Antibody-conjugated 8-sulfonyl-benzazepine compounds and their uses

Abstract

The present invention provides immunoconjugates of formula (I) comprising an antibody linked by conjugation to one or more 8-sulfonyl-2-aminobenzazepine derivatives. The present invention also provides 8-sulfonyl-2-aminobenzazepine derivative intermediate compositions comprising reactive functional groups. Such intermediate compositions are suitable substrates for the formation of the present immunoconjugates via a linker or linking moiety. The present invention further provides methods of treating cancer using the present immunoconjugates.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This non-provisional application claims the benefit of priority to U.S. Provisional Application No. 63 / 308,275, filed February 9, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application has been submitted in XML format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The above XML copy, created on Jan. 24, 2023, is named 17019.023WO1-TW1 and is 13,070 bytes in size.

[0003] The present invention relates generally to immunoconjugates comprising an antibody conjugated to one or more 8-sulfonyl-benzazepine molecules. [Background technology]

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

[0005] The present invention is generally directed to an immunoconjugate comprising an antibody selected from anti-PD-L1, anti-HER2, anti-CEA, and anti-TROP2, covalently attached by a linker to one or more 8-sulfonyl-benzazepine TLR agonist moieties having the formula: [ka] In the formula, R 1 , R 2 , R 3 , and R 4 is attached to L. The various substituents are defined herein.

[0006] Another aspect of the invention is a method of preparing an immunoconjugate by conjugation of one or more 8-sulfonyl-benzazepine-linker compounds with an antibody selected from anti-PD-L1, anti-HER2, anti-CEA, and anti-TROP2.

[0007] Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate comprising an antibody selected from anti-PD-L1, anti-HER2, anti-CEA, and anti-TROP2 covalently attached by a linker to one or more 8-sulfonyl-benzazepine moieties, and one or more pharma- ceutically acceptable diluents, vehicles, carriers, or excipients.

[0008] Another aspect of the invention is 8-sulfonyl-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 selected from anti-PD-L1, anti-HER2, anti-CEA, and anti-TROP2, covalently attached by a linker to one or more 8-sulfonyl-benzazepine moieties.

[0010] Another aspect of the invention is the use of an immunoconjugate comprising an antibody selected from anti-PD-L1, anti-HER2, anti-CEA, and anti-TROP2 covalently attached by a linker to one or more 8-sulfonyl-benzazepine moieties, in the treatment of disease, particularly cancer. [Brief description of the drawings]

[0011] [Figure 1] Graph of secreted TNFα (tumor necrosis factor alpha) cytokine levels following incubation of various concentrations of immunoconjugate IC-3, comparative CIC-1, and naked antibody TROP2 with co-cultures of cancer cells and cDC-enriched primary cell isolates. [Diagram 2]Graph of secreted TNFa (tumor necrosis factor alpha) cytokine levels after incubation of various concentrations of immunoconjugate IC-3, comparative CIC-2, and naked antibody TROP2 with co-cultures of cancer cells and cDC-enriched primary cell isolates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

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

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

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

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

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

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

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

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

[0021] "Antibody" refers to a polypeptide that contains an antigen-binding region (including complementarity determining regions (CDRs)) derived from an immunoglobulin gene or a fragment thereof. The term "antibody" specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa) connected by disulfide bonds. Each chain is composed of structural domains called immunoglobulin domains. These domains are characterized by size and function, e.g., the variable domains or regions on the light and heavy chains (V and V, respectively). L and V H ), and constant domains or regions on the light and heavy chains (C L and C H) are classified into different categories. 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 each to a light chain by disulfide bonds. The resulting tetramer has two identical halves that together form a Y-shaped shape. 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.

[0022] 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 with at least one antigen-binding region that targets a specific antigen. In some embodiments, the targeting monoclonal antibody is a bispecific antibody with 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).

[0023] In some embodiments, the antibody construct is an antigen-binding antibody "fragment," which comprises at least the antigen-binding region of an antibody, either alone or together with other components that together constitute the antibody construct. For example, (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region, and (iii) a V fragment consisting of a single arm of an antibody. L and V H (iv) Fab' fragments resulting from disrupting the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) two domains of the Fv fragment (i.e., V and VF) linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain. L and V HMany different types of antibody "fragments" are known in the art, including single-chain Fvs (scFvs), which are monovalent molecules consisting of (i) an antigen-binding domain and (ii) an Fc domain. In some embodiments, an antibody construct refers to an antibody or fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0024] 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 to PD-L1. In yet another embodiment, an antibody fragment is part of a bispecific T cell engager (BiTE) that includes a domain that binds CD1 or CD3 and a linker.

[0025] In some embodiments, the antibody construct comprises an Fc domain. In certain embodiments, the antibody construct is an antibody. In certain embodiments, the antibody construct is a fusion protein. The antigen-binding domain can be a single chain variable fragment (scFv). A single chain variable fragment (scFv) is a truncated Fab fragment comprising a variable (V) domain of an antibody heavy chain linked to a 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-CEA antibody, each variable region comprising CDR1, CDR2, and CDR3.

[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 methods (Junutula, et al., (2008b) Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; US2012 / 0121615; WO2009 / 052249). The cysteine ​​residue provides site-specific conjugation of adjuvants such as TLR agonists to the antibody via reactive cysteine ​​thiol groups at the engineered cysteine ​​sites, but does not interfere with immunoglobulin folding and assembly or alter antigen binding and effector functions. 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), which 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 that binds IgG, (2) FcαR that binds IgA, and (3) FcεR that binds IgE. The FcγR family includes several members, such as FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). Fcγ receptors have different affinities for IgG and for IgG subclasses (e.g., IgG1, IgG2, IgG3, IgG4).

[0029] The "identity" of a nucleic acid or amino acid sequence referred to herein can 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-Coffee, ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity search). Sequence alignment algorithms are described, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009); Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge The percent sequence identity is calculated, for example, as follows: 100×[(identical positions) / min(TG A , T.G. B)], where TG A and T.G. B T.G. A and T.G. B is the sum of the number of residues and internal gap positions of peptide sequences A and B in the alignment that minimizes (see, e.g., Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0030] An "antibody construct" or "binding agent" comprises Ig heavy and light chain variable region polypeptides that together form an antigen-binding site. The heavy and light chain variable regions are each polypeptides that comprise three complementarity determining regions (CDR1, CDR2, and CDR3) linked by framework regions. The antibody construct can be any of the various types of binding agents known in the art that comprise Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment," or a T-cell receptor.

[0031] "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).

[0032] "Biobetter" refers to an approved antibody construct that is an improvement over a previously approved antibody construct such as atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and labetuzumab. A biobetter can have one or more modifications (e.g., an altered glycan profile, or a unique epitope) relative to the previously approved antibody construct.

[0033] "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 having the same molecular formula and intramolecular bonds, but a different 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.

[0034] 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 D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), and D-leucine. (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0035] Naturally occurring amino acids include those formed in proteins by post-translational modifications, such as citrulline (Cit).

[0036] 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, either in the L- or D-configuration, that function in a manner similar to naturally occurring 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, an amino group), but has a modified side group or peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a similar manner to a natural amino acid.

[0037] "Linker" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linking moiety can serve to covalently bond an adjuvant moiety to an antibody construct in an immunoconjugate.

[0038] "Linking moiety" refers to a functional group that covalently bonds two or more moieties in a compound or material. For example, a linking moiety can serve to covalently link an adjuvant moiety to an antibody in an immunoconjugate. Useful bonds for connecting linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0039] "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.

[0040] Wavy line ( [ka] ) represents the point of attachment of a particular chemical moiety. [ka] ), it is understood that the chemical moiety can be used in both ways, that is, read from left to right or right to left. In some embodiments, the two wavy lines ( [ka] ) are to be considered to be used reading from left to right.

[0041] "Alkyl" refers to a straight chain (linear) or branched saturated aliphatic radical having the indicated number of carbon atoms. 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, -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 (-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 (-C(CH3)2CH2CH3), 3-methyl-1-butyl (-CH2CH2CH(CH3)2 ... H2CH2CH2CH2CH2CH3), 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 (-C(CH3)(CH2CH3)2), Examples of "substituted alkyl" groups include butyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, 1-heptyl, and 1-octyl. 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.

[0042] The term "alkyldiyl" refers to a divalent alkyl radical. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), and the like. Alkyldiyl groups are sometimes referred to as "alkylene" groups.

[0043] "Alkenyl" refers to a straight (linear) or branched, unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon double bond, sp2. Alkenyl can contain from 2 to about 12 or more carbon atoms. Alkenyl groups are radicals having "cis" and "trans" orientations, 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 substituted or unsubstituted. "Substituted alkenyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0044] 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-), and the like.

[0045] "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.

[0046] The terms "alkynylene" or "alkynyldiyl" refer to a divalent alkynyl radical.

[0047] The terms "carbocycle", "carbocyclyl", "carbocycle", and "cycloalkyl" refer to saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assemblies containing 3 to 12 ring atoms or the indicated number of atoms. Saturated monocyclic 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 carbocyclic groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene.

[0048] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0049] "Aryl" means an aromatic ring system having 6 to 20 carbon atoms (C6-C8) derived by the removal of a hydrogen atom from a carbon atom of a parent aromatic ring system. 20 ) refers to a monovalent aromatic hydrocarbon radical. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl, which has a methylene linking group. Some aryl groups, such as phenyl, naphthalene, or biphenyl, have 6 to 12 ring members. Other aryl groups, such as phenyl or naphthyl, have 6 to 10 ring members.

[0050] "Arylene" or "aryldiyl" means an aryl group of 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 in the exemplary structures as "Ar". 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 as described herein.

[0051] 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, where at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally substituted independently with one or more substituents described below. The heterocycle may be a monocycle of 3 to 7 members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicycle 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.

[0052] 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 three 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 one or more ring atoms are optionally substituted independently with one or more substituents as described. Five- and six-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl.

[0053] The term "heteroaryl" refers to a 5-, 6-, or 7-membered monovalent aromatic radical, 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.

[0054] 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.

