryl and C1-C 12 alkyl, or two R 5together form a 5- or 6-membered heterocyclyl ring, R 5a is C6~C 20 Aryl and C1-C 20 Heteroaryl; LQ 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)-; 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] wherein AA is independently selected from a natural or unnatural amino acid side chain, or AA and one or more of the adjacent nitrogen atoms form a 5-membered proline amino acid ring, and the wavy line indicates the point of attachment; Cyc is an element that contains F, Cl, NO2, -OH-, OCH3 and the structure: [ka] Glucuronic acid having C6-C, optionally substituted with one or more selected groups 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 to 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 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 F, Cl, NO2 and SO3 - is selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, substituted with one or more groups independently selected from 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, -CH2CH2C H3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH , -CH(OH)CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(C H3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -C OCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COC H3, -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) n Substituted with one or more groups independently selected from H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0177] An exemplary embodiment of the 2-amino-4-carboxamido-benzazepine linker compound of formula II is a and R1b Optionally substituted C6 to C 20 Aryl, C2-C9 heterocyclyl and C1-C 20 heteroaryl.

[0178] In an exemplary embodiment of the 2-amino-4-carboxamido-benzazepine linker compound of formula II, R 1a Optionally substituted C6-C 20 aryl, R 1b This includes H.

[0179] In an exemplary embodiment of the 2-amino-4-carboxamido-benzazepine linker compound of formula II, X 2 and X 3 is a bond, and R 2 and R 3 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 ) independently selected from 2.

[0180] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compound of formula II include X 2 is a bond and R 2 C1~C 12 It includes being alkyl.

[0181] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compound of formula II include X 3 is O and R3 -(C1~C 12 Alkyldiyl)-N(R 5 )-*.

[0182] An exemplary embodiment of the 2-amino-4-carboxamido-benzazepine linker compound of formula II is 3 is -CH2CH2CH2NH-.

[0183] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compound of formula II include where L is -C(=O)-PEG-C(=O)-.

[0184] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compounds of formula II include those in which AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0185] An exemplary embodiment of a 2-amino-4-carboxamido-benzazepine linker compound of formula II includes AA1 or AA2 with adjacent nitrogen atoms forming a 5-membered ring proline amino acid.

[0186] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compound of formula II include those where AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(CH6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2.

[0187] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compound of formula II include where AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0188] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compounds of formula II are those in which Q is [ka] The present invention includes being selected from the following:

[0189] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compounds of formula II include where Q is phenoxy substituted with one or more F.

[0190] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compounds of formula II include where Q is 2,3,5,6-tetrafluorophenoxy.

[0191] Exemplary embodiments of the 2-amino-4-carboxamido-benzazepine linker compounds of formula II include where Q is 2,3,5,6-tetrafluoro,4-sulfonate-phenoxy.

[0192] An exemplary embodiment of the 2-amino-4-carboxamido-benzazepine linker compound of formula II has formula IIa: [ka]

[0193] Exemplary embodiments of 2-amino-4-carboxamido-benzazepine linker compounds are selected from Table 2. Each compound was characterized by mass spectrometry and shown to have the mass indicated. [Table 32] [Table 33-1] [Table 33-2] [Table 33-3] [Table 33-4] [Table 33-5] [Table 33-6] [Table 33-7] [Table 33-8]

[0194] immune complex An exemplary embodiment of the immunoconjugate is one or more 2-amino-4-carboxamido-benzazepine (2Am4CBza) moieties covalently attached by a linker and represented by formula I: [ka] or a pharma- ceutically acceptable salt thereof, Ab is an antibody that binds to a target selected from the group consisting of PD-L1, HER2, CEA, or TROP2; p is an integer from 1 to 8; X 2 and X 3 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 1a , R 1b and R 2 are independently H, C1 to 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, or R 1a and R 1bforms a 5- or 6-membered heterocyclyl ring, R 3 teeth, -(C1~C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-C(=O)*; -(C1~C 12 Alkyldiyl)-N(R 5 )-C(=O)O-(C3~C 12 Carbocyclyldiyl)-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-(C1~C 20 Heteroaryldiyl)-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-(C1~C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-*; -(C1~C 12 Alkyldiyl)-N(R 5 )-S(O2)-*; -(C1~C 12 Alkyldiyl)-OC(=O)-(C2-C9 heterocyclyldiyl)-*; -(C1~C 12 Alkyldiyl)-O-*; -(C1~C 12 Alkyldiyl)-(C3~C 12 Carbocyclyldiyl)-*; -(C1~C 12 Alkyldiyl)-(C6~C 20 Alkyldiyl)-*; -(C1~C 12 Alkyldiyl)-(C6~C 20 Aryl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C2-C9 heterocyclyldiyl)-(C1-C 12Alkyldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-N(R 5 )-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-*; -(C1~C 12 Alkyldiyl)-(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C3~C 12 Carbocyclyldiyl)-*; -(C3~C 12 Carbocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C3~C 12 Carbocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C3~C 12 Carbocyclyldiyl)-NR 5 -C(=NR 5a )-N(R 5 )-*; -(C6~C 20 Aryldiyl)-*; -(C6~C 20 Aryldiyl)-N(R 5 )-*; -(C6~C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C6~C 20 Aryldiyl)-(C1-C 12Alkyldiyl)-(C2-C 20 Heterocyclyldiyl)-*; -(C6~C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-C(=NR 5a )-N(R 5 )-*; -(C2~C 20 Heterocyclyldiyl)-*; -(C2-C9 heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C2-C9 heterocyclyldiyl)-N(R 5 )-C(=NR 5a )-N(R 5 )-*; -(C1~C 20 Heteroaryldiyl)-*; -(C1~C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*; -(C1~C 20 Heteroaryldiyl)-(C1-C 12 alkyldiyl)-O-*; and -(C1~C 20 Heteroaryldiyl)-N(R 5 )-C(=NR 5a )-N(R 5 )-*, The asterisk * indicates the attachment site of the linker L. Or, R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring, 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 R5 the groups taken together form a 5- or 6-membered heterocyclyl ring; R 5a is C6~C 20 Aryl and C1-C 20 Heteroaryl; 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(R 6 )-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)-; 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] wherein AA is independently selected from a natural or unnatural amino acid side chain, or AA and one or more of the adjacent nitrogen atoms form a 5-membered proline amino acid ring, and the wavy line indicates the point of attachment; Cyc is an element that contains F, Cl, NO2, -OH-, OCH3 and the structure: [ka] Optionally substituted with one or more groups selected from glucuronic acid having the formula: 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 to 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 taken together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1, 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, -CH2CH2C H3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH , -CH(OH)CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(C H3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -C OCH(OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COC H3, -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) n Substituted with one or more groups independently selected from H, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0195] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to PD-L1.

[0196] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is selected from the group consisting of atezolizumab, durvalumab and avelumab, or a biosimilar or biobetter thereof.

[0197] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to HER2.

[0198] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is selected from the group consisting of trastuzumab and pertuzumab, or a biosimilar or biobetter thereof.

[0199] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to CEA.

[0200] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is labetuzumab, or a biosimilar or biobetter thereof.

[0201] Exemplary embodiments of the immunoconjugate of Formula I include those in which the antibody is an antibody construct having an antigen-binding domain that binds to TROP2.

[0202] An exemplary embodiment of the immunoconjugate of Formula I includes where the Trop2 antibody is a monoclonal antibody.

[0203] An exemplary embodiment of the immunoconjugate of formula I is 1a and R 1b Optionally substituted C6 to C 20 Aryl, C2-C9 heterocyclyl and C1-C 20 heteroaryl.

[0204] In an exemplary embodiment of the immunoconjugate of Formula I, R 1aOptionally substituted C6-C 20 aryl, R 1b This includes H.

[0205] In an exemplary embodiment of the immunoconjugate of formula I, X 2 and X 3 is a bond, and R 2 and R 3 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 ) independently selected from 2.

[0206] Exemplary embodiments of the immunoconjugate of formula I include X 2 is a bond and R 2 C1~C 12 It includes being alkyl.

[0207] Exemplary embodiments of the immunoconjugate of formula I include X 3 is O and R 3 -(C1~C 12 Alkyldiyl)-N(R 5 )-*.

[0208] An exemplary embodiment of the immunoconjugate of formula I is 3 is -CH2CH2CH2NH-.

[0209] Exemplary embodiments of the immunoconjugate of Formula I include where L is -C(=O)-PEG-C(=O)-.

[0210] Exemplary embodiments of the immunoconjugate of formula I include those in which L comprises PEG, n is 10, and m is 1.

[0211] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0212] An exemplary embodiment of the immunoconjugate of Formula I includes AA1 or AA2 with adjacent nitrogen atoms forming a five-membered ring proline amino acid.

[0213] Exemplary embodiments of the immunoconjugate of formula I include those in which AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2.

[0214] Exemplary embodiments of the immunoconjugate of formula I include where AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0215] An exemplary embodiment of the immunoconjugate has Formula Ia: [ka]

[0216] An exemplary embodiment of the immunoconjugate of formula Ia is 1a Optionally substituted C6 to C 20 Aryl, C2-C9 heterocyclyl, and C1-C 20 Heteroaryl is one of the groups selected from the group consisting of aryl, aryl, and heteroaryl.

[0217] An exemplary embodiment of the immunoconjugate of formula Ia is 1a is pyrimidinyl or pyridyl.

[0218] An exemplary embodiment of the immunoconjugate of formula Ia is2 is a bond and R 2 C1~C 12 It includes being alkyl.

[0219] An exemplary embodiment of the immunoconjugate of formula Ia is 3 -(C1~C 12 Alkyldiyl)-N(R 5 )-*.

[0220] An exemplary embodiment of the immunoconjugate of formula Ia is 3 -R 3 -L, [ka] wherein the wavy line indicates the point of attachment to the N.

[0221] The present invention includes all reasonable and operable combinations and permutations of features of the embodiments of Formula I.

[0222] In certain embodiments, the immunoconjugate compounds of the present invention include those with immune stimulatory activity. The antibody-drug conjugates of the present invention can selectively deliver an effective dose of a 2-amino-4-carboxamido-benzazepine drug to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated 2-amino-4-carboxamido-benzazepine.

[0223] Drug loading is represented by p, the number of 2Am4CBza moieties per antibody in the immunoconjugate of formula I. Drug (2Am4CBza) loading can range from 1 to about 8 drug moieties (D) per antibody. The immunoconjugate of formula I comprises a mixture or population 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 aspects, 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 aspects, p and n are equal (i.e., p=n=1, 2, 3, 4, 5, 6, 7 or 8, or some range therebetween). Exemplary antibody-drug conjugates of formula I include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine ​​residues are already present in the antibody that form intrachain disulfide bonds without the use of genetic engineering, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation of the antibody to generate one or more free cysteine ​​residues.

[0224] For some 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 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 2Am4CBza 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.

[0225] The loading of the immunoconjugate (drug / antibody ratio) can be controlled in different ways, as well as by, for example, (i) limiting the molar excess of 2Am4CBza 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.

[0226] It is understood that if more than one nucleophilic group on an antibody reacts with a drug, the resulting product is a mixture of antibody-drug conjugate compounds with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay, which is specific for the antibody and specific for the drug. Individual immune complex 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 No. 627, American Association for (See Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004.) In certain embodiments, homogeneous immune complexes having a single loading value may be isolated from a complex mixture by electrophoresis or chromatography.

[0227] Exemplary embodiments of immunoconjugates of formula I are selected from the immunoconjugates of Tables 3a and 3b. In co-cultures of cancer cells and cDC-enriched primary cell isolates, certain immunoconjugates of Tables 3a and 3b induce secretion of the cytokine IL-12p70, which is associated with mounting an immune response to cancer. Immunoconjugates targeting Clostridium difficile toxin B and including Bezlotox (bezlotocumab) IC-28 and IC-33 were examined as isotype non-tumor binding controls. In vitro immunoconjugate activity was evaluated according to the method of Example 203. [Table 34] [Table 35-1] [Table 35-2]

[0228] Pharmaceutical Compositions of Immunoconjugates The invention provides compositions, e.g., pharma- ceutical or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates as described herein and optionally a carrier therefor, e.g., a pharma- ceutical or pharmacologically acceptable carrier. The immunoconjugates may be of the same or different composition, i.e., a composition may comprise immunoconjugates with the same number of adjuvants linked to the same position on the antibody construct, and / or may comprise immunoconjugates with the same number of 2Am4CBza adjuvants linked to different positions on the antibody construct, with different numbers of adjuvants linked to the same position on the antibody construct, or with different numbers of adjuvants linked to different positions on the antibody construct.

[0229] In an exemplary embodiment, the composition comprising immunoconjugate compounds comprises a mixture of immunoconjugate compounds, and the average drug (2Am4CBza) loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5.

[0230] The compositions of the immune complexes of the invention can have an average adjuvant to antibody construct ratio of about 0.4 to about 10. One of skill in the art will recognize that the number of 2Am4CBza adjuvants conjugated to the antibody construct can vary from immune complex to immune complex in a composition comprising multiple immune complexes of the invention, and thus the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average. This can 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.

[0231] The average number of adjuvant moieties per antibody (DAR) in preparing immunoconjugates from conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the immunoconjugates in the composition in terms of p can also be determined. In some cases, separation, purification, and characterization of homogeneous 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.

[0232] 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 prepared for parenteral administration, such as IV 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 will generally comprise a solution of the immunoconjugate dissolved in a pharma- ceutically acceptable carrier. Among the acceptable vehicles and solvents that may be used are water and isotonic solutions of one or more salts, such as sodium chloride, for example, 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 substances. 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, isotonicity agents, etc., required to approximate physiological conditions, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0233] The composition may contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition may 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 the immunoconjugate in the solution formulation for injection ranges from about 0.1% (w / w) to about 10% (w / w).

[0234] Methods for Treating Cancer with Immunoconjugates - Patent application The present invention provides a method for treating cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate described herein (e.g., a composition described herein) to a subject in need thereof, e.g., a subject having cancer and in need of cancer treatment. The method comprises administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3.

[0235] 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, as well as hematological disorders, such as leukemias and lymphoid malignancies. In another aspect, the immunoconjugate is provided for use as a medicament. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual, comprising administering to the individual an effective amount of the immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0236] In a further aspect, the invention provides for 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 to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0237] Carcinomas are malignant tumors that arise from epithelial tissue. Epithelial cells line the outer surfaces of the body, line internal cavities, and form the lining of glandular tissue. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer that begins in glandular (secretory) cells, e.g., breast, pancreas, lung, prostate, stomach, gastroesophageal junction, and colon); adrenal cortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian carcinoma; 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, etc. Carcinomas can be found in the prostate, pancreas, colon, brain (usually as a secondary metastasis), lung, breast, skin, etc. In some embodiments, a method for treating non-small cell lung cancer comprises administering an immunoconjugate comprising an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof). In some embodiments, a method for treating breast cancer comprises administering an immunoconjugate comprising an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof). In some embodiments, a method for treating triple-negative breast cancer comprises administering an immunoconjugate comprising an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof).

[0238] 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, hemangioid fibrous histiocytoma, chondromyxoid fibroma, skeletal chondrosarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, desmoplastic small round cell tumor, dermatofibrosarcoma protuberans, endometrial stromal tumor, Ewing's sarcoma, fibromatosis (desmoid), infantile fibromatosis, gastrointestinal stromal tumor, giant cell tumor of bone, giant cell tumor of tendon sheath, inflammatory myofibroblastic tumor, uterine fibroid, leiocytoma, lipoblastoma, typical lipoma, spindle cell or pleomorphic lipoma, atypical lipoma, chondroid lipoma, well-differentiated liposarcoma, myxoid / round cell liposarcoma, pleomorphic liposarcoma, myxoid malignant fibrous histiocytoma, high grade malignant fibrous histiocytoma, myxofibrosarcoma, malignant peripheral nerve tumor, myxoid fibrosarcoma ... These include, but are not limited to, schwannoma, mesothelioma, neuroblastoma, osteochondroma, 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, synovial sarcoma, malignant peripheral nerve sheath tumor, neurofibroma, pleomorphic adenoma of soft tissue, and tumors derived from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and nerve sheath cells.

[0239] Sarcomas are rare types of cancer that arise in cells of mesenchymal origin, such as in bone, or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissues. Different types of sarcomas are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to, Askin tumor; botryoid sarcoma; chondrosarcoma; Ewing sarcoma; malignant hemangioendothelioma; malignant nerve sheath tumor; 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; angiosarcoma (more commonly called "angiosarcoma"); Kaposi's sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neuroblastoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0240] Teratomas are a type of germ cell tumor that can contain several different types of tissue, including, for example, hair, muscle, and bone (e.g., can include tissue derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm).Teratomas most commonly occur in the ovaries in women, the testes in men, and the coccyx in children.

[0241] 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 may also begin in other pigmented tissues, such as in the eye or in the intestine.

[0242] 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 a neuroendocrine carcinoma of the skin. In some embodiments, the method for treating Merkel cell carcinoma comprises administering an immunoconjugate comprising an antibody construct (e.g., atezolizumab, durvalumab, avelumab, biosimilars thereof, or biobetters thereof) that can bind to PD-L1. In some embodiments, the Merkel cell carcinoma has metastasized when administration is performed.

[0243] Leukemias are cancerous cells that originate in blood-forming tissues, such as the bone marrow, and cause the production and entry of large numbers of abnormal blood cells into the bloodstream. For example, leukemias can occur in cells from the bone marrow that would 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 myelogenous 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 leukemias include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0244] Lymphoma is a cancer that begins in the cells of the immune system. For example, lymphoma can arise in bone marrow-derived cells that would 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.

[0245] 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 indolent (slow-growing) and aggressive (fast-growing) cancers. There are currently 61 recognized types of NHL. Examples of non-Hodgkin's lymphomas include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, hematologic immunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small 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, treatment-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0246] Brain tumors include cancers of brain tissue. Examples of brain tumors include, but are not limited to, gliomas (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningiomas, pituitary adenomas, and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas).

[0247] The immunoconjugates of the present invention can be used in therapy either alone or in combination with other agents. For example, the immunoconjugates of the present invention 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.

[0248] The immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized therapy. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0249] Atezolizumab, durvalumab, avelumab, their biosimilars, and their biobetters are known to be useful in the treatment of cancer, particularly breast cancer, especially triple negative (tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma. The immunoconjugates described herein can be used in the treatment of the same types of cancer as atezolizumab, durvalumab, avelumab, their bioanalogs, and their biobetters, especially breast cancer, especially triple negative (tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma.

[0250] The immunoconjugate is administered to a subject in need thereof in a therapeutically effective amount using any suitable dosing regimen, such as those utilized for atezolizumab, durvalumab, avelumab, their biosimilars, and their biobetters. For example, the method can include administering the immunoconjugate to provide the subject with a dose of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can range from about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of the immunoconjugate can be outside of these ranges depending on the particular conjugate and the type and severity of the cancer being treated. The frequency of administration can 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.

[0251] In another aspect, the invention provides a method for preventing cancer. The method includes 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 the particular cancer to be prevented. For example, the method can include administering the immunoconjugate to provide the subject with a dose of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can range from about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of the immunoconjugate can be outside of these ranges depending on the particular conjugate and the type and severity of the cancer being treated. The frequency of administration can 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.

[0252] Some embodiments of the present invention provide a method for treating cancer as described above, where the cancer is breast cancer.Breast cancer can originate from various regions of the breast, and various types of breast cancer have been 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; triple negative (tests negative for estrogen receptor, progesterone receptor, and excess HER2 protein) breast cancer. In some embodiments, methods for treating breast cancer include administering an immunoconjugate comprising an antibody construct capable of binding to HER2 (e.g., trastuzumab, pertuzumab, a biosimilar thereof, or a biobetter thereof) and an antibody construct capable of binding to PD-L1 (e.g., atezolizumab, durvalumab, avelumab, a biosimilar thereof, or a biobetter thereof). In some embodiments, methods for treating colon, lung, renal, pancreatic, gastric, and esophageal cancer include administering an immunoconjugate comprising an antibody construct capable of binding to CEA, or tumors that overexpress CEA (e.g., labetuzumab, a biosimilar, or a biobetter thereof).