[0055] The heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked, where possible. By way of example and not limitation, the carbon-linked heterocycle or heteroaryl is bonded at the 2-, 3-, 4-, 5-, or 6-position of pyridine, the 3-, 4-, 5-, or 6-position of pyridazine, the 2-, 4-, 5-, or 6-position of pyrimidine, the 2-, 3-, 5-, or 6-position of pyrazine, the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole, the 2-, or 3-position of aziridine, the 2-, 3-, or 4-position of azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline.

[0056] 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.

[0057] The terms "halo" or "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0058] 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 within the carbonyl-containing moiety.

[0059] 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).

[0060] The terms "treat", "treatment", and "treating" refer to any indicia of success in the treatment or amelioration of 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 the symptom, reducing the frequency or duration of the symptom or condition, or, in some circumstances, preventing the onset of the symptom. Treatment or amelioration of the symptom can be based on any objective or subjective parameter, including the results of a physical examination.

[0061] 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.

[0062] 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, including both primary and metastatic tumors).

[0063] "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.

[0064] "HER2" refers to the protein human epidermal growth factor receptor 2.

[0065] "HER2 expression" refers to a cell that has a HER2 receptor on its surface. For example, a cell may have about 20,000 to about 50,000 HER2 receptors on its surface. As used herein, "HER2 overexpression" refers to a cell 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%.

[0066] The "pathology" of cancer includes all phenomena that compromise the well-being of the patient, including, but not limited to, abnormal or uncontrolled cell proliferation, metastasis, interference with the normal function of neighboring cells, release of abnormal levels of cytokines or other secretory products, suppressed or exacerbated inflammatory or immunological responses, neoplasms, premalignancies, malignant tumors, and invasion of surrounding or distant tissues or organs, such as lymph nodes.

[0067] 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.

[0068] 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 the departure of cancer cells from the original tumor site, as well as the several stages of the process in which cancer cells migrate and / or invade other parts of the body.

[0069] The phrases "effective amount" and "therapeutically effective amount" refer to the dose or amount of a substance, such as an immunoconjugate, that produces the therapeutic effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one of 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 ed., 1999). th Edition (McGraw-Hill, 2006), and The Science and Practice of Pharmacy, 22 ndEdition, (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).

[0070] 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 treatment, "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.

[0071] 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.

[0072] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or implantation of a sustained release device, e.g., a mini-osmotic pump, into the subject.

[0073] The terms "about" and "approximately" used herein to modify a numerical value indicate a close range around that numerical value. Thus, when "X" is a value, "about X" or "approximately X" indicates a value of 0.9X to 1.1X, such as 0.95X to 1.05X, or 0.99X to 1.01X. Reference to "about X" or "approximately X" specifically indicates at least the values ​​X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "approximately X" are intended to teach and provide written descriptive support for the limitations of the claims, such as "0.98X".

[0074] antibody 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.

[0075] 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.

[0076] Several antibodies targeting PD-L1, including atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™), have been developed for the treatment of cancer. Nevertheless, there remains a need for new PD-L1 antibody constructs, including agents that bind to PD-L1 with high affinity and effectively prevent PD-L1 / PD-1 signaling, and agents that can deliver therapeutic payloads to PD-L1-expressing cells. In addition, there is a need for novel PD-L1 binding agents for the treatment of autoimmune and infectious diseases.

[0077] Methods are provided for delivering a TLR agonist payload to a cell expressing PD-L1, comprising administering to the cell or a mammal comprising the cell an immunoconjugate comprising an anti-PD-L1 antibody covalently linked to a linker that is covalently linked to one or more TLR agonist moieties.

[0078] Also provided are methods for enhancing or reducing or inhibiting an immune response in a mammal, and methods for treating a disease, disorder, or condition in a mammal that responds to PD-L1 inhibition, comprising administering to the mammal the PD-L1 immunoconjugate.

[0079] 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.

[0080] 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. In addition, diabodies, triabodies, or tetrabodies, which are dimers, trimers, or tetramers of polypeptide chains, respectively, can be provided, which are composed of V and V+ domains 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 to H , 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, generating bispecific bis-scFv fragments capable of binding to two different epitopes. Fab dimers (Fab2) and Fab trimers (Fab3) can be generated using genetic engineering methods to create multispecific constructs based on Fab fragments.

[0081] 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).

[0082] 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.

[0083] 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.

[0084] Several anti-HER2 monoclonal antibodies have been approved and are in clinical development (Costa, RLB et al (2020) Breast Cancer 6 (10): 1-11).

[0085] In certain embodiments, the immunoconjugates of the invention comprise an anti-HER2 antibody, such as those prepared by the method of Example 201. In one embodiment of the invention, the anti-HER2 antibody of the immunoconjugates 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 US5821337, which is specifically incorporated herein by reference. These antibodies comprise human framework regions with the complementarity determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also known as trastuzumab and is commercially available under the trade name HERCEPTIN™ (Genentech, Inc.).

[0086] Trastuzumab (CAS 180288-69-1, huMAb4D5-8, rhuMAb HER2, HERCEPTIN®, Genentech, Inc.) binds to the extracellular domain of HER2 (US5677171, US5821337, US6054297, US6165464, US6339142, US6407213, US6639055, US6719971, US6800738, US7074404, Coussens et al (1985) Science 230:1132-9, Slamon et al (1989) Science 244:707-12, Slamon et al (2001) New It is a recombinant DNA-derived IgG1 kappa, monoclonal antibody that is a humanized version of a murine anti-HER2 antibody (4D5) that selectively binds with high affinity to HER2 (Engl. J. Med. 344:783-792).

[0087] 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.

[0088] 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 US7862817. 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 HER2 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.

[0089] 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.

[0090] Margetuximab (MGAH22, MARGENZA™, MacroGenics, Inc.), CAS Registry Number 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.

[0091] 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).

[0092] 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.

[0093] 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 also used as a blood marker for many carcinomas. Labetuzumab (CEA-CIDE™, Immunomedics, CAS Registry Number 219649-07-7), also known as MN-14 and hMN14, is a humanized IgG1 monoclonal antibody that is being investigated for the treatment of colorectal cancer (Blumenthal, R. et al (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analog (labetuzumab govitecan, IMMU-130) targets carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and has been studied in patients with recurrent or refractory metastatic colorectal cancer (Sharkey, R. et al, (2018), Molecular Cancer Therapeutics 17(1):196-203; Cardillo, T. et al (2018) Molecular Cancer Therapeutics 17(1):150-160). In one embodiment of the present invention, the CEA-targeting antibody construct or antigen-binding domain comprises the variable light chain (VL kappa) of hMN-14 / labetuzumab, as disclosed in US6676924, which is incorporated herein by reference for this purpose.

[0094] In an exemplary embodiment, the immunoconjugate of the present invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds TROP2. Tumor-associated calcium signal transducer 2 (TROP-2) 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). TROP2 is an intracellular calcium signal transducer that is differentially expressed in many cancers and signals cells for self-renewal, proliferation, invasion, and survival. TROP2 is considered a stem cell marker and 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 is of prognostic importance. 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.

[0095] 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. The existence of a cell membrane protein involved in immune resistance common to human trophoblast cells and cancer cells has been previously suggested (Faulk WP, et al., Proc. Natl. Acad. Sci. 75(4): 1947-1951 (1978)). We identified an antigen molecule recognized by a monoclonal antibody against a cell membrane protein in a human choriocarcinoma cell line and designated TROP2 as one of the molecules expressed in human trophoblast cells (Lipinski M, et al., Proc. Natl. Acad. Sci. 78(8), 5147-5150 (1981)). This molecule was also named as tumor antigen GA733-1 (Linnenbach AJ, et al., Proc. Natl. Acad. Sci. 86(1), 27-31(1989)), recognized by mouse monoclonal antibody GA733 obtained by immunization with gastric cancer cell lines, or as 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 TROP2 gene was cloned and all of these molecules were confirmed to be the same molecule (Fornaro M, et al., Int. J. Cancer, 62(5), 610-618(1995)). The DNA and amino acid sequences of hTROP2 are published in public databases and can be referenced, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0096] In response to such information suggesting a link with cancer, several anti-hTROP2 antibodies have been established and their antitumor effects have been studied. Among these antibodies, for example, non-binding 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 together with cytotoxic drugs (WO2003 / 074566, WO2011 / 068845, WO2013 / 068946, US7999083) have been disclosed. However, the strength or scope of their activity is still insufficient, and there is an unmet medical need to target hTROP2 for therapy.

[0097] TROP2 expression in cancer cells correlates with drug resistance. Several strategies target TROP2 on cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, etc. In vitro and preclinical studies using these various therapeutic treatments have significantly suppressed tumor cell growth both in vitro and in vivo in mice. Clinical studies are exploring the potential application of TROP2 as both a prognostic biomarker and a therapeutic target to reverse resistance.

[0098] 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 (US2016 / 0297890, WO2015 / 098099).

[0099] In one embodiment of the present invention, the TROP2 targeting antibody construct or antigen binding domain comprises the light chain CDRs (complementarity determining regions) of hRS7 (humanized RS7) (US7238785, incorporated herein by reference).

[0100] In one embodiment of the invention, the TROP2-targeting antibody construct or antigen-binding domain comprises a light chain CDR (complementarity determining region) or light chain framework (LFR) sequence selected from SEQ ID NOs: 1-7. [Table 1]

[0101] In one embodiment of the present invention, the TROP2-targeting antibody construct or antigen-binding domain comprises a heavy chain CDR (complementarity determining region) or heavy chain framework (HFR) sequence selected from SEQ ID NOs: 8-14. [Table 2]

[0102] In some embodiments, the antibody in the immune complex contains a modified Fc region, where the modification modulates binding of the Fc region to one or more Fc receptors.

[0103] 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 US9676863, which is incorporated herein. An "Fc linker" may be used to link the IgG to the TGFβRII extracellular domain. The Fc linker may 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 an alanine.