[0253] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8. EXAMPLES

[0254] Preparation of 2-amino-4-carboxamido-benzazepine compounds (2Am4CBza) and intermediates Example 1 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-(phenylcarbamoyl)-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, 2Am4CBza-L-1 [ka] Preparation of ethyl 2-amino-8-(phenylcarbamoyl)-3H-benzo[b]azepine-4-carboxylate, 2Am4CBza-1b To a solution of ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate, 2Am4CBza-1a (2 g, 6.47 mmol, 1 eq.) and aniline (3.01 g, 32.3 mmol, 2.95 mL, 5 eq.) in DMF (20 mL), Pd(OAc)2 (218 mg, 970 umol, 0.15 eq.), DPPF (610 mg, 1.10 mmol, 0.17 eq.) and TEA (3.27 g, 32.3 mmol, 4.50 mL, 5 eq.) were added under N2. The suspension was degassed under vacuum and purged with carbon monoxide gas (CO) several times. The mixture was stirred at 80° C. under CO (50 psi) for 12 h. The reaction mixture was filtered and concentrated under reduced pressure. The residue was diluted with H2O (60 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (30 mL x 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) and then (SiO2, EtOAc / MeOH = 1 / 0 to 10 / 1) to give 2Am4CBza-1b (0.82 g, 2.35 mmol, 36.28% yield) as a yellow solid.

[0255] Preparation of 2-amino-8-(phenylcarbamoyl)-3H-1-benzazepine-4-carboxylic acid, 2Am4CBza-1c To a solution of 2Am4CBza-1b (810 mg, 2.32 mmol, 1 equiv) in EtOH (8 mL) was added a solution of LiOH.H2O (486.40 mg, 11.59 mmol, 5 equiv) in H2O (2 mL). The mixture was stirred at 20 °C for 3 h, then adjusted to pH = 6-7 with 1N HCl and concentrated under vacuum. The residue was purified by preparative HPLC (column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B%: 5%-35%, 10 min) to give 2Am4CBza-1c (180 mg, 560.17 umol, 24.16% yield) as a white solid. 1 H NMR (DMSO-d 4, 400MHz)δ12.43(s,1H),10.49(s,1H),10.09(s,1H),9.07(br s,1H),7.99(d,J=8.4Hz,1H),7.96-7.93(m,2H),7.87-7.83(m,1H),7.79 (d,J=7.6Hz,2H),7.38(t,J=7.6Hz,2H),7.16-7.11(m,1H),3.52(s,2H).

[0256] Preparation of tert-butyl N-[2-[[2-amino-8-(phenylcarbamoyl)-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, 2Am4CBza-1 To a mixture of 2Am4CBza-1c (100 mg, 311 umol, 1 eq.) and tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (88.3 mg, 405 umol, 1.3 eq.) in DCM (3 mL) and DMA (1.5 mL), EDCI (238 mg, 1.24 mmol, 4 eq.) was added at 25° C. under N2, then stirred at 25° C. for 1 h. The mixture was concentrated under vacuum to remove DCM. The residue was diluted with H2O (10 mL), then the pH of the mixture was adjusted to about 8 with aqueous NaHCO3, and extracted with ethyl acetate (20 mL*3). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um, mobile phase: [water (0.1% TFA)-ACN]; B%: 15%~45%, 8 min) to obtain 2Am4CBza-1 (80mg, 153umol, yield 49.28%) as a white solid. 1 H NMR(MeOD,400MHz)δ8.03-7.93(m,2H),7.77(d,J=8.0Hz,1H),7.74-7.67(m,2H),7.52(s,1H),7.39(t,J=8.0Hz,2H),7.24-7.14(m,1H),3. 95(t,J=5.2Hz,2H),3.76(t,J=7.2Hz,2H),3.43(s,2H),3.27(t,J=5.2Hz,2H),1.78(sxt,J=7.2Hz,2H),1.37(s,9H),1.00(t,J=7.2Hz,3H).

[0257] Preparation of 2-amino-N4-(2-aminoethoxy)-N8-phenyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-1a To a mixture of 2Am4CBza-1 (80 mg, 153 umol, 1 equiv) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 3 mL, 78.0 equiv) in one portion at 25° C., then stirred for 1 h at 25° C. The mixture was concentrated under vacuum to give 2Am4CBza-L-1a (70 mg, 152.85 umol, 99.66% yield, HCl) as a white solid.

[0258] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, PNC-PEG10-CO2tBu [ka] To a mixture of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, HO-PEG10-CO2tBu (1 g, 1.70 mmol, 1.0 equiv.) and (4-nitrophenyl)carbonochloridate (378 mg, 1.87 mmol, 1.1 equiv.) in DCM (20 mL) was added pyridine (202 mg, 2.56 mmol, 206 uL, 1.5 equiv.) at 0° C. The mixture was stirred at 25° C. for 2 h. The pH of the mixture was adjusted to about 4 with 1 M HCl. The residue was poured into ice water (w / w=1 / 1) (100 mL) and stirred for 10 min. The aqueous phase was extracted with DCM (50 mL x 3). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0:0 / 1, ethyl acetate / methanol = 1 / 0:2 / 1) to give PNC-PEG10-CO2tBu (650 mg, 865 umol, 50.73% yield) as a colorless oil. 1 H NMR(MeOD,400MHz)δ8.38-8.27(m,2H),7.54-7.45(m,2H),4.47-4.42(m,2H),3.80-3.53(m,40H),2.53-2.44(m,2H),1.50-1.41(m,9H).

[0259] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-(phenylcarbamoyl)-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 2Am4CBza-L-1b To a mixture of 2Am4CBza-L-1a (70 mg, 153 umol, 1 equiv, HCl) and PNC-PEG10-CO2tBu (126 mg, 168 umol, 1.1 equiv) in DMF (2.5 mL), Et3N (38.7 mg, 382 umol, 2.5 equiv) was added under N2 at 25° C., then stirred for 1 h at 25° C. The mixture was filtered and purified by prep-HPLC (column: Phenomenex luna C18 100*40 mm*5 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-55%, 8 min) to give 2Am4CBza-L-1b (70 mg, 67.7 umol, 44.28% yield) as a colorless oil.

[0260] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-(phenylcarbamoyl)-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 2Am4CBza-L-1c To a mixture of 2Am4CBza-L-1b (70 mg, 67.7 umol, 1 equiv.) in CH3CN (1 mL) and HO (1 mL), TFA (77.2 mg, 677 umol, 10 equiv.) was added under N2 at 25°C, then stirred at 80°C for 2 h. The pH of the mixture was adjusted to about 5 with aqueous NaHCO3 at 0°C, then extracted with DCM / i-PrOH=3 / 1 (10 mL*3). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum to give 2Am4CBza-L-1c (65 mg, 66.46 umol, 98.18% yield) as a pale yellow oil.

[0261] Preparation of 2Am4CBza-L-1 To a mixture of 2Am4CBza-L-1c (60 mg, 61.34 umol, 1 eq.) and sodium; 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate, STP (49.3 mg, 184 umol, 3 eq.) in DCM (2 mL) and DMA (0.5 mL), EDCI (35.3 mg, 184 umol (micromol), 3 eq.) was added under N2 at 25°C, then stirred for 1 h at 25°C. The mixture was concentrated under vacuum and then purified by preparative HPLC (column: Phenomenex Luna80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 8 min) to give 2Am4CBza-L-1 (40 mg, 33.16 umol, 54.06% yield) as a white solid. 1 H NMR(MeOD,400MHz)δ8.06-7.95(m,2H),7.73(dd,J=5.0,7.6Hz,3H),7.49-7.43(m,1H),7.45(s,1H) ),7.38(t,J=8.0Hz,2H),7.23-7.13(m,1H),3.98(t,J=5.0Hz,2H),3.85(t,J=5.6Hz,2H),3.80(br d,J=4.4Hz,2H),3.75(t,J=7.2Hz,2H),3.66-3.54(m,38H),3.51-3.42(m ,4H),2.96(t,J=6.0Hz,2H),1.77(sxt,J=7.2Hz,2H),1.00(t,J=7.2Hz,3H ).HPLC:99.47%(220nm),99.31%(254nm).LC / MS[M+H]1206.4(calculated value);LC / MS[M+H]1206.7(measured value).LC / MS[M+H]1206.4(calculated value);LC / MS[M+H]1206.7(measured value).

[0262] Example 4 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-[(3S)-3-anilinopiperidine-1-carbonyl]-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, 2Am4CBza-L-4 [ka] [ka] Preparation of tert-butyl (3S)-3-anilinopiperazine-1-carboxylate, 2Am4CBza-L-4b To a solution of tert-butyl (3S)-3-aminopiperazine-1-carboxylate, 2Am4CBza-L-4a (1.00 g, 4.99 mmol, 1.0 equiv) and bromobenzene (780 mg, 4.99 mmol, 526 uL, 1.0 equiv) in toluene (10.0 mL) was added sodium tert-butoxide, NaOtBu (580 mg, 5.99 mmol, 1.2 equiv), 2-(2-dicyclohexylphosphanylphenyl)-N,N-dimethyl-aniline (140 mg, 349 umol, 0.07 equiv), and tris(dibenzylideneacetone)dipalladium(0), Pd2(dba)3, CAS Registry Number 51364-51-3 (230 mg, 249 umol, 0.05 equiv) at 20 °C under N2. The mixture was stirred at 100 °C for 24 h. The mixture was diluted with water (30 mL) and extracted with EtOAc (30 mL × 3). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash silica flash column, eluent of 0-50% ethyl acetate / petroleum ether gradient: 45 mL / min) to give 2Am4CBza-L-4b (1.2 g, 4.34 mmol, 86.9% yield) as a pale yellow solid. 1H NMR(CDCl3,400MHz)δ7.18(dd,J=7.6,8.4Hz,2H),6.77-6.60(m,3H),4.09-3.95(m,1H),3.83-3.62(m,2H),3.45-3.33(m,1H),3.09(br d,J=9.6Hz,1H),2.98-2.78(m,1H),2.04-1.94(m,1H),1.80-1.71(m,1H),1.6 5-1.53(m,2H),1.46(s,9H).LC / MS[M+H]277.2(calculated value);LC / MS[M+H]277.2(measured value).