[0104] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0105] In some embodiments, the antibody in the immunoconjugate is a cysteine ​​engineered antibody that provides for site-specific conjugation of adjuvants, labels, or drug moieties via cysteine ​​substitution at sites where the engineered cysteine ​​is available for conjugation but does not perturb immunoglobulin folding and assembly, or antigen binding and effector function (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; US7521541; US7723485; US2012 / 0121615; WO2009 / 052249). Cysteine ​​engineered antibodies can be conjugated to 8-sulfonyl-2-aminobenzazepine adjuvant moieties as 8-sulfonyl-2-aminobenzazepine-linker compounds with uniform stoichiometry (e.g., for antibodies with a single engineered cysteine ​​site, up to two 8-sulfonyl-2-aminobenzazepine moieties per antibody).

[0106] 8-Sulfonyl-2-aminobenzazepine adjuvant compounds The immunoconjugates of the present invention include an 8-sulfonyl-2-aminobenzazepine adjuvant moiety. The adjuvant moieties described herein are compounds that induce an immune response (i.e., immunostimulants). Generally, the adjuvant moieties described herein are TLR agonists. TLRs are type I transmembrane proteins involved in the initiation of innate immune responses in vertebrates. TLRs recognize 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 engaged (e.g., by natural stimuli or synthetic TLR agonists), TLRs initiate a signaling cascade that leads to activation of nuclear factor-κB (NF-κB) via the adaptor protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of IL-1 receptor-associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF-receptor associated factor 6 (TRAF6), which leads to 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 the 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 IRF5 and IRF7, while the TRIF-dependent pathway also activates the NF-κB pathway.

[0107] 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 myeloid origin, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 can detect the presence of "foreign" single-stranded RNA within cells as a means of responding to viral invasion. Treatment of TLR8-expressing cells with 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.

[0108] An exemplary 8-sulfonyl-2-aminobenzazepine compound of the invention (8SO2Bz) was synthesized, purified, characterized by mass spectrometry, and shown to have the expected mass. Additional experimental procedures are found in the Examples. Activity against HEK293 NFKB reporter cells expressing human TLR7 or human TLR8 was measured according to Example 202. Certain 8-sulfonyl-2-aminobenzazepine compounds demonstrate surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0109] 8-Sulfonyl-2-aminobenzazepine-linker compounds The immunoconjugates of the present invention are prepared by conjugation of an antibody with 8-sulfonyl-2-aminobenzazepine-linker compound 8SO2Bz-L. The 8-sulfonyl-2-aminobenzazepine-linker compound comprises an 8-sulfonyl-2-aminobenzazepine (8SO2Bz) moiety covalently attached to a linker unit. The linker unit comprises functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. The linker unit comprises a polyethyleneoxy (PEG) group. The linker unit comprises a reactive functional group that reacts with, 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 a reactive electrophilic functional group of the 8SO2Bz-L compound to form an immunoconjugate. Also, for example, a cysteine ​​thiol of an antibody reacts with a maleimide or bromoacetamide group of the 8SO2Bz-L linker compound to form an immunoconjugate.

[0110] Suitable reactive electrophilic functional groups (Q in formula II) for the 8SO2Bz-L linker compounds include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N-hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive), carbodiimides (amine and carboxyl reactive), hydroxymethylphosphines (amine reactive), maleimides (thiol reactive), halogenated acetamides such as N-iodoacetamide (thiol reactive), aryl azides (primary amine reactive), fluorinated aryl azides (reactive via hydrocarbon (CH) insertion), pentafluorophenyl (PFP) esters (amine reactive), terafluorophenyl (TFP) esters (amine reactive), imidoesters (amine reactive), isocyanates (hydroxyl reactive), vinyl sulfones (thiol, amine, and hydroxyl reactive), pyridyl disulfides (thiol reactive), and benzophenone derivatives (reactive via C-H bond insertion). Additional reagents include those described in Hermanson, Bioconjugate Techniques, 2 nd Edition, Academic Press, 2008.

[0111] The present invention provides solutions to limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers may be unstable in the bloodstream, thereby releasing 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. In other words, bloodstream stability and intracellular release are usually inversely related. In addition, 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 generally correlates positively to the number of equivalents of adjuvant / drug moiety and its derivatives that are 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 immunoconjugate yields and make process scale-up difficult.

[0112] Exemplary embodiments include 8-sulfonyl-2-aminobenzazepine-linker compounds of formula II: [ka] During the ceremony, R 1 , R 2 , R 3 , and R 4 are independent, H, C1-C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6-C 20 Aryl, C2-C9 heterocyclyl, and C1-C 20 heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally selected from the group consisting of: -(C1-C 12 Alkyldiyl)-N(R5 )-*, -(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C1-C 12 Alkyldiyl)-OR 5 , -(C3-C 12 carbocyclyl), -(C3-C 12 Carbocyclyl)-*, -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*, -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C3-C 12 Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*, -(C6-C 20 aryl), -(C6-C 20 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 )2, -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -C(=NR 5a)N(R 5 )-*, -(C2-C 20 heterocyclyl), -(C2-C 20 Heterocyclyl)-*, -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*, -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C2-C9 heterocyclyl)-C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*, -(C2-C9 heterocyclyl)-NR 5 -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -(C2-C9 heterocyclyl)-(C6-C 20 Aryldiyl)-*, -(C1-C 20 Heteroaryl), -(C1-C 20 Heteroaryldiyl)-*, -(C1-C 20 Heteroaryl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -(C1-C 20 Heteroaryl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C1-C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C1-C 20 Heteroaryl)-N(R5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -C(=O)-*, -C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -C(=O)-(C2-C 20 Heterocyclyldiyl)-*, -C(=O)N(R 5 )2, -C(=O)N(R 5 )-*, -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-N(R 5 )C(=O)R 5 , -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-N(R 5 )C(=O)N(R 5 )2, -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-N(R 5 )CO2R 5 , -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 )2, -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 , -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 5 (C2-C5 heteroaryl), -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-N(R 5 )-*, -C(=O)NR5 -(C1-C 20 Heteroaryldiyl)-*, -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -*, -N(R 5 )2, -N(R 5 )-*, -N(R 5 )C(=O)R 5 , -N(R 5 )C(=O)-*, -N(R 5 )C(=O)N(R 5 )2, -N(R 5 )C(=O)N(R 5 )-*, -N(R 5 )CO2R 5 , -NR 5 C(=NR 5a )N(R 5 )2, -NR 5 C(=NR 5a )N(R 5 )-*, -NR 5 C(=NR 5a )R 5 , -N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -N(R 5 )-(C2-C5 heteroaryl), -N(R 5)-S(=O)2-(C1-C 12 alkyl), -O-(C1-C 12 alkyl), -O-(C1-C 12 Alkyldiyl)-N(R 5 )2, -O-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -OC(=O)N(R 5 )2, -OC(=O)N(R 5 )-*, -O-(R 5 )-*, -OR 5 , -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -*, and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 or substituted with one or more groups selected from: or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring, X 1 , X 2 , X 3 , and X 4 are independently bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, and S(O)2N(R 5 ) selected from the group consisting of R 5 are independent, H, C6-C 20 Aryl, C3-C12 Carbocyclyl, C6-C 20 Aryldiyl, C1-C 12 Alkyl, and C1-C 12 or two R 5 the groups taken together form a 5- or 6-membered heterocyclyl ring, R 5a But, C6-C 20 Aryl and C1-C 20 Heteroaryl; where the asterisk * indicates the binding site of L and R 1 , R 2 , R 3 , and R 4 One of the is bonded to L, L, 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(R6 )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)-, 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(PEG-COH)-PEG-N(R 5 )-, Q-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(PEG-CO2H)-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-N(PEG-CO2H)-PEG-C(=O)-, Q-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(PEG-CO2H)-PEG-C(=O)-, Q-(CH2) m -C(=O)N(R 6 )-PEG-C(═O)-PEP-, and Q-(CH2) m -C(=O)N(R 6 )-PEG-SS-(C1-C 12 the linker being selected from the group consisting of: -OC(=O)-, R 6 is 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 3. The method of claim 2, wherein the PEP has the formula: [ka] having wherein AA are independently selected from natural or unnatural amino acid side chains, 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 optionally has F, Cl, NO2, -OH, -OCH3, and the structure: [ka] Glucuronic acid having the formula: 20 Aryldiyl and C1-C 20 heteroaryldiyl; R 7 But -CH(R 8 )O-, -CH2-, -CH2N(R 8 )-, and -CH(R8 )OC(=O)-(wherein, R 8 is H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and -C(=O)N(R 9 )2, wherein R 9 are independent, H, C1-C 12 Alkyl, and -(CH2CH2O) n -(CH2) m -OH, wherein m is an integer from 1 to 5 and 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, -CH2 CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3) 2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN , -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH( OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2 , -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n 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.

[0113] An exemplary embodiment of the 8-sulfonyl-2-aminobenzazepine-linker compound of formula II is where Q is [ka] The present invention includes being selected from the following:

[0114] Exemplary embodiments of the 8-sulfonyl-2-aminobenzazepine-linker compounds of formula II include where Q is phenoxy substituted with one or more F.

[0115] Exemplary embodiments of the 8-sulfonyl-2-aminobenzazepine-linker compounds of formula II include where Q is 2,3,5,6-tetrafluorophenoxy.

[0116] Exemplary embodiments of 8-sulfonyl-2-aminobenzazepine-linker (8SO2BzL) compounds are selected from Table 2a. Each compound is synthesized, purified, characterized by mass spectrometry, and shown to have the indicated mass. Additional experimental procedures are found in the Examples. The 8-sulfonyl-2-aminobenzazepine-linker compounds of Table 2a demonstrate surprising and unexpected properties of TLR8 agonist selectivity that may predict useful therapeutic activity for treating cancer and other disorders. The 8-sulfonyl-2-aminobenzazepine-linker intermediate, a compound of formula II in Table 2a, is used to conjugate with an antibody by the method of Example 201 to form the immunoconjugate of Table 3a. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12]

[0117] The comparative compounds in Table 2b have activated ester, tetrafluorophenyl, or sulfotetrafluorophenyl groups that react with lysine residues of antibodies to form immunoconjugates with amide bonds between the antibody and the TLR-agonist-linker moiety according to Example 201. [Table 5]

[0118] 8-Sulfonyl-benzazepine immunoconjugates Immunostimulatory antibody complexes, i.e., immune complexes, target TLR7 / 8 agonists into tumors to activate tumor-infiltrating myeloid cells and initiate broad innate and adaptive anti-tumor immune responses (Ackerman, et al., (2021) Nature Cancer 2:18-33).