[0263] Preparation of (3S)-N-phenylpiperidin-3-amine, 2Am4CBza-L-4c To a solution of 2Am4CBza-L-4b (1.20 g, 4.34 mmol, 1.0 equiv) in EtOAc (5.00 mL) was added HCl / EtOAc (4 M, 18.0 mL, 17.0 equiv). The mixture was stirred at 20° C. for 1 h. The mixture was concentrated to give 2Am4CBza-L-4c (900 mg, 3.61 mmol, 83.1% yield, 2HCl) as a white solid. 1 H NMR(MeOD,400MHz)δ7.59-7.52(m,2H),7.49-7.41(m,3H),3.94(tt,J=4.0,10.8Hz,1H),3.62-3.54(m,1H),3.40(d,J=12.0Hz,1H),3.20 (t,J=11.6Hz,1H),3.04(dt,J=3.2,12.4Hz,1H),2.26-2.07(m,2H),1.93-1.78(m,2H).LC / MS[M+H]177.2(calculated value);LC / MS[M+H]177.2(measured value).

[0264] Preparation of 2Am4CBza-L-4e To a solution of 2-amino-4-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine-8-carboxylic acid, 2Am4CBza-L-4d (250 mg, 559 umol, 1.0 equiv) in DMF (1.00 mL) was added HATU (210 mg, 559 umol, 1.0 equiv), DIEA (290 mg, 2.24 mmol, 390 uL, 4.0 equiv) and (3S)-N-phenylpiperidin-3-amine, 2Am4CBza-L-4c (130 mg, 603 umol, 1.08 equiv, HCl) then stirred at 0 °C for 1 h. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%~50%, 8 min) to give 2Am4CBza-L-4e (160mg, 222umol, yield 39.7%, TFA) as a white solid. 1 H NMR(MeOD,400MHz)δ7.76-7.38(m,3H),7.36-7.15(m,2H),7.11-6.90(m,2H),6 .63-6.42(m,2H),3.95-3.92(m,2H),3.89-3.61(m,4H),3.60-3.32(m,4H),3.2 8-3.23(m,2H),3.08-2.96(m,1H),2.20-1.94(m,2H),1.84-1.67(m,4H),1.38( s,9H),0.99(t,J=7.6Hz,3H).LC / MS[M+H]605.3(calculated value);LC / MS[M+H]605.3(measured value).

[0265] Preparation of 2-amino-N-(2-aminoethoxy)-8-[(3S)-3-anilinopiperidine-1-carbonyl]-N-propyl-3H-1-benzazepine-4-carboxamide, 2Am4CBza-L-4f To a solution of 2Am4CBza-L-4e (90.0 mg, 125 umol, 1.0 equiv, TFA) in EtOAc (5.00 mL) was added HCl / EtOAc (4M, 10.0 mL, 319.0 equiv). The mixture was stirred at 20° C. for 1 h and then concentrated to give 2Am4CBza-L-4f (78.0 mg, crude, 2HCl) as a white solid. LC / MS [M+H] 505.3 (calculated); LC / MS [M+H] 505.3 (observed).

[0266] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-[(3S)-3-anilinopiperidine-1-carbonyl]-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 2Am4CBza-L-4g To a solution of 2Am4CBza-L-4f (70.0 mg, 121 umol, 1.0 equiv, 2Hcl) in DMF (1.00 mL) was added Et3N (50.0 mg, 484 umol, 70.0 uL, 4.0 equiv) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy] ...

[0267] The organic layer was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give 2Am4CBza-L-4g (200 mg, crude) as a pale yellow oil. LC / MS [M+H] 1117.6 (calculated); LC / MS [M+H] 1117.6 (observed).

[0268] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-[(3S)-3-anilinopiperidine-1-carbonyl]-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 2Am4CBza-L-4h To a solution of 2Am4CBza-L-4g (200 mg, 179 umol, 1.0 equiv) in CH3CN (1.00 mL) and HO (1.00 mL), TFA (200 mg, 1.79 mmol, 130 uL, 10.0 equiv) was added and then stirred at 80 °C for 1 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-40%, 8 min) to give 2Am4CBza-L-4h (50 mg, 42.5 umol, 23.7% yield, TFA) as a colorless oil. LC / MS [M+H] 1061.5 (calculated); LC / MS [M+H] 1061.5 (observed).

[0269] Preparation of 2Am4CBza-L-4 To a solution of 2Am4CBza-L-4h (40.0 mg, 34.0 umol, 1.0 equiv, TFA) and 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonic acid (33.5 mg, 136 umol, 4.0 equiv) in DCM (2.00 mL) and DMA (0.20 mL), EDCI (30.0 mg, 136 umol, 4.0 equiv) was added and then stirred at 20 °C for 1 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15% ~ 50%, 8 min) to give 2Am4CBza-L-4 (21 mg, 14.9 umol, 43.9% yield, TFA) as a white solid. 1H NMR(MeOD,400MHz)δ7.67-7.24(m,5H),7.17-6.96(m,2H),6.71-6.41(m,2H) ,3.99-3.94(m,2H),3.90-3.80(m,6H),3.77-3.71(m,4H),3.65-3.58(m,38H ),3.54-3.47(m,4H),2.97(t,J=6.0Hz,2H),2.22-1.95(m,2H),1.85-1.68(m ,4H),1.03-0.97(m,3H).LC / MS[M+H]1289.5(calculated value);LC / MS[M+H]1289.5(measured value).

[0270] Example 6 Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (2-((2-amino-8-(dimethylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-6 [ka] Preparation of tert-butyl (2-((2-amino-8-(dimethylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-6 2-Amino-4-((2-((tert-butylcarbonyl)amino)ethoxy)(propyl)carbamoyl)-3H-benzo[b]azepine-8-carboxylic acid, 2Am4CBza-L-12d, was reacted with dimethylamine and HATU to give 2Am4CBza-L-6a. LC / MS [M+H] 474.27 (calculated); LC / MS [M+H] 474.45 (observed).

[0271] Preparation of 2-amino-N4-(2-aminoethoxy)-N8,N8-dimethyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-6b 2Am4CBza-L-6a was reacted with methanesulfonic acid to give 2Am4CBza-L-6b. LC / MS [M+H] 374.22 (calculated); LC / MS [M+H] 374.36 (observed).

[0272] Preparation of 2Am4CBza-L-6 To a solution of 2Am4CBza-L-6b in DMF, DIEA and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl) carbonate were added and then stirred at 25°C for 1 hour. The mixture was quenched with TFA until pH=about 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water(TFA)-ACN]; B%: 5%-35%, 8 min) to give 2Am4CBza-L-6. LC / MS [M+H] 1038.52 (calculated); LC / MS [M+H] 1038.63 (observed).

[0273] Example 7 Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (2-((2-amino-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-7 [ka] Preparation of tert-butyl (2-((2-amino-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-7a 2-Amino-4-((2-((tert-butoxycarbonyl)amino)ethoxy)(propyl)carbamoyl)-3H-benzo[b]azepine-8-carboxylic acid, 2Am4CBza-L-12d, was reacted with pyrrolidine and HATU to give 2Am4CBza-L-7a. LC / MS [M+H] 500.29 (calculated); LC / MS [M+H] 500.48 (observed).

[0274] Preparation of 2-amino-N-(2-aminoethoxy)-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamide, 2Am4CBza-L-7b 2Am4CBza-L-7a was reacted with methanesulfonic acid to give 2Am4CBza-L-7b. LC / MS [M+H] 400.23 (calculated); LC / MS [M+H] 400.39 (observed).

[0275] Preparation of 2Am4CBza-L-7 To a solution of 2Am4CBza-L-7b in DMF, DIEA and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl) carbonate were added and then stirred at 25°C for 1 hour. The mixture was quenched with TFA until pH=about 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water(TFA)-ACN]; B%: 5%-35%, 8min) to give 2Am4CBza-L-7. LC / MS [M+H] 1064.54 (calculated); LC / MS [M+H] 1064.67 (observed).

[0276] Example 8 Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (3-(2-amino-8-(dimethylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, 2Am4CBza-L-8 [ka] Preparation of tert-butyl (3-(2-amino-8-(dimethylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, 2Am4CBza-L-8b 2-Amino-4-((3-((tert-butoxycarbonyl)amino)propyl)(propyl)carbamoyl)-3H-benzo[b]azepine-8-carboxylic acid, 2Am4CBza-L-8a, was reacted with dimethylamine and HATU to give 2Am4CBza-L-8b. LC / MS [M+H] 472.29 (calculated); LC / MS [M+H] 472.56 (observed).

[0277] Preparation of 2-amino-N4-(3-aminopropyl)-N8,N8-dimethyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-8c 2Am4CBza-L-8b was reacted with methanesulfonic acid to give 2Am4CBza-L-8c. LC / MS [M+H] 372.24 (calculated); LC / MS [M+H] 372.34 (observed).

[0278] Preparation of 2Am4CBza-L-8 To a solution of 2Am4CBza-L-8c in DMF, DIEA and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl) carbonate were added and then stirred at 25°C for 1 hour. The mixture was quenched with TFA until pH=about 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; yl:[water(TFA)-ACN]; B%:5%~35%, 8min) to give 2Am4CBza-L-8. LC / MS [M+H] 1036.54 (calculated); LC / MS [M+H] 1036.60 (observed).

[0279] Example 9 Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (3-(2-amino-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, 2Am4CBza-L-9 [ka] Preparation of tert-butyl 3-(2-amino-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, 2Am4CBza-L-9a 2-Amino-4-((3-((tert-butoxycarbonyl)amino)propyl)(propyl)carbamoyl)-3H-benzo[b]azepine-8-carboxylic acid, 2Am4CBza-L-8a, was reacted with pyrrolidine and HATU to give 2Am4CBza-L-9a. LC / MS [M+H] 498.31 (calculated); LC / MS [M+H] 498.44 (observed).

[0280] Preparation of 2-amino-N-(3-aminopropyl)-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamide, 2Am4CBza-L-9b 2Am4CBza-L-9a was reacted with methanesulfonic acid to give 2Am4CBza-L-9b. LC / MS [M+H] 398.26 (calculated); LC / MS [M+H] 398.37 (observed).

[0281] Preparation of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (3-(2-amino-N-propyl-8-(pyrrolidine-1-carbonyl)-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate To a solution of 2Am4CBza-L-9b in DMF, DIEA and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl) carbonate were added and then stirred at 25°C for 1 hour. The mixture was quenched with TFA until pH=about 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water(TFA)-ACN]; B%: 5%-35%, 8min) to give 2Am4CBza-L-9. LC / MS [M+H] 1062.56 (calculated); LC / MS [M+H] 1062.20 (observed).

[0282] Example 10 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-[(3R)-3-anilinopiperidine-1-carbonyl]-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, 2Am4CBza-L-10 [ka] [ka] Preparation of tert-butyl (3R)-3-anilinopiperidine-1-carboxylate, 2Am4CBza-L-10b To a solution of tert-butyl (3R)-3-aminopiperidine-1-carboxylate, 2Am4CBza-L-10a (1 g, 4.99 mmol, 1.0 equiv.) and bromobenzene (784 mg, 4.99 mmol, 526 uL, 1.0 equiv.) in toluene (10 mL), 2-(2-dicyclohexylphosphanylphenyl)-N,N-dimethyl-aniline (138 mg, 349 umol, 0.07 equiv.), sodium 2-methylpropan-2-olate, sodium tert-butoxide (576 mg, 5.99 mmol, 1.2 equiv.) and Pd2(dba)3 (228 mg, 250 umol, 0.05 equiv.) were added under N2. The suspension was degassed under vacuum and purged with N2 several times, then stirred at 100 °C for 10 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was diluted with HO (30 mL) and extracted with EtOAc (30 mL × 3), and the combined organic phase was washed with brine (15 mL), dried over anhydrous NaSO, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography (eluted with petroleum ether:ethyl acetate = 1:0 to 0:1) to give 2Am4CBza-L-10b (900 mg, 3.26 mmol, 65.2% yield) as a colorless oil. 1H NMR(CDCl3,400MHz)δ7.22-7.17(m,2H),6.77-6.67(m,3H),4.09-3.96(m,1H),3.77-3.70(m,1H),3.42-3.34(m,1H),3.12-2.82(m,1H),3 .02-2.82(m,1H),2.04-1.98(m,1H),1.79-1.71(m,1H),1.63-1.52(m,4H),1.46(s,9H).LC / MS[M+H]277.2(calculated value);LC / MS[M+H]277.2(measured value).

[0283] Preparation of (3R)-N-phenylpiperidin-3-amine, 2Am4CBza-L-10c To a solution of 2Am4CBza-L-10b (900 mg, 3.26 mmol, 1.0 equiv) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 16.3 mL, 20.0 equiv) and then stirred for 2 h at 20° C. The reaction mixture was concentrated in vacuo to give 2Am4CBza-L-10c (650 mg, 2.61 mmol, 80.1% yield, 2HCl) as a white solid. 1 H NMR(CDCl3,400MHz)δ7.52-7.47(m,2H),7.43-7.32(m,3H),3.96-3.85(m,1H),3.56(br d,J=12.0Hz,1H),3.43-3.36(m,1H),3.19-3.10(m,1H),3.03(dt,J=2.8,12.2Hz,1H),2.25-2.08(m,2H),1.92-1.77(m,2H).

[0284] Preparation of tert-butyl N-[2-[[2-amino-8-[(3R)-3-anilinopiperidine-1-carbonyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, 2Am4CBza-L-10d To a solution of 2-amino-4-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine-8-carboxylic acid, 2Am4CBza-L-4d (250 mg, 560 umol, 1.0 equiv), HATU (234 mg, 616 umol, 1.1 equiv) and DIEA (290 mg, 2.24 mmol, 390 uL, 4.0 equiv) in DMF (3 mL) was added (3R)-N-phenylpiperidin-3-amine (209 mg, 840 umol, 1.5 equiv, 2HCl) under N2 at 0° C. The mixture was stirred at 20° C. for 3 h. The reaction mixture was filtered and concentrated under vacuum. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%~45%, 8 min) to obtain 2Am4CBza-L-10d (220mg, 306umol, yield 54.7%, TFA) as a white solid. 1 H NMR(MeOD,400MHz)δ7.58-7.52(m,1H),7.52-7.43(m,1H),7.43-7.38(m,1H),7.36-7.30(m,1H),7.24-7.16(m,1H),6.97(br t,J=7.2Hz,1H),6.91-6.76(m,1H),6.58-6.49(m,1H),6.44(br d,J=7.4Hz,1H),3.93(br s,2H),3.84-3.57(m,4H),3.55-3.35(m,4H),3.28-3.23(m,2H),3.07-2.97(m,1H),2.22-1.97(m,2H) ,1.86-1.65(m,4H),1.38(s,9H),1.01-0.96(m,3H).LC / MS[M+H]605.3(calculated value);LC / MS[M+H]605.3(measured value).

[0285] Preparation of 2-amino-N-(2-aminoethoxy)-8-[(3R)-3-anilinopiperidine-1-carbonyl]-N-propyl-3H-1-benzazepine-4-carboxamide, 2Am4CBza-L-10e To a solution of 2Am4CBza-L-10d (220 mg, 364 umol, 1.0 equiv) in EtOAc (3 mL) was added HCl / EtOAc (4 M, 1.82 mL, 20.0 equiv) and then stirred at 20° C. for 2 h. The reaction mixture was concentrated under vacuum to give 2Am4CBza-L-10e (170 mg, 294 umol, 80.9% yield, 2HCl) as a white solid. LC / MS [M+H] 505.3 (calculated); LC / MS [M+H] 505.2 (observed).

[0286] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-[(3R)-3-anilinopiperidine-1-carbonyl]-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 2Am4CBza-L-10f To a solution of 2Am4CBza-L-10e (90 mg, 156 umol, 1.0 equiv, 2HCl) and DIEA (80.6 mg, 623 umol, 109 uL, 4.0 equiv) in DMF (2 mL) was added tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (117 mg, 156 umol, 1.0 equiv) at 0° C., then stirred at 20° C. for 2 h. The reaction mixture was filtered and concentrated in vacuo, the residue was diluted with HO (10 mL) and extracted with EtOAc (10 mL×3), the combined organic phase was washed with brine (10 mL×3), dried over anhydrous NaSO and concentrated in vacuo to give 2Am4CBza-L-10f (160 mg, 143 umol, 91.9% yield) as a white solid. LC / MS [M+H] 1117.6 (calculated); LC / MS [M+H] 1117.6 (observed).

[0287] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-amino-8-[(3R)-3-anilinopiperidine-1-carbonyl]-3H-1-benzoazepine-4-carbonyl]-propyl-amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 2Am4CBza-L-10g To a solution of 2Am4CBza-L-10f (130 mg, 116 umol, 1.0 equiv) in DCM (2 mL) was added methanesulfonic acid (111 mg, 1.16 mmol, 82.8 uL, 10.0 equiv) and stirred at 20 °C under N2 for 2 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80 * 30 mm * 3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 8 min) to give 2Am4CBza-L-10g (80 mg, 75.4 umol, 64.8% yield) as a light colorless oil. LC / MS [M+H] 1061.6 (calculated); LC / MS [M+H] 1061.6 (observed).

[0288] Preparation of 2Am4CBza-L-10 To a solution of 2Am4CBza-L-10g (70 mg, 65.9 umol, 1.0 equiv.) and sodium 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (70.7 mg, 264 umol, 4.0 equiv.) in DCM (1.5 mL) and DMA (0.5 mL), EDCI (50.6 mg, 264 umol, 4.0 equiv.) was added and then stirred at 20°C for 1 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; B%: 15%-45%, 8 min) to give 2Am4CBza-L-10 (25.1 mg, 19.5 umol, 29.5% yield) as a white solid. 1H NMR(MeOD-d4,400MHz)δ7.73-7.63(m,1H),7.60-7.42(m,3H),7.41-7.25(m,3H),7 .15-7.03(m,1H),6.72-6.60(m,1H),4.00-3.94(m,2H),3.90-3.82(m,4H),3.74(br t,J=7.2Hz,2H),3.65-3.57(m,40H),3.51(br d,J=4.0Hz,2H),3.45-3.38(m,2H),3.27-3.14(m,2H),3.00-2.96(m,2H),2.21-2.08(m,1H), 1.81-1.69(m,4H),0.99(t,J=7.6Hz,3H).LC / MS[M+H]1289.5(calculated value);LC / MS[M+H]1289.5(measured value).

[0289] Example 11 Synthesis of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl N-[2-[[2-amino-8-[(3R)-3-anilinopiperidine-1-carbonyl]-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, 2Am4CBza-L-11 [ka] To a solution of 2-amino-N-(2-aminoethoxy)-8-[(3R)-3-anilinopiperidine-1-carbonyl]-N-propyl-3H-1-benzazepine-4-carboxamide, 2Am4CBza-L-10e (50.0 mg, 86.5 umol, 1.0 equiv, 2Hcl) in DMF (1.00 mL) was added DIEA (50.0 mg, 346 umol, 60.0 uL, 4.0 equiv) and 2 -[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl(4-nitrophenyl)carbonate (70.0 mg, 86.5 umol, 1.0 equiv.) was added and then stirred at 20° C. for 1 h. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; B%: 15%-40%, 8 min) to give 2Am4CBza-L-11 (23 mg, 17.9 umol, yield 20.7%, TFA) as a pale yellow oil. 1 H NMR(MeOD,400MHz)δ7.75-7.16(m,4H),7.07-6.79(m,4H),6.62-6.39(m,2H),4. 17(s,2H),4.04-3.85(m,4H),3.81-3.72(m,4H),3.72-3.56(m,38H),3.56-3.51 (m,4H),3.48-3.34(m,6H),3.17-3.05(m,1H),2.26-1.93(m,2H),1.85-1.65(m, 4H),1.00(t,J=7.6Hz,3H).LC / MS[M+H]1169.6(calculated value);LC / MS[M+H]1169.6(measured value).