[0119] An exemplary embodiment of the immunoconjugate comprises an antibody covalently attached to one or more 8-sulfonyl-2-aminobenzazepine moieties by a linker and has Formula I: Ab-[LD] p I or a pharma- ceutically acceptable salt thereof; During the ceremony, Ab is the antibody, p is an integer from 1 to 8; L is the linker, D is a compound of the formula: [ka] and wherein the 8-sulfonyl-2-aminobenzazepine moiety has the formula: R 1 , R 2 , R 3 , and R 4 are independent, H, C1-C 12 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 12 Carbocyclyl, C6-C 20 Aryl, C2-C9 heterocyclyl, and C1-C 20 heteroaryl, where alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally selected from the group consisting of: -(C1-C 12 Alkyldiyl)-N(R 5 )-*, -(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C1-C 12 Alkyldiyl)-OR 5 , -(C3-C 12 carbocyclyl), -(C3-C 12 Carbocyclyl)-*, -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*, -(C3-C 12 Carbocyclyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C3-C 12 Carbocyclyl)-NR 5 -C(=NR 5 )NR 5 -*, -(C6-C 20 aryl), -(C6-C 20 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 )2, -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C2-C 20 heterocyclyl), -(C2-C 20 Heterocyclyl)-*, -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-NR 5 -*, -(C2-C9 heterocyclyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C2-C9 heterocyclyl)-C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -(C2-C9 heterocyclyl)-NR 5 -C(=NR 5a )NR 5 -*, -(C2-C9 heterocyclyl)-NR 5 -(C6-C 20 Aryldiyl)-(C1-C 12 Alkyldiyl)-N(R5 )-*, -(C2-C9 heterocyclyl)-(C6-C 20 Aryldiyl)-*, -(C1-C 20 Heteroaryl), -(C1-C 20 Heteroaryl)-*, -(C1-C 20 Heteroaryl)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -(C1-C 20 Heteroaryl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -(C1-C 20 Heteroaryl)-NR 5 -C(=NR 5a )N(R 5 )-*, -(C1-C 20 Heteroaryl)-N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -C(=O)-*, -C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -C(=O)-(C2-C 20 Heterocyclyldiyl)-*, -C(=O)N(R 5 )2, -C(=O)N(R 5 )-*, -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-N(R 5 )C(=O)R 5 , -C(=O)N(R 5 )-(C1-C 12 Alkyldiyl)-N(R 5 )C(=O)N(R 5 )2, -C(=O)NR 5-(C1-C 12 Alkyldiyl)-N(R 5 )CO2R 5 , -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-N(R 5 )C(=NR 5a )N(R 5 )2, -C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 C(=NR 5a )R 5 , -C(=O)NR 5 -(C1-C8 alkyldiyl)-NR 5 (C2-C5 heteroaryl), -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-N(R 5 )-*, -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-*, -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -C(=O)NR 5 -(C1-C 20 Heteroaryldiyl)-(C2-C 20 Heterocyclyldiyl)-C(=O)NR 5 -(C1-C 12 Alkyldiyl)-NR 5 -*, -N(R 5 )2, -N(R 5 )-*, -N(R 5 )C(=O)R 5 , -N(R 5 )C(=O)-*, -N(R 5 )C(=O)N(R5 )2, -N(R 5 )C(=O)N(R 5 )-*, -N(R 5 )CO2R 5 , -NR 5 C(=NR 5a )N(R 5 )2, -NR 5 C(=NR 5a )N(R 5 )-*, -NR 5 C(=NR 5a )R 5 , -N(R 5 )C(=O)-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -N(R 5 )-(C2-C5 heteroaryl), -N(R 5 )-S(=O)2-(C1-C 12 alkyl), -O-(C1-C 12 alkyl), -O-(C1-C 12 Alkyldiyl)-N(R 5 )2, -O-(C1-C 12 Alkyldiyl)-N(R 5 )-*, -OC(=O)N(R 5 )2, -OC(=O)N(R 5 )-*, -O-(R 5 )-*, -OR 5 , -S(=O)2-(C2-C 20 Heterocyclyldiyl)-*, -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-N(R 5 )2, -S(=O)2-(C2-C20 Heterocyclyldiyl)-(C1-C 12 Alkyldiyl)-NR 5 -*, and -S(=O)2-(C2-C 20 Heterocyclyldiyl)-(C1-C 12 or substituted with one or more groups selected from: or R 2 and R 3 together form a 5- or 6-membered heterocyclyl ring, X 1 , X 2 , X 3 , and X 4 are independently bond, C(=O), C(=O)N(R 5 ), O, N(R 5 ), S, S(O)2, and S(O)2N(R 5 ) selected from the group consisting of R 5 are independent, H, C6-C 20 Aryl, C3-C 12 Carbocyclyl, C6-C 20 Aryldiyl, C1-C 12 Alkyl, and C1-C 12 or two R 5 the groups taken together form a 5- or 6-membered heterocyclyl ring, R 5a But, C6-C 20 Aryl and C1-C 20 Heteroaryl; where the asterisk * indicates the binding site of L and R 1 , R 2 , R 3 , and R 4 One of the is bonded to L, L, -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)-, -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(PEG-COH)-PEG-N(R 5 )-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(PEG-CO2H)-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-N(PEG-CO2H)-PEG-C(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(=O)N(PEG-CO2H)-PEG-C(=O)-, -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-C(═O)-PEP-, and -Succinimidyl-(CH2) m -C(=O)N(R 6 )-PEG-SS-(C1-C 12 the linker being selected from the group consisting of: -OC(=O)-, R 6 is 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 3. The method of claim 2, wherein the PEP has the formula: [ka] having wherein AA are independently selected from natural or unnatural amino acid side chains, 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 optionally has F, Cl, NO2, -OH, -OCH3, and the structure: [ka] Glucuronic acid having the formula: 20 Aryldiyl and C1-C 20 heteroaryldiyl; R 7 But -CH(R 8 )O-, -CH2-, -CH2N(R 8 )-, and -CH(R 8 )OC(=O)-(wherein, R 8 is H, C1-C6 alkyl, C(=O)-C1-C6 alkyl, and -C(=O)N(R 9 )2, wherein R 9 are independent, H, C1-C 12 Alkyl, and -(CH2CH2O) n -(CH2) m -OH, wherein m is an integer from 1 to 5 and 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, -CH2 CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3) 2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, -C(CH3)2CN , -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH( OH)CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, -NHC(=NH)H, -NHC(=NH)CH3, -NHC(=NH)NH2 , -NHC(=O)NH2, -NO2, =O, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O) n -(CH2) m CO2H, -O(CH2CH2O) n 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.

[0120] Exemplary embodiments of the immunoconjugate of formula I include X 1 is a bond and R 1 Including that H.

[0121] Exemplary embodiments of the immunoconjugate of formula I include X 2 is a bond and R 2 is C1-C8 alkyl.

[0122] Exemplary embodiments of the immunoconjugate of formula I include X 2 and X 3 are each bonds, and R 2 and R 3 are independently C1-C8 alkyl, -O-(C1-C 12 Alkyl), -(C1-C 12 Alkyldiyl)-OR 5 , -(C1-C8 alkyldiyl)-N(R 5 )CO2R 5 , -(C1-C 12 alkyl)-OC(O)N(R 5 )2, -O-(C1-C 12 Alkyl)-N(R 5 )CO2R 5 , and -O-(C1-C 12 alkyl)-OC(O)N(R 5 )2.

[0123] An exemplary embodiment of the immunoconjugate of formula I is 2 is C1-C8 alkyl, R 3 -(C1-C8 alkyldiyl)-N(R 5 )CO2R 4 This includes being.

[0124] An exemplary embodiment of the immunoconjugate of formula I is 2 is -CH2CH2CH3, and R 3 is selected from -CH2CH2CH2NHCO2(t-Bu), -OCH2CH2NHCO2(cyclobutyl), and -CH2CH2CH2NHCO2(cyclobutyl).

[0125] An exemplary embodiment of the immunoconjugate of formula I is 2 and R 3are each independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH, and -CH2CH2CH2OH.

[0126] An exemplary embodiment of the immunoconjugate of formula I is 2 and R 3 are each -CH2CH2CH3.

[0127] An exemplary embodiment of the immunoconjugate of formula I is 2 is -CH2CH2CH3, and R 3 is -OCH2CH3.

[0128] Exemplary embodiments of the immunoconjugate of formula I include X 3 -R 3 but, [ka] The compound is selected from the group consisting of:

[0129] Exemplary embodiments of the immunoconjugate of formula I include X 4 is a bond and R 4 Including that H.

[0130] An exemplary embodiment of the immunoconjugate of formula I is 1 is attached to L.

[0131] An exemplary embodiment of the immunoconjugate of formula I is 2 or R 3 is attached to L.

[0132] Exemplary embodiments of the immunoconjugate of formula I include X 3 -R 3 -L, [ka] is selected from the group consisting of Here the wavy line indicates the point of attachment to N.

[0133] An exemplary embodiment of the immunoconjugate of formula I is 4 C1-C 12 It includes being alkyl.

[0134] An exemplary embodiment of the immunoconjugate of formula I is 4 Ga-(C1-C 12 Alkyldiyl)-N(R 5 )-*, where the asterisk * indicates the attachment site of L.

[0135] Exemplary embodiments of the immunoconjugate of Formula I include where L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0136] An exemplary embodiment of the immunoconjugate of Formula I includes where L is attached to a cysteine ​​thiol of the antibody.

[0137] Exemplary embodiments of the immunoconjugates of Formula I include those in which, for PEG, m is 1 or 2 and n is an integer from 2 to 10, or n is 10.

[0138] An exemplary embodiment of the immunoconjugate of formula I is an immunoconjugate of formula I, wherein L comprises PEP, PEP is a dipeptide, and has the formula: [ka] This includes having

[0139] Exemplary embodiments of the immunoconjugate of formula I include where AA1 and AA2 are independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2, or where AA1 and AA2 form a five-membered proline amino acid ring.

[0140] Exemplary embodiments of the immunoconjugate of formula I include where AA1 is -CH(CH3)2 and AA2 is -CH2CH2CH2NHC(O)NH2.

[0141] Exemplary embodiments of the immunoconjugate of Formula I include those in which AA1 and AA2 are independently selected from GlcNAc aspartic acid, -CH2SO3H, and -CH2OPO3H.

[0142] An exemplary embodiment of the immunoconjugate of Formula I is wherein the PEP has the formula: [ka]

[0023] wherein AA1 and AA2 are independently selected from the side chains of naturally occurring amino acids.

[0143] An exemplary embodiment of the immunoconjugate of formula I is an immunoconjugate wherein L comprises PEP, PEP is a tripeptide, and has the formula: [ka] This includes having

[0144] An exemplary embodiment of the immunoconjugate of formula I is an immunoconjugate wherein L comprises PEP, PEP is a tetrapeptide, and has the formula: [ka] This includes having

[0145] An exemplary embodiment of the immunoconjugate of formula I is AA1 is selected from the group consisting of Abu, Ala, and Val; AA2 is selected from the group consisting of Nle(O-Bzl), Oic, and Pro; AA3 is selected from the group consisting of Ala and Met(O)2; AA4 is selected from the group consisting of Oic, Arg(NO2), Bpa, and Nle(O-Bzl).

[0146] Exemplary embodiments of the immunoconjugate of Formula I include those in which L comprises PEP, wherein PEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val, Ala-Ala-Pro-Ala, Ala-Ala-Pro-Val, and Ala-Ala-Pro-Nva.

[0147] An exemplary embodiment of the immunoconjugate of formula I is one in which L comprises PEP, wherein PEP has the structure: [ka] The present invention includes being selected from the following:

[0148] An exemplary embodiment of the immunoconjugate of formula I is one in which L has the structure: [ka] is selected from Here, the wavy line is R 5 The bond to is shown.

[0149] The present invention includes all reasonable combinations and permutations of features of the embodiments of Formula I.

[0150] In certain embodiments, the immunoconjugate compounds of the present invention include those with immunostimulatory activity. The immunoconjugates of the present invention selectively deliver effective doses of 8-sulfonyl-2-aminobenzazepine (8SO2Bz) drugs to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated 8SO2Bz.

[0151] Each of the immunoconjugates in Tables 3a and 3b was prepared according to the methods of Example 201, purified by HPLC, and characterized by mass spectrometry. [Table 6-1] [Table 6-2] [Table 6-3] [Table 7]

[0152] Figure 1 shows a graph of secreted TNFα (tumor necrosis factor alpha) cytokine levels after incubation of various concentrations of immunoconjugate IC-3, comparative CIC-1, and naked antibody TROP2 with co-cultures of cancer cells and cDC-enriched primary cell isolates. Figure 2 shows a graph of secreted TNFα (tumor necrosis factor alpha) cytokine levels after incubation of various concentrations of immunoconjugate IC-3, comparative CIC-2, and naked antibody TROP2 with co-cultures of cancer cells and cDC-enriched primary cell isolates.

[0153] Secreted cytokine levels in the supernatants were determined using the LegendPlex cytokine bead array kit. TROP2-targeting immunoconjugates IC-2, CIC-1, and CIC-2 induce secretion of the cytokine TNFα (alpha), which is associated with the initiation of immune responses against cancer, demonstrating activation of myeloid cells when exposed to TROP2-expressing tumor cells. 8-sulfonyl-2-aminobenzazepine immunoconjugate IC-3 stimulated higher levels of TNFα than comparative immunoconjugates CIC-1 and CIC-2. Notably, the 8-sulfonyl-2-aminobenzazepine payload represents a more efficient payload, reducing molecular weight and hydrophobicity while providing increased activity. Naked antibody TROP2 does not induce myeloid activation, demonstrating a dependency on the TLR7 / 8 activating payload.

[0154] Drug loading is represented by p, which is the number of 8-sulfonyl-2-aminobenzazepine (8SO2Bz) moieties per antibody in the immunoconjugate of formula I, as measured in the exemplary immunoconjugates of Table 3a (DAR). Drug (8SO2Bz) loading can range from 1 to about 8 drug moieties (D) per antibody. The immunoconjugates of formula I contain 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, or a range therebetween, such as 1 to 8 or 2 to 5. In any such aspect, p and n are equal (i.e., p=n=1, 2, 3, 4, 5, 6, 7, or 8, or some range therebetween). Exemplary immunoconjugates of formula I include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine ​​residues are already present in the antibody that form intra- and inter-chain disulfide bonds (native disulfide groups) without the use of genetic engineering, in which case the existing free reduced cysteine ​​residues may 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.

[0155] For some immune complexes, 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 reactively 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 8SO2Bz-linker compound of formula II. In certain embodiments, higher drug loading, e.g., p>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug complexes. In certain embodiments, the average drug loading of the immune complex 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.

[0156] The loading of the immunoconjugate (drug / antibody ratio) can be controlled in different ways, for example, by (i) limiting the molar excess of 8SO2Bz-linker intermediate compound relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction denaturation conditions for optimized antibody reactivity.

[0157] It should be understood that if more than one nucleophilic group on an antibody reacts with a drug, the resulting product is a mixture of immunoconjugate compounds with a distribution of one or more drug moieties attached to the antibody. The average number 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 can 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 Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). (See, Proceedings of the AACR Research, 2004 Annual Meeting, March 27-31, 2004, Volume 45, March 2004.) In certain embodiments, homogenous immune complexes having a single loading value may be isolated from a complex mixture by electrophoresis or chromatography.

[0158] In vitro assessment of immunoconjugate activity can be carried out according to the methods of Example 203.

[0159] Composition of the immunoconjugate The present invention provides compositions, e.g., pharma- ceutical or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates described herein and, optionally, a carrier therefor, e.g., a pharma- ceutical or pharmacologically acceptable carrier. The immunoconjugates may be the same or different in the composition, i.e., the composition may include immunoconjugates having the same number of adjuvants linked to the same position on the antibody construct, and / or immunoconjugates having the same number of 8-sulfonyl-2-aminobenzazepine (8SO2Bz) adjuvants linked to different positions on the antibody construct, immunoconjugates having different numbers of 8SO2Bz adjuvants linked to the same position on the antibody construct, or having different numbers of 8SO2Bz adjuvants linked to different positions on the antibody construct.

[0160] In an exemplary embodiment, the composition comprising the immunoconjugate compounds comprises a mixture of immunoconjugate compounds, wherein the average drug (8SO2Bz) loading (DAR) per antibody in the mixture of immunoconjugate compounds is about 2 to about 5.

[0161] The compositions of the immune complexes of the invention can have an average adjuvant to antibody construct ratio (DAR) of about 0.4 to about 10. One of skill in the art will recognize that the number of 8-sulfonyl-2-aminobenzazepine adjuvants conjugated to the antibody constructs 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, which 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, including conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the immune complexes in the composition in terms of p can also be determined. In some cases, separation, purification, and characterization of homogeneous immune complexes with a certain value of p from other drug-loaded immune complexes can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0162] 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 can 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.

[0163] 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 injectable solution formulation ranges from about 0.1% (w / w) to about 10% (w / w).

[0164] Immunoconjugate therapy for cancer The present invention provides a method for treating cancer. The method includes administering a therapeutically effective amount of an immunoconjugate (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 includes administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 3a.

[0165] It is contemplated that the immunoconjugates of the invention may be used to treat a variety of hyperproliferative diseases or disorders characterized, for example, by overexpression of tumor antigens. Exemplary hyperproliferative disorders include benign or malignant solid tumors, as well as hematological disorders, such as leukemia and lymphoid malignancies.

[0166] 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.

[0167] 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.

[0168] 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, and the like. Carcinomas can be found in the prostate, pancreas, colon, brain (usually as a secondary metastasis), lung, breast, and skin. In some embodiments, a method for treating non-small cell lung cancer comprises administering an immunoconjugate containing an antibody construct capable of binding to a tumor-associated antigen.

[0169] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include alveolar soft part sarcoma, angiomatoid fibrous histiocytoma, chondromyo-oxidizing 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 fibrosarcoma, gastrointestinal stromal tumor, giant cell tumor of bone, tenosynovial giant cell tumor, inflammatory myofibroblastic tumor, uterine leiomyoma, leiomyosarcoma, lipoblastoma, typical lipoma, spindle cell or pleomorphic lipoma, atypical lipoma, chondrolipoma, 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, perineural tumor, mesothelioma, neuroblastoma, osteochondroma, osteosarcoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, benign or malignant nerve sheath tumor, synovial sarcoma, Evans' tumor, nodular fasciitis, desmoid-type fibromatosis, solitary fibrous tumor, dermatofibrosarcoma protuberans (DFSP), angiosarcoma, epithelioid hemangioendothelioma, tenosynovial giant cell tumor (TGCT), pigmented villonodular synovitis (PVNS), fibrous dysplasia, myxofibrosarcoma, fibrosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, neurofibroma, pleomorphic adenoma of soft tissue, and neoplasms derived from fibroblasts, myofibroblasts, histiocytes, vascular / endothelial cells, and nerve sheath cells.

[0170] Sarcoma is a rare type of cancer that arises 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 sarcoma are based on where the cancer forms. For example, osteosarcoma arises in bone, liposarcoma arises in fat, and rhabdomyosarcoma arises in muscle. Examples of sarcomas include, but are not limited to, primitive neuroectodermal tumors (PNETs) of the thoracic and pulmonary regions (Askin tumors), sarcoma botryoides, chondrosarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcomas (e.g., alveolar soft tissue sarcoma, angiosarcoma, cystic sarcoma fjordosarcoma protoverans (DFSP), desmoid tumor, anaplastic small round cell tumor, epithelial sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrous sarcoma, digestive gastrointestinal stromal tumor (GIST), vascular carcinoma, angiosarcoma (more commonly referred to as "angiosarcoma"), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, synovial sarcoma, and undifferentiated polyplastic sarcoma).

[0171] Teratomas are a type of germ cell tumor that may contain several different types of tissue (e.g., tissue derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas most commonly occur in the ovaries in women, the testes in men, and the coccyx in children.