[0290] Example 12 Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (2-((2-amino-8-carbamoyl-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-12 [ka] Preparation of tert-butyl N-[2-[(2-amino-8-bromo-3H-1-benzazepine-4-carbonyl)-propyl-amino]oxyethyl]carbamate, 2Am4CBza-L-12b To a mixture of 2-amino-8-bromo-3H-1-benzazepine-4-carboxylic acid, 2Am4CBza-L-12a (8.08 g, 28.7 mmol, 1.0 equiv.) and DMA (50 mL) in DCM (50 mL) was added methanesulfonic acid (2.76 g, 28.7 mmol, 2.05 mL, 1.0 equiv.), tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (6.9 g, 31.6 mmol, 1.1 equiv.) and EDCI (22.0 g, 115 mmol, 4.0 equiv.) at 0° C. under N2, then stirred at this temperature for 2 h. The mixture was concentrated to remove DCM and diluted with water (100 mL). The pH of the mixture was then adjusted to about 8 by adding aqueous Na2CO3 solution. The aqueous phase was extracted with EtOAc (100 mL×3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was triturated with MTBE (15 mL) and petroleum ether (100 mL) at 0° C. for 0.5 h to give 2Am4CBza-L-12b (11 g, 22.85 mmol, 79.52% yield) as a yellow solid. 1 H NMR(DMSO-d6,400MHz)δ7.27(d,J=8.4Hz,1H),7.14(d,J=2.0Hz,1H),7.08-7 .01(m,2H),7.01-6.87(m,2H),6.85-6.82(m,1H),3.80(t,J=5.6Hz,2H),3.57 (t,J=7.2Hz,2H),3.09-3.05(m,2H),2.76(s,2H),1.67-1.56(m,2H),1.31(s, 9H),0.87(t,J=7.6Hz,3H).LC / MS[M+H]481.1(calculated value);LC / MS[M+H]481.1(measured value).

[0291] Preparation of methyl 2-amino-4-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine-8-carboxylate, 2Am4CBza-L-12c To a solution of 2Am4CBza-L-12b (8 g, 16.6 mmol, 1.0 equiv) in MeOH (100 mL) was added Pd(dppf)Cl2 (1.22 g, 1.66 mmol, 0.1 equiv) and Et3N (5.04 g, 49.9 mmol, 6.94 mL, 3.0 equiv) under N2. The suspension was degassed under vacuum and purged with CO several times. The mixture was stirred at 80 °C under CO (50 psi) for 12 h. The mixture was filtered and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, ethyl acetate / MeOH = 1 / 0, 10 / 1) to give 2Am4CBza-L-12c (5 g, 10.86 mmol, 65.33% yield) as a yellow solid. 1 H NMR(MeOD,400MHz)δ7.81(d,J=1.6Hz,1H),7.63(dd,J=1.6,8.0Hz,1H),7.46(d,J=8.0Hz,1H),7.27(s,1H),3.94-3.86(m,5H),3.73-3.69(m,2H) ),3.31(s,2H),3.23(t,J=5.2Hz,2H),1.81-1.70(m,2H),1.35(s,9H),0.97(t,J=7.6Hz,3H).LC / MS[M+H]461.2(calculated value);LC / MS[M+H]461.1(measured value).

[0292] Preparation of 2-amino-4-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine-8-carboxylic acid, 2Am4CBza-L-12d To a mixture of 2Am4CBza-L-12c (4 g, 8.69 mmol, 1.0 equiv) in THF (30 mL) and H2O (30 mL) was added LiOH.H2O (547 mg, 13.0 mmol, 1.5 equiv) in one portion at 25 °C and then stirred at this temperature for 2 h. The mixture was quenched with aqueous HCl (1 M) until pH = approx. 6. The mixture was then diluted with water (30 mL) and extracted with EtOAc (50 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The mixture was purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 5%~40%, 8 min) to give 2Am4CBza-L-12d (3.3g, 7.39mmol, yield 85.09%) as a yellow solid. 1 H NMR(MeOD,400MHz)δ8.13(s,1H),7.92(d,J=7.6Hz,1H),7.59(d,J=8.0Hz,1H),7.46(s,1H),3.97-3.92(m,2H),3.75(t,J=7.2Hz,2H),3.31- 3.30(m,2H),3.26(t,J=5.2Hz,2H),1.83-1.71(m,2H),1.35(s,9H),0.99(t,J=7.6Hz,3H).LC / MS[M+H]447.2(calculated value);LC / MS[M+H]447.2(measured value).

[0293] Preparation of tert-butyl (2-((2-amino-8-carbamoyl-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-12e 2Am4CBza-L-12d was reacted with ammonium chloride and HATU to give 2Am4CBza-L-12e. LC / MS [M+H] 446.24 (calculated); LC / MS [M+H] 446.43 (observed).

[0294] Preparation of 2-amino-N4-(2-aminoethoxy)-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-12f 2Am4CBza-L-12e was reacted with hydrochloric acid in ethyl acetate to give 2Am4CBza-L-12f. LC / MS [M+H] 346.19 (calculated); LC / MS [M+H] 346.35 (observed).

[0295] Preparation of 2Am4CBza-L-12 To a solution of 2Am4CBza-L-12f in DMF, DIEA and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl) carbonate were added and then stirred at 25°C for 1 hour. The mixture was quenched with TFA until pH=about 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8 min) to give 2Am4CBza-L-12. LC / MS [M+H] 1010.49 (calculated); LC / MS [M+H] 1010.71 (observed).

[0296] Example 13 Synthesis of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (3-(2-amino-8-(phenylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate, 2Am4CBza-L-13 [ka] Preparation of 2-amino-N4-(2-aminoethoxy)-N8-phenyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-13b tert-Butyl (2-((2-amino-8-(phenylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 2Am4CBza-L-13a, was reacted with methanesulfonic acid to give 2Am4CBza-L-13b. LC / MS [M+H] 422.22 (calculated); LC / MS [M+H] 422.39 (observed).

[0297] Preparation of 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (3-(2-amino-8-(phenylcarbamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)propyl)carbamate To a solution of 2Am4CBza-L-13b in DMF, DIEA and 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl (4-nitrophenyl) carbonate were added and then stirred at 25°C for 1 hour. The mixture was quenched with TFA until pH=about 6. The mixture was then filtered and purified by preparative HPLC (column: Phenomenex Luna80*30mm*3um; mobile phase: [water(TFA)-ACN]; B%: 5%-35%, 8 min) to give 2Am4CBza-L-13. LC / MS [M+H] 1086.52 (calculated); LC / MS [M+H] 1086.73 (observed).

[0298] Example 17 Synthesis of 2-amino-N4-((40-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,39-dioxo-8,11,14,17,20,23,26,29,32,35-decaoxa-3,5,38-triazatetracontyl)oxy)-N8,N8-dimethyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-17 [ka] Preparation of tert-butyl (32-isocyanato-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, 2Am4CBza-L-17b To a solution of tert-butyl (32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, 2Am4CBza-L-17a (0.15 g, 0.25 mmol, 1 equiv.) in DCM was added TEA (0.348 ml, 2.5 mmol, 10 equiv.), followed by phosgene (0.892 ml, 0.25 mmol, 1 equiv. as a 1.4 M solution in toluene). The reaction mixture was monitored by LCMS, concentrated, and purified by reverse phase HPLC to give 2Am4CBza-L-17b (78 mg, 0.125 mmol, 50%). LC / MS [M+H] 627.37 (calculated); LC / MS [M+H] 627.64 (observed).

[0299] Preparation of tert-butyl (39-(2-amino-8-(dimethylcarbamoyl)-3H-benzo[b]azepine-4-carbonyl)-34-oxo-3,6,9,12,15,18,21,24,27,30,38-undecaoxa-33,35,39-triazadtetracontyl)carbamate, 2Am4CBza-L-17c To a mixture of 2-amino-N4-(2-aminoethoxy)-N8,N8-dimethyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-6b (1 eq.) and 2Am4CBza-L-17b (1 eq.) in DMF was added TEA (10 eq.). The reaction was stirred at room temperature, then diluted with water and purified by reverse phase HPLC to give 2Am4CBza-L-17c. LC / MS [M+H] 1000.58 (calculated); LC / MS [M+H] 1000.91 (observed).

[0300] Preparation of 2-amino-N4-((37-amino-4-oxo-8,11,14,17,20,23,26,29,32,35-decaoxa-3,5-diazaheptatriacontyl)oxy)-N8,N8-dimethyl-N4-propyl-3H-benzo[b]azepine-4,8-dicarboxamide, 2Am4CBza-L-17d 2Am4CBza-L-17c (48 mg, 0.052 mmol, 1 equiv.) was dissolved in minimal TFA. After 15 min, the reaction mixture was concentrated to give 2Am4CBza-L-17d as the TFA salt. LC / MS [M+H] 900.53 (calculated); LC / MS [M+H] 900.83 (observed).

[0301] Preparation of 2Am4CBza-L-17 To a solution of 2Am4CBza-L-17c (1 eq.) in DMF was added TEA (12.8 eq.) followed by 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (1.28 eq.). The reaction mixture was concentrated, diluted with 1% TFA in water and purified by reverse phase HPLC to give 2Am4CBza-L-17. LC / MS [M+H] 1037.54 (calculated); LC / MS [M+H] 1037.84 (observed).