[0172] 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 the eye or intestine.

[0173] Merkel cell carcinoma is a rare type of skin cancer that usually appears as flesh-colored or bluish-red nodules on the face, head or neck. Merkel cell carcinoma is also called cutaneous neuroendocrine carcinoma. In some embodiments, the method for treating Merkel cell carcinoma includes, for example, administering an immunoconjugate containing an antibody construct that can bind to CEA (e.g., labetuzumab, its biosimilar, or its biobetter). In some embodiments, the Merkel cell carcinoma has metastasized when administration is performed.

[0174] 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).

[0175] Lymphoma is a cancer that develops in 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.

[0176] 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, therapy-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0177] Brain tumors include any cancer 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).

[0178] 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.

[0179] The immunoconjugates of the invention (and any additional therapeutic agents) may be administered by any suitable means, including oral, parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, e.g., injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0180] The immunoconjugate is administered in any therapeutically effective amount to a subject in need thereof using any suitable dosing regimen, such as those utilized for labetuzumab, its biosimilars, and its biobetters. For example, the method can include administering the immunoconjugate to provide a dose of about 100 ng / kg to about 50 mg / kg to the subject. The immunoconjugate dose 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 immunoconjugate dose can be about 100, 200, 300, 400, or 500 μg / kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The immunoconjugate dose 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.

[0181] In another aspect, the present invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition described above) to a subject. In certain embodiments, the subject is susceptible to the particular cancer to be prevented.

[0182] Some embodiments of the present invention provide a method for treating the above cancers, where the cancer is breast cancer. Breast cancer can arise 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 ductal carcinoma in situ, invasive ductal carcinoma (e.g., tubular, medullary, mucinous, papillary, or cribriform), lobular carcinoma in situ, invasive lobular carcinoma, inflammatory breast cancer, and other forms of breast cancer, such as triple-negative (test negative for estrogen receptor, progesterone receptor, and excess HER2 protein) breast cancer. In some embodiments, the method for treating breast cancer comprises administering an immunoconjugate containing an antibody construct capable of binding to a tumor-associated antigen (TAA) or tumors that overexpress a TAA.

[0183] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8.

[0184] In some embodiments, a therapeutically effective amount of the immunoconjugate is administered to a patient in need of treatment for cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, or breast cancer. The Merkel cell carcinoma can be metastatic Merkel cell carcinoma. The breast cancer can be triple negative breast cancer. The esophageal cancer can be gastroesophageal junction adenocarcinoma. EXAMPLES

[0185] Example L-1 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]sulfonyl]benzoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, 8SO2BzL-1 [ka] [ka] Preparation of 8-Bromo-N-ethoxy-N-propyl-2-(tritylamino)-3H-1-benzazepine-4-carboxamide, 8SO2BzL-1b To a mixture of 8-bromo-2-(tritylamino)-3H-1-benzazepine-4-carboxylic acid, 8SO2BzL-1a (1 g, 1.91 mmol, 1 equiv.) and N-ethoxypropan-1-amine (320 mg, 2.29 mmol, 1.2 equiv., HCl) in DCM (15 mL) and DMA (5 mL) was added EDCI (1.10 g, 5.73 mmol, 3.0 equiv.) in one portion at 25 °C, followed by stirring at 25 °C for 0.5 h. The mixture was concentrated to remove DCM. The residue was then diluted with aqueous NaHCO3 until the pH was 8-9. The mixture was extracted with EtOAc (30 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate=1 / 0, 3 / 1) to give 8SO2BzL-1b (0.7 g, 1.15 mmol, 60.21% yield) as a white solid. 1 H NMR(CDCl3,400 MHz)δ 7.38-7.15 (m,15H),7.06-6.94(m,2H),6.72(s,1H),6.16 (s,1H),4.03-3.83(m,2H),3.73(t,J=7.2 Hz,2H),2.78(s,2H),1.89-1.64(m,2H),1.25(t,J = 7.2 Hz,3H),0.99(t,J=7.2 Hz,3H).LC / MS[M+H]608.2(calculated value);LC / MS[M+H]608.2(observed value).

[0186] Preparation of methyl 4-[[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-1-benzazepin-8-yl]sulfanyl]benzoate, 8SO2BzL-1c A mixture of 8SO2BzL-1b (0.35 g, 575 umol, 1.0 equiv.) and methyl 4-sulfanylbenzoate (116 mg, 690 umol, 1.2 equiv.) in DMF (4 mL) was treated with dicyclohexyl[2',4',6'-tris)propan-2-yl)[1,1'-biphenyl]phosphane, XPhos, CAS Registry Number 564483-18-7, Huang, X., et al (2003) J. Am. Chem. Soc. 125(22):6653-6655, Bruno, N. C. et al, (2013) Chemical Science,4(3):916-920 (82.3 mg, 173 umol, 0.3 equiv), Cs2CO3 (375 mg, 1.15 mmol, 2.0 equiv), and [2-(2-aminophenyl)phenyl]-chloro-palladium; dicyclohexyl-[3-(2,4,6-triisopropylphenyl)phenyl]phosphane, Pd-Xphos-G2, CAS Registry Number 1310584-14-5 (226 mg, 288 umol, 0.5 equiv) were added in one portion under N2 at 25°C, then stirred at 120°C for 12 hours. The mixture was diluted with water (20 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate=1 / 0, 3 / 1) to give 8SO2BzL-1c (0.25 g, 359.26 umol, 62.47% yield). LC / MS [M+H] 696.3 (calculated); LC / MS [M+H] 696.2 (observed).

[0187] Preparation of methyl 4-[[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-1-benzazepin-8-yl]sulfonyl]benzoate, 8SO2BzL-1d To a mixture of 8SO2BzL-1c (0.25 g, 359 umol, 1.0 equiv) in DCM (2 mL), THF (2 mL), and H2O (2 mL) was added potassium peroxymonosulfate, KHSO5, oxone (663 mg, 1.08 mmol, 3.0 equiv) in one portion at 25 °C, then stirred at 25 °C for 12 h. The mixture was diluted with water and extracted with EtOAc (20 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 1 / 1) to give 8SO2BzL-1d (0.2 g, 274.78 umol, 76.48% yield) as a yellow oil. LC / MS [M+H] 728.27 (calculated); LC / MS [M+H] 728.2 (observed).

[0188] Preparation of methyl 4-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]sulfonyl]benzoate, 8SO2BzL-1e To a mixture of 8SO2BzL-1d (0.1 g, 137 umol, 1.0 equiv) in DCM (4 mL) was added TFA (313 mg, 2.75 mmol, 203 uL, 20.0 equiv) in one portion at 25° C., then stirred at 50° C. for 12 h. The mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (0.1% TFA)-acetonitrile, ACN]; B%: 15%-35%, 8 min) to give 8SO2BzL-1e (0.046 g, 94.74 umol, 68.96% yield) as a white solid. 1H NMR(CDCl3,400 MHz) δ8.21(d,J = 8.4 Hz,2H),8.10(d,J = 8.4 Hz,2H),8.04(s,1H),7.95-7.87(m,1H),7.57(d,J = 8.0 Hz,1H),7.49(s,1H),6.97(d,J = 1.2 Hz,1H),3.95(s,3H),3.93-3.82(m,2H),3.70(t,J = 7.2 Hz,2H),3.23(s,2H),1.82-1.66(m,2H),1.21(t,J = 7.2 Hz,3H),0.96(t,J = 7.2 Hz,3H).LC / MS[M+H] 485.16 (calculated); LC / MS [M+H] 486.1 (observed).

[0189] Preparation of 4-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]sulfonyl]benzoic acid, 8SO2BzL-1f To a mixture of 8SO2BzL-e (0.24 g, 494 umol, 1.0 equiv) in MeOH (2 mL), HO (2 mL), and THF (2 mL) was added LiOH.HO (62.2 mg, 1.48 mmol, 3.0 equiv) in one portion at 25 °C, then stirred at 25 °C for 2 h. The mixture was quenched with HCl (1 M) to adjust the pH to 6-7, and the aqueous phase was extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to give 8SO2BzL-1f (0.23 g, crude) as a yellow oil. 1 H NMR(DMSO,400 MHz)δ 8.16-8.06(m,4H),7.61-7.54(m,2H),7.49(dd,J = 1.6,8.0 Hz,1H),7.11(s,1H),3.82(q,J = 7.2 Hz,2H),3.59(t,J = 7.2 Hz,2H),2.89(s,2H),1.70-1.55(m,2H),1.01(t,J = 7.2 Hz,3H),0.89(t,J = 7.2 Hz,3H).LC / MS [M+H]472.1(calculated value);LC / MS[M+H]472.1(observed value).

[0190] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]sulfonyl]benzoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 8SO2BzL-1g To a mixture of 8SO2BzL-1f (0.2 g, 424 umol, 1.0 equiv) in DMF (4 mL) was added tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (248 mg, 424 umol, 1.0 equiv), DIEA (164 mg, 1.27 mmol, 222 uL, 3.0 equiv), and azabenzotriazole tetramethyluronium hexafluorophosphate, HATU, CAS Registry Number 148893-10-1 (177 mg, 467 umol, 1.1 equiv) in one portion at 25 °C, then stirred at 25 °C for 0.5 h. The mixture was diluted with water and extracted with EtOAc (30 mL×3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to give 8SO2BzL-1g (0.5 g, crude) as a yellow oil. LC / MS [M+H] 1039.5 (calculated); LC / MS [M+H] 1039.5 (observed).

[0191] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-8-yl]sulfonyl]benzoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 8SO2BzL-1h To a mixture of 8SO2BzL-1g (0.5 g, 481 umol, 1.0 equiv) in CH3CN (1 mL) and HO (3 mL) was added TFA (439 mg, 3.85 mmol, 285 uL, 8.0 equiv) in one portion at 25 °C, 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 Luna 80 × 30 mm × 3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-35%, 8 min) to give 8SO2BzL-1h (0.15 g, 152.57 umol, 31.71% yield) as a yellow oil. 1 H NMR(MeOD,400 MHz) δ 8.14-8.08(m,2H),8.06-7.95(m,4H),7.78(d,J = 8.4 Hz,1H),7.40(s,1H),3.94(q,J = 6.8 Hz,2H),3.75-3.70(m,4H),3.65-3.50(m,40H),3.40(s,2H),2.53(t,J = 6.4 Hz,2H),1.82-1.69(m,2H),1.16(t,J = 7.2 Hz,3H),0.98(t,J = 7.2 Hz,3H).LC / MS[M+H]982.45(calculated value);LC / MS[M+H]983.4(observed value).