[0302] Example 19 Synthesis of 2-amino-N4-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-N8,N8-dimethyl-N4-propyl-3H-1-benzazepine-4,8-dicarboxamide, 2Am4CBza-L-19 [ka] Preparation of ethyl 8-bromo-2-(tritylamino)-3H-1-benzazepine-4-carboxylate, 2Am4CBza-L-19b To a solution of ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate, 2Am4CBza-L-19a (3 g, 9.70 mmol, 1 equiv.) in DCM (20 mL) was added TrtCl (4.06 g, 14.6 mmol, 1.5 equiv.) and Et3N (2.95 g, 29.1 mmol, 4.05 mL, 3 equiv.). The mixture was stirred at 50° C. for 12 h. The resulting mixture was poured into ice water (w / w=1 / 1) (30 mL) and stirred for 10 min. The aqueous phase was extracted with DCM (20 mL×3). The combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was triturated with MTBE at 25 °C for 5 min to give 2Am4CBza-L-19b (4 g, 7.25 mmol, 74.75% yield) as a yellow solid. LC / MS [M+H] 551.1 (calculated); LC / MS [M+H] 551.1 (observed).

[0303] Preparation of ethyl 8-(dimethylcarbamoyl)-2-(tritylamino)-3H-1-benzazepine-4-carboxylate, 2Am4CBza-L-19c A mixture of 2Am4CBza-L-19b (2 g, 3.63 mmol, 1 equiv. eq), N-methylmethanamine, dimethylamine (1.48 g, 18.2 mmol, 1.66 mL, 5 equiv., HCl), Et3N (3.67 g, 36.3 mmol, 5.05 mL, 10 equiv.), Pd(dppf)Cl2 (266 mg, 363 umol, 0.1 equiv.) in DMF (30 mL) was degassed and purged with carbon monoxide, CO (3.63 mmol, 1 equiv.) three times, then the mixture was stirred at 80° C. for 12 h under CO (50 psi) atmosphere. The mixture was poured into ice water (w / w=1 / 1) (30 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with brine (20 mL x 3), dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 1 / 1) to give 2Am4CBza-L-19c (900 mg, 1.66 mmol, 45.61% yield) as a yellow solid. LC / MS [M+H] 544.2 (calculated); LC / MS [M+H] 544.4 (observed).

[0304] Preparation of 8-(dimethylcarbamoyl)-2-(tritylamino)-3H-1-benzazepine-4-carboxylic acid, 2Am4CBza-L-19d To a solution of 2Am4CBza-L-19c (800 mg, 1.47 mmol, 1 equiv.) in EtOH (15 mL) and HO (8 mL) was added LiOH.HO (247 mg, 5.89 mmol, 4 equiv.). The mixture was stirred at 20 °C for 12 h. The pH of the mixture was adjusted to about 6 with HCl (1 M) and then concentrated under vacuum to remove EtOH. Water (20 mL) was added and the desired white solid precipitated from the mixture was filtered to give 2Am4CBza-L-19d (750 mg, 1.45 mmol, 98.85% yield) as a white solid. 1H NMR(400MHz,DMSO-d6)δ8.19(s,1H),7.69(s,1H),7.35(d,J=8.4Hz,1H),7.29-7.19(m,12H),7.17-7.09(m,3H),6.83(dd,J=1. 6,1.6Hz,1H),6.35(d,J=1.6Hz,1H),3.00(s,2H),2.91(s,3H),2.80(s,3H).LC / MS[M+H]516.2(calculated value);LC / MS[M+H]516.4(measured value).

[0305] Preparation of 2-amino-8-(dimethylcarbamoyl)-3H-1-benzazepine-4-carboxylic acid, 2Am4CBza-L-19e To Am4CBza-L-19d (750 mg, 1.45 mmol, 1 equiv) in DCM (10 mL) was added TFA (1.66 g, 14.6 mmol, 1.08 mL, 10 equiv), then stirred at 50° C. for 12 h. The mixture was concentrated under vacuum. The crude product was triturated with MTBE at 20° C. for 5 min to give 2Am4CBza-L-19e (550 mg, 1.42 mmol, 97.62% yield, TFA) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ7.90(s,1H),7.73(d,J=8.0Hz,1H),7.44-7.36(m,2H),3.50 (s,2H),3.00(s,3H),2.93(s,3H).LC / MS[M+H]274.1(calculated value);LC / MS[M+H]274.1(measured value).

[0306] Preparation of 2Am4CBza-L-19 To a solution of 2Am4CBza-L-19e (52.3 mg, 191 umol, 0.8 equiv) and 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(propylaminooxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (200 mg, 239 umol, 1.0 equiv, MsOH) in DCM (3 mL) and DMA (1 mL) was added EDCI (229 mg, 1.20 mmol, 5.0 equiv). The mixture was stirred at 25° C. for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was filtered and purified by preparative HPLC (TFA condition; column: Phenomenex Luna80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 10%~35%, 8 min) to obtain 2Am4CBza-L-19 (59.3mg, 59.6umol, yield 24.9%) as a pale yellow oil. 1 H NMR(MeOD,400MHz)δ7.65(d,J=8.4Hz,1H),7.48-7.44(m,2H),7.36(s,1H),6.89(s,2H) ),4.17(s,2H),4.07-4.03(m,2H),3.75(t,J=7.2Hz,2H),3.68-3.53(m,40H),3.50-3. 45(m,2H),3.44-3.35(m,4H),3.28-3.23(m,2H),3.13(s,3H),3.05(s,3H),1.78(sxt, J=7.2Hz,2H),1.01(t,J=7.6Hz,3H).LC / MS[M+H]995.5(calculated value);LC / MS[M+H]995.5(measured value).

[0307] Example 201: Preparation of immune complexes (IC) To prepare ricin-conjugated immunoconjugates, the antibodies are 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), which is then used to adjust the eluate to a concentration of approximately 1-10 mg / ml, respectively, and then sterile filtered. The antibody is pre-heated to 20-30°C and immediately mixed with 2-20 (e.g., 7-10) molar equivalents of tetrafluorophenyl (TFP) or sulfonate tetrafluorophenyl (sulfo-TFP) ester, 2-amino-4-carboxamido-benzazepine linker (2Am4CBza-L) compound of formula II dissolved at a concentration of 5-20 μM in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA). The reaction is allowed to proceed for approximately 16 hours at 30°C and the immune complex (IC) is separated from the reactants by passage over two successive G-25 desalting columns or Zeba™ spin desalting columns equilibrated in phosphate buffered saline (PBS) at pH 7.2 to obtain the immune complex (IC) of Table 3a and Table 3b. The adjuvant-antibody ratio (DAR) is determined by liquid chromatography-mass spectrometry using a C4 reverse-phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, Mass.) interfaced with a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0308] 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 with a 2-4 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 conjugation buffer. The concentration of the buffer-exchanged antibodies was adjusted to approximately 5-20 mg / ml using conjugation buffer and sterile filtered. Maleimido-2Am4CBza-L compounds are dissolved in either dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5-20 mM. For conjugation, the antibody is mixed with 10-20 molar equivalents of maleimide-2Am4CBza-L. In some cases, additional DMA or DMSO up to 20% (v / v) was added to improve the solubility of maleimide-2Am4CBza-L 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-2Am4CBza-L using two consecutive Zeba™ spin desalting columns. The columns are pre-equilibrated with phosphate buffered saline (PBS), pH 7.2. The adjuvant-antibody ratio (DAR) is estimated by liquid chromatography mass spectrometry using a C4 reversed-phase column of an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0309] For conjugation, the antibody can be dissolved in an aqueous buffer system known in the art that does not adversely affect the stability or antigen binding specificity of the antibody. Phosphate buffered saline can be used. The 2Am4CBza-L compound is dissolved in a solvent system that includes at least one polar aprotic solvent described elsewhere herein. In some such embodiments, the 2Am4CBza-L is dissolved in Tris buffer pH 8 (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 within these ranges, for example, about 5 mM to about 50 mM or about 10 mM to about 30 mM. In some embodiments, the 2-amino-4-carboxamide-benzazepine linker intermediate is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide), acetonitrile, or another suitable dipolar aprotic solvent.

[0310] Alternatively, an equivalent excess of 2Am4CBza-L solution may be diluted and combined with the antibody solution in the conjugation reaction. The 2Am4CBza-L solution may be preferably 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 equivalents of 2Am4CBza-L intermediate to antibody may 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. Completion of the reaction can be suitably monitored by methods known in the art, such as LC-MS. The conjugation reaction is usually completed within a range of about 1 hour to about 16 hours. After the reaction is completed, a reagent can be added to the reaction mixture to stop the reaction. When the antibody thiol group reacts with a thiol-reactive group, such as maleimide, of the 2Am4CBza-L linker intermediate, the unreacted antibody thiol group can react with a capping reagent. An example of a suitable capping reagent is ethylmaleimide.

[0311] After conjugation, the immune complex 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, chromatofractionation, ultrafiltration, centrifugal ultrafiltration, tangential filtration, and combinations thereof. For example, prior to purification, the immune complex can be diluted with 20 mM sodium succinate, pH 5, etc. The diluted solution is applied to a cation exchange column, followed by washing, for example, with at least 10 column volumes of 20 mM sodium succinate, pH 5. The immune complex can be suitably eluted with a buffer such as PBS.

[0312] Example 202 HEK reporter assay HEK293 reporter cells expressing human TLR7 or human TLR8 were purchased from Invivogen, and cell growth and experiments were performed according to the vendor protocol. Briefly, cells were grown to 80-85% confluence in DMEM supplemented with 10% FBS, Zeocin, and Blasticidin at 5% CO2. Cells were then plated at 4 × 10 in 96-well flat plates with HEK detection medium and substrate containing immunostimulatory 2-amino-4-carboxamido-benzoazepine compounds (such as those in Table 1). 4 Cells were seeded / well. Activity was measured at wavelengths of 620-655 nm using a plate reader.

[0313] Example 203: Evaluation of Immunoconjugate Activity In Vitro This example demonstrates that the immunoconjugates of the invention, including those in Tables 3a and 3b, are effective in inducing immune activation and are therefore useful in the treatment of cancer.

[0314] 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 cocktail (Stem Cell Technologies, Vancouver, Canada) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123 and HLA-DR. Immature APCs with purity of >90% were then purified by negative selection using EASYSEP® human monocyte enrichment kit (Stem Cell Technologies) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123 and HLA-DR, without CD16 deletion.

[0315] b) Myeloid APC activation assay: 2×10 5 APCs are incubated in 96-well plates (Corning, Corning, NY) containing 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 (ICs) of the invention (prepared according to the examples above), including those in Tables 3a and 3b. Cell-free supernatants are analyzed 18 hours later via ELISA to measure TNFα secretion as a readout for the proinflammatory response.

[0316] c) PBMC activation assay: Human peripheral blood mononuclear cells were isolated from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. PBMCs were incubated in co-culture with CEA-expressing tumor cells (e.g., MKN-45, HPAF-II) at an effector to target cell ratio of 10:1 in 96-well plates (Corning, Corning, NY). Cells were stimulated with various concentrations of unconjugated (naked) antibodies and immunoconjugates of the invention (prepared according to the examples above). Cell-free supernatants were analyzed by cytokine bead array using the LegendPlex™ kit according to the manufacturer's guidelines (BioLegend®, San Diego, CA).

[0317] 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 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 the EASYSEP™ human bone marrow DC enrichment kit (Stem Cell Technologies).

[0318] e) cDC activation assay: 8×10 4APCs were co-cultured with tumor cells expressing ISAC target antigens at an effector (cDC) to target (tumor cell) ratio of 10:1. Cells were incubated with various concentrations of the indicated immunoconjugates of the invention (prepared according to the above examples) in 96-well plates (Corning, Corning, NY) containing RPMI-1640 medium supplemented with 10% FBS, where indicated. After approximately 18 hours of overnight incubation, cell-free supernatants were collected and analyzed for cytokine secretion (including TNFα) using BioLegend LEGENDPLEX cytokine bead arrays.

[0319] Activation of myeloid cell types can be measured using various screening assays in addition to the described assays utilizing various myeloid populations. These may 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 toward an immunosuppressive state (also called myeloid-derived suppressor cells or MDSCs). MDSC-polarized cells include monocytes differentiated toward an immunosuppressive state, such as M2aΜΦ (IL4 / IL13), M2cΜΦ (IL10 / TGFb), GM-CSF / IL6 MDSC, and 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 may also include primary cells present in dissociated tumor cell suspensions (Discovery Life Sciences).

[0320] Assessment of activation of the described populations of bone marrow cells can be performed as monocultures or as cocultures with cells expressing the antigen of interest to which immune complexes (ICs) can bind via the CDR region of the antibody. After 18-48 h of incubation, activation can be assessed by upregulation of cell surface costimulatory molecules using flow cytometry or by measurement of secreted inflammatory cytokines. For cytokine measurements, cell-free supernatants are harvested and analyzed by cytokine bead arrays (e.g., LegendPlex from Biolegend) using flow cytometry.

[0321] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each such reference was individually and specifically indicated to be incorporated by reference and as if set forth in its entirety herein.

Claims

1. 2, comprising an antibody covalently attached by a linker to one or more 2-amino-4-carboxamido-benzazepine moieties, comprising formula Ia: or a pharma- ceutically acceptable salt thereof; Ab is an antibody; p is an integer from 1 to 8; X ２ is 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 １ａ and R ２ 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 or R １ａ and R １ｂ forms a 5- or 6-membered heterocyclyl ring, R ３ teeth, - (C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C １ ~C １２ alkyldiyl)-N(R ５ )-C(=O)*; - (C １ ~C １２ alkyldiyl)-N(R ５ )-C(=O)O-(C ３ ~C １２ Carbocyclyldiyl)-*; - (C １ ~C １２ alkyldiyl)-N(R ５ )-(C １ ~C ２０ Heteroaryldiyl)-*; - (C １ ~C １２ alkyldiyl)-N(R ５ )-(C １ ~C ２０ Heteroaryldiyl)-(C １ ~C １２ Alkyldiyl)-*; - (C １ ~C １２ alkyldiyl)-N(R ５ )-S(O ２ ) - *; - (C １ ~C １２ alkyldiyl)-OC(=O)-(C ２ ~C ９ Heterocyclyldiyl)-*; - (C １ ~C １２ alkyldiyl)-O-*; - (C １ ~C １２ Alkyldiyl)-(C ３ ~C １２ Carbocyclyldiyl)-*; - (C １ ~C １２ Alkyldiyl)-(C ６ ~C ２０ Alkyldiyl)-*; - (C １ ~C １２ Alkyldiyl)-(C ６ ~C ２０ Aryl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C １ ~C １２ Alkyldiyl)-(C ２ ~C ９ Heterocyclyldiyl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C １ ~C １２ Alkyldiyl)-(C １ ~C ２０ Heteroaryldiyl)-N(R ５ ) - *; - (C １ ~C １２ Alkyldiyl)-(C １ ~C ２０ Heteroaryldiyl)-*; - (C １ ~C １２ Alkyldiyl)-(C １ ~C ２０ Heteroaryldiyl)-(C １ ~C １２ Alkyldiyl)-*; - (C １ ~C １２ Alkyldiyl)-(C １ ~C ２０ Heteroaryldiyl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C ３ ~C １２ Carbocyclyldiyl)-*; - (C ３ ~C １２ Carbocyclyldiyl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C ３ ~C １２ Carbocyclyldiyl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C ３ ~C １２ Carbocyclyldiyl)-NR ５ -C(=NR ５ａ )-N(R ５ ) - *; - (C ６ ~C ２０ Aryldiyl)-*; - (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 ) -C(=NR ５ａ )-N(R ５ ) - *; - (C ２ ~C ２０ Heterocyclyldiyl)-*; - (C ２ ~C ９ Heterocyclyldiyl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C ２ ~C ９ Heterocyclyldiyl)-N(R ５ ) -C(=NR ５ａ )-N(R ５ ) - *; - (C １ ~C ２０ Heteroaryldiyl)-*; - (C １ ~C ２０ Heteroaryldiyl)-(C １ ~C １２ alkyldiyl)-N(R ５ ) - *; - (C １ ~C ２０ Heteroaryldiyl)-(C １ ~C １２ alkyldiyl)-O-*; and - (C １ ~C ２０ Heteroaryldiyl)-N(R ５ ) -C(=NR ５ａ )-N(R ５ )-*; The asterisk * indicates the attachment site of the linker L. Or, R ２ and R ３ together form a 5- or 6-membered heterocyclyl ring, R ５ are independently H, 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; L is, -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 2015-04-2016 10:00:00 (1998-2016) -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)-; 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: having PEP has the formula: wherein AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA and the adjacent nitrogen atom form a 5-membered proline amino acid ring, and the wavy line indicates the point of attachment; Cyc is F, Cl, NO ２ , -OH-, OCH ３ and structure: Optionally substituted with one or more groups selected from glucuronic acid having the formula: 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 is 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 are independently 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; 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(OH)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, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H.

2. The immune complex of claim 1, wherein the antibody is an antibody construct having an antigen-binding domain that binds to PD-L1, HER2, CEA, or TROP2.

3. 3. The immunoconjugate of claim 2, wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, labetuzumab and sacituzumab.

4. R １ａ is an optionally substituted C ６ ~C ２０ Aryl, C ２ ~C ９ Heterocyclyl and C １ ~C ２０ The immunoconjugate of any one of claims 1 to 3, wherein the immunoconjugate is selected from the group consisting of heteroaryl.

5. R １ａ is optionally substituted C ６ ~C ２０ aryl, R １ｂ The immune complex according to any one of claims 1 to 3, wherein

6. R ３ is C １ ~C ８ Alkyl, -(C １ ~C １２ Alkyldiyl)-OR ５ , -(C １ ~C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , and -(C １ ~C １２ alkyl)-OC(O)N(R ５ ) ２ The immune complex according to any one of claims 1 to 3, selected from the group consisting of:

7. X ２ is a bond, R ２ is C １ ~C １２ The immunoconjugate of any one of claims 1 to 3, which is alkyl.

8. R ３ Ha-(C １ ~C １２ alkyldiyl)-N(R ５ 4. The immune complex according to claim 1, wherein said immunoconjugate is selected from the group consisting of 1)-*.

9. R ３ Ha-CH ２ CH ２ CH ２ The immune complex of claim 8, which is NH-.

10. The immunoconjugate of claim 8, wherein L is -C(=O)-PEG-C(=O)-.

11. The immunoconjugate of any one of claims 1 to 3, wherein L comprises PEG.

12. The immune complex of claim 11, wherein n is 10 and m is 1.

13. A.A. １ and A.A. ２ The immunoconjugate of any one of claims 1 to 3, wherein:

14. A.A. １ or A.A. ２ The immunoconjugate of claim 13 , wherein, together with the adjacent nitrogen atom, forms a five-membered ring proline amino acid.

15. A.A. １ and A.A. ２ is H, -CH ３ , -CH(CH ３ ) ２ , -CH ２ (C ６ H ５ ), -CH ２ CH ２ CH ２ CH ２ N.H. ２ , -CH ２ CH ２ CH ２ NHC (NH) NH ２ , -CHCH(CH ３ ) CH ３ , -CH ２ SO ３ H, and -CH ２ CH ２ CH ２ NHC(O)NH ２ The immunoconjugate of claim 13, independently selected from the group consisting of:

16. A.A. １ HA -CH(CH ３ ) ２ and A.A. ２ Ha-CH ２ CH ２ CH ２ NHC(O)NH ２ The immune complex of claim 15, 17. A 2-amino-4-carboxamido-benzazepine linker compound selected from:

18. 20. An immunoconjugate prepared by conjugating an antibody with the 2-amino-4-carboxamido-benzazepine linker compound of claim 17.

19. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 3 and a pharma- ceutically acceptable diluent, vehicle, carrier or excipient.

20. 4. A medicament for treating cancer comprising a therapeutically effective amount of the immunoconjugate of any one of claims 1 to 3, wherein the cancer is selected from bladder cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

21. The pharmaceutical of claim 20, wherein the cancer is susceptible to a proinflammatory response induced by TLR7 and / or TLR8 agonism.

22. The pharmaceutical composition of claim 20, wherein the cancer is triple-negative breast cancer, metastatic Merkel cell carcinoma, or gastroesophageal junction adenocarcinoma.

23. A method for preparing an immunoconjugate of formula Ia according to any one of claims 1 to 3, wherein the 2-amino-4-carboxamido-benzazepine linker compound according to claim 17 is conjugated to an antibody.

Images