[0192] Preparation of 8SO2BzL-1 To a mixture of 8SO2BzL-1h (0.15 g, 152.57 umol, 1.0 equiv) in DCM (3 mL) and DMA (0.5 mL), sodium; 2,3,5,6-tetrafluoro-4-hydroxy-benzenesulfonate (164 mg, 610 umol, 4.0 equiv) and EDCI (146 mg, 763 umol, 5.0 equiv) were added in one portion at 25° C., then stirred at 25° C. for 0.5 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%-40%, 8 min) to give 8SO2BzL-1 (58.9 mg, 48.63 umol, 31.87% yield) as a light yellow solid. 1H NMR(MeOD,400 MHz) δ 8.11-8.06(m,2H),8.05-7.99(m,3H),7.95(dd,J = 2.0,8.4 Hz,1H),7.77(d,J = 8.4 Hz,1H),7.39(s,1H),3.93(q,J = 7.2 Hz,2H),3.86(t,J = 6.0 Hz,2H),3.71(t,J = 7.2 Hz,2H),3.67-3.53(m,34H),3.53-3.48(m,6H),3.42(s,2H),2.97(t,J = 6.0 Hz,2H),1.83-1.65(m,2H),1.15(t,J = 7.2 Hz,3H),0.97(t,J = 7.2 Hz,3H).LC / MS [M+H] 1211.4(calculated value);LC / MS [M+H] 1211.3(observed value).

[0193] Example L-2 Synthesis of 2-amino-4-[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]ethoxycarbonylamino]ethoxy-propyl-carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-2 [ka] Preparation of 2-amino-4-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-2b To a mixture of tert-butyl N-[2-[(2-amino-8-benzylsulfanyl-3H-1-benzazepine-4-carbonyl)-propyl-amino]oxyethyl]carbamate, 8SO2BzL-2a (0.15 g, 286 umol, 1.0 equiv.) in HO (0.15 mL) and AcOH (0.5 mL), N-chlorosuccinimide, NCS (153 mg, 1.14 mmol, 4.0 equiv.) was added in one portion at 25 °C and then stirred at 25 °C for 1 h. The mixture was diluted with aqueous NaHCO3 to adjust the pH to 7-8. The mixture was then extracted with EtOAc (10 mL × 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8 min) to give 8SO2BzL-2b (11.5 mg, 23.83 umol, 8.34% yield) as a yellow solid. 1 H NMR(DMSO,400 MHz)δ 11.87(s,1H),9.78(s,1H),8.81(s,1H),7.66(s,1H),7.55(s,2H),7.34-7.27(m,1H),3.92-3.78(m,2H),3.63(t,J = 7.2 Hz,2H),3.31(s,2H),3.16-3.01(m,2H),1.70-1.60(m,2H),1.36(s,9H),0.89(t,J = 7.2 Hz,3H).LC / MS[M+H]483.2(calculated value);LC / MS[M+H]483.1(observed value).

[0194] Preparation of 2-amino-4-[2-aminoethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-2c To a solution of 8SO2BzL-2b (150 mg, 310.85 umol, 1 equiv) in EtOAc (10.0 mL) was added HCl / EtOAc (4 M, 20.0 mL, 257 equiv) and then stirred at 25° C. for 1 h. The mixture was concentrated to give 8SO2BzL-2c (200 mg, crude) as a white solid. LC / MS [M+H] 383.1 (calculated); LC / MS [M+H] 383.2 (observed).

[0195] Preparation of 8SO2BzL-2 To a solution of 8SO2BzL-2c (70.0 mg, 167.11 umol, 1 equiv, HCl) in DMF (1.00 mL) was added DIEA (90.0 mg, 668 umol, 120 uL, 4 equiv) and 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]ethyl (4-nitrophenyl)carbonate (70.0 mg, 83.5 umol, 0.5 equiv) and then stirred at 0° C. for 1 hour. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 1%-30%, 8 min) to give 8SO2BzL-2 (15 mg, 12.92 umol, 7.73% yield, TFA) as a pale yellow oil. 1 H NMR(MeOD,400 MHz) δ7.89-7.77(m,2H),7.66(d,J = 8.0 Hz,1H),7.42(s,1H),6.89(s,2H),4.17(s,2H),3.97(br t,J = 4.8 Hz,2H),3.86-3.79(m,2H),3.75(t,J = 7.2 Hz,2H),3.66-3.58(m,38H),3.56-3.49(m,4H),3.42-3.35(m,4H),1.81-1.73(m,2H),1.00(t,J = 7.6 Hz,3H).LC / MS [M+H] 1047.4(calculated value);LC / MS [M+H] 1047.7 (observed value).

[0196] Example L-3 Synthesis of 2-amino-8-(N-(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)-N-methylsulfamoyl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-3 [ka] Preparation of 2-amino-N-ethoxy-8-((4-methoxybenzyl)thio)-N-propyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-3b To a mixture of 2-amino-8-bromo-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-3a (0.96 g, 1.99 mmol) and 4-methoxy-α-toluenethiol (0.37 g, 2.39 mmol) in dioxane (10 mL) was added 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, Xantphos, CAS Registry Number 161265-03-8 (0.06 g, 0.10 mmol), Pd2(dba)3 (0.05 g, 0.05 mmol) followed by triethylamine (0.56 mL, 3.99 mmol). The mixture was heated to reflux for 1 h and then cooled. The solvent was removed by evaporation and the crude product was purified by silica gel chromatography using a gradient of 1-10% MeOH / DCM over 12 column volumes to give 8SO2BzL-3b (0.72 g, 82%).

[0197] Preparation of 2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine-8-sulfonyl chloride, 8SO2BzL-3c To a solution of 8SO2BzL-3b (0.72 g, 1.64 mmol) in acetonitrile / water (9:1) (10 mL) was added N-chlorosuccinimide (0.66 g, 4.91 mmol) in small portions at 10° C. After the addition was complete, stirring was continued for an additional 20 min to give a crude product solution of 8SO2BzL-3c, which was used directly in the next step.

[0198] Preparation of tert-butyl (2-((2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)sulfonyl)-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34-yl)carbamate, 8SO2BzL-3d An aliquot of 8SO2BzL-3c (1.00 mL, 0.16 mmol) was added dropwise to a stirred mixture of tert-butyl (5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34-yl)carbamate (0.12 g, 0.19 mmol) and triethylamine (0.09 mL, 0.64 mmol) in acetonitrile (3 mL). After 15 min, the reaction was concentrated and purified by reverse phase chromatography using a gradient of 10-90% ACN / water (+0.1% TFA) over 10 min to give 8SO2BzL-3d (0.08 g, 51%).

[0199] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-N-methylsulfamoyl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide hydrochloride, 8SO2BzL-3e To a solution of 8SO2BzL-3d (0.08 g, 0.08 mmol) in ACN (3 mL) was added aqueous HCl (6 M, 3 mL) and the mixture was stirred at room temperature for 45 min. The solvent was removed and the isolated syrup was azeotroped with ACN (3 mL) to give 8SO2BzL-3e HCl salt (0.06 g, 85%) as an opaque white film.

[0200] Preparation of 8SO2BzL-3 To a solution of 8SO2BzL-3e HCl (0.06 g, 0.07 mmol) in DMF (3 mL) was added triethylamine (0.04 mL, 0.28 mmol). 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.02 g, 0.08 mmol) was added in small portions. After the addition was complete, acetic acid (9 uL) was added and the solvent was removed by vacuum. After purification by reverse phase HPLC, 8SO2BzL-3 (0.03 g, 48%) was obtained as a clear oil after evaporation of the solvent. LC / MS [M+H] 1001.48 (calculated); LC / MS [M+H] 1074.88 (observed).

[0201] Example L-4 Synthesis of 2-amino-8-(N-(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)sulfamoyl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-4 [ka] Preparation of tert-butyl (32-((2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine)-8-sulfonamido)-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, 8SO2BzL-4b To a solution of tert-butyl (32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, Boc-amino-PEG10-amine (0.10 g, 0.16 mmol) and DIPEA (0.14 mL, 0.80 mmol) in DMF (4 mL) was added a solution of 2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine-8-sulfonyl chloride, 8SO2BzL-4a (0.16 M, 1.00 mL, 0.16 mmol) in DMF. After 20 min, the reaction was concentrated and then purified by reverse phase HPLC using a gradient of 10-90% ACN / water over 10 min to give 8SO2BzL-4b (0.07 g, 47%) after removal of the solvent.

[0202] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)sulfamoyl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, SO2BzL-4c To a solution of 8SO2BzL-4b (0.07 g, 0.07 mmol) in acetonitrile (3 mL) was added aqueous HCl (6 M, 3 mL) and the mixture was stirred at room temperature for 45 min. The solvent was removed and the isolated syrup was azeotroped with ACN (3 mL) to give 8SO2BzL-4c HCl salt (0.06 g, 82%) as an opaque white film.

[0203] Preparation of 8SO2BzL-4 To a solution of 8SO2BzL-4c HCl (0.06 g, 0.06 mmol) in DMF (3 mL) was added triethylamine (0.03 mL, 0.25 mmol). 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.02 g, 0.07 mmol) was added in small portions. After the addition was complete, acetic acid (9 uL) was added and the solvent was removed in vacuo. After purification by reverse phase HPLC, 8SO2BzL-4 (0.04 g, 69%) was obtained after removal of the solvent. LC / MS [M+H] 987.45 (calculated); LC / MS [M+H] 987.86 (observed).

[0204] Example L-5 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-sulfamoyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-5 [ka] Preparation of tert-butyl (2-((2-amino-8-((4-methoxybenzyl)thio)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-b To a mixture of tert-butyl (2-((2-amino-8-bromo-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-5a (0.96 g, 1.99 mmol), and 4-methoxy-α-toluenethiol (0.37 g, 2.39 mmol) in dioxane (10 mL) was added Xantphos (0.06 g, 0.10 mmol), Pd2(dba)3 (0.05 g, 0.05 mmol), followed by triethylamine (0.56 mL, 3.99 mmol). The mixture was heated to reflux for 1 h and then cooled. The solvent was removed by evaporation and the crude product was purified by silica gel chromatography using a gradient of 1-10% MeOH / DCM over 12 column volumes to give 8SO2BzL-5b (0.78 g, 71%) as a yellow solid.

[0205] Preparation of tert-butyl (2-((2-amino-8-(chlorosulfonyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-5c To a solution of 8SO2BzL-5b (0.78 g, 1.41 mmol) in acetonitrile / water (9:1, 10 mL) was added N-chlorosuccinimide (0.56 g, 4.22 mmol) in three equal portions at 0° C. After the addition was complete, the product 8SO2BzL-5c was used as is without further purification.

[0206] Preparation of tert-butyl (2-((2-amino-N-propyl-8-sulfamoyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-5d To a solution of 8SO2BzL-5c (2.00 mL, 0.28 mmol) in acetonitrile / water (9:1) was added ammonium hydroxide solution (0.20 mL, 1.66 mmol) at 0° C. After 10 min, the solvent was removed and the crude product was purified by reverse-phase HPLC using a gradient of 10-90% acetonitrile / water to give 8SO2BzL-5d (0.06 g, 41%) as a yellow film after evaporation of the solvent.

[0207] Preparation of 8SO2BzL-5e A solution of 8SO2BzL-5e (0.06 g, 0.11 mmol) in acetonitrile (2 mL) and 6 N HCl (2.00 mL, 12.00 mmol) was stirred at room temperature for 45 min. The solvent was removed in vacuo to give 8SO2BzL-5e (0.05 g, 101%) as the HCl salt.

[0208] Preparation of 8SO2BzL-5 To a solution of 8SO2BzL-5e (0.04 g, 0.10 mmol) in DMF (4 mL) was added triethylamine (0.06 mL, 0.40 mmol) at room temperature. To this mixture was added a solution 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(4-nitrophenyl)carbonate, PNPC-PEG10-Mal (0.08 g, 0.10 mmol) in DMF (2 mL). After 20 min, acetic acid (6 uL) was added and the reaction was concentrated in vacuo and purified by reverse phase HPLC using a gradient of 10-90% ACN / water (+0.1% TFA) over 10 min to give 8SO2BzL-5 (0.04 g, 37%) after concentration of pure fractions. LC / MS [M+H] 1046.45 (calculated); LC / MS [M+H] 1046.88 (observed).

[0209] Example L-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-(N,N-dimethylsulfamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-6 [ka] Preparation of tert-butyl N-[2-[(2-amino-8-benzylsulfanyl-3H-1-benzazepine-4-carbonyl)-propyl-amino]oxyethyl]carbamate, 8SO2BzL-6b To a mixture of tert-butyl N-[2-[(2-amino-8-bromo-3H-1-benzazepine-4-carbonyl)-propyl-amino]oxyethyl]carbamate, 8SO2BzL-6a (0.5 g, 1.04 mmol, 1.0 equiv.), and benzyl thiol, benzyl mercaptan, phenylmethanethiol, BnSH, CAS Registry Number 100-53-8 (155 mg, 1.25 mmol, 146.05 uL, 1.2 equiv.) in dioxane (15 mL), 4,5-bis(diphenylphosphatase) was added. phino)-9,9-dimethylxanthene, Xantphos, CAS Registry Number 161265-03-8 (120 mg, 208 umol, 0.2 equiv.) tris)dibenzylideneacetone)dipalladium, Pd2(dba)3, CAS Registry Number 51364-51-3 (190 mg, 208 umol, 0.2 equiv.), and diisopropylethylamine, DIEA (268 mg, 2.08 mmol, 362 uL, 2.0 equiv.) were added in one portion under N2 at 25°C, then stirred at 110°C for 2 hours. The mixture was diluted with water (20 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The mixture was further purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 25%-55%, 8 min) to give 8SO2BzL-6b (0.5 g, 952.97 umol, 91.75% yield) as a yellow solid. 1H NMR(MeOD,400 MHz) δ 7.49(d,J = 8.4 Hz,1H),7.43-7.38(m,3H),7.36-7.22(m,5H),4.30(s,2H),3.91(t,J = 5.2 Hz,2H),3.73(t,J = 7.2 Hz,2H),3.32(s,2H),3.24(t,J = 5.2 Hz,2H),1.81-1.70(m,2H),1.34(s,9H),0.98(t,J = 7.2 Hz,3H).LC / MS [M+H] 525.2(calculated value);LC / MS [M+H]525.2 (observed value).

[0210] Preparation of tert-butyl N-[2-[[2-amino-8-(dimethylsulfamoyl)-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, 8SO2BzL-6c To a solution of 8SO2BzL-6b (50.0 mg, 95.30 umol, 1 equiv) in CH3CN (1.00 mL) and HO (0.10 mL) was added AcOH (60.0 mg, 953 umol, 50.0 uL, 10 equiv), N-chlorosuccinimide, NCS (50.0 mg, 381 umol, 4 equiv) at 25°C, then stirred at this temperature for 10 minutes, followed by the addition of N-methylmethanamine; hydrochloride, dimethylamine HCl (80.0 mg, 953 umol, 10 equiv) and DIEA (250 mg, 1.91 mmol, 330 uL, 20 equiv). The mixture was stirred at 0°C for an additional hour. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 10%-40%, 8 min) to give 8SO2BzL-6c (12 mg, 23.55 umol, 24.71% yield) as a white solid. 1H NMR(MeOD,400 MHz) δ7.92-7.72(m,3H),7.50(s,1H),3.94(t,J = 5.2 Hz,2H),3.75(t,J = 7.2 Hz,2H),3.44(s,2H),3.26(br t,J = 5.2 Hz,2H),2.77(s,6H),1.77(sxt,J = 7.2 Hz,2H),1.37(s,9H),0.99(t,J = 7.2 Hz,3H).LC / MS [M+H] 510.2(calculated value);LC / MS[M+H]510.3(observed value).

[0211] Preparation of 2-amino-N-(2-aminoethoxy)-8-(N,N-dimethylsulfamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide hydrochloride, 8SO2BzL-6d A solution of 8SO2BzL-6c (0.04 g, 0.08 mmol) in acetonitrile (2 mL) and 6 N HCl (1.41 mL, 8.46 mmol) was stirred at room temperature for 45 min. The solvent was removed in vacuo to give 8SO2BzL-6d (0.04 g, 100%).

[0212] Preparation of 8SO2BzL-6 To a solution of 8SO2BzL-6d (0.04 g, 0.10 mmol) in DMF (4 mL) was added triethylamine (0.04 mL, 0.28 mmol) at room temperature. To this mixture was added a solution 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(4-nitrophenyl)carbonate (0.06 g, 0.07 mmol) in DMF (2 mL). After 20 min, acetic acid (6 uL) was added and the reaction was concentrated in vacuo and purified by reverse phase HPLC using a gradient of 10-90% ACB / water (+0.1% TFA) over 10 min to give 8SO2BzL-6 (0.04 g, 51%) after concentration of pure fractions. LC / MS [M+H] 1074.48 (calculated); LC / MS [M+H] 1074.90 (observed).

[0213] Example L-9 Synthesis of 2-amino-8-(N-(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)sulfamoyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-9 [ka] Preparation of 2-amino-8-((4-methoxybenzyl)thio)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-9b To a mixture of 2-amino-8-bromo-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-9a (0.96 g, 1.99 mmol) and 4-methoxy-α-toluenethiol (0.37 g, 2.39 mmol) in dioxane (10 mL) was added Xantphos (0.06 g, 0.10 mmol), Pd2(dba)3 (0.05 g, 0.05 mmol) followed by triethylamine (0.56 mL, 3.99 mmol). The mixture was heated to reflux for 1 h and then cooled. The solvent was removed by evaporation and the crude product was purified by silica gel chromatography using a gradient of 1-10% MeOH / DCM over 12 column volumes to give 8SO2BzL-9b (0.69 g, 79%).

[0214] Preparation of 2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-sulfonyl chloride, 8SO2BzL-9c To a solution of 8SO2BzL-9b (0.68 g, 1.55 mmol) in acetonitrile / water (9:1) (10 mL) was added N-chlorosuccinimide, NCS (0.62 g, 4.66 mmol) in three portions at 10° C. After complete addition, stirring for an additional 20 min afforded 8SO2BzL-9c.

[0215] Preparation of tert-butyl (32-((2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine)-8-sulfonamido)-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, 8SO2BzL-9d An aliquot of 8SO2BzL-9c obtained previously (1.00 mL, 0.15 mmol) was added dropwise to a stirred mixture of tert-butyl (32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate (0.11 g, 0.18 mmol) and triethylamine (0.08 mL, 0.60 mmol) in acetonitrile (3 mL). After 15 min, the reaction was concentrated and purified by reverse phase chromatography using a gradient of 10-90% ACN / water (+0.1% TFA) over 10 min to give 8SO2BzL-9d (0.07 g, 51%).

[0216] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)sulfamoyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-9e To a solution of 8SO2BzL-9d (0.07 g, 0.08 mmol) in acetonitrile (3 mL) was added aqueous HCl (6 M, 3 mL) and the mixture was stirred at room temperature for 45 min. The solvent was removed and the isolated syrup was azeotroped with acetonitrile (3 mL) to give 8SO2BzL-9e (0.06 g, 86%) as the HCl salt as an opaque white film.

[0217] Preparation of 8SO2BzL-9 To a solution of 8SO2BzL-9e HCl (0.06 g, 0.06 mmol) in DMF (3 mL) was added triethylamine (0.03 mL, 0.25 mmol). 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.02 g, 0.07 mmol) was added in small portions. After the addition was complete, acetic acid (9 uL) was added and the solvent was removed in vacuo. After purification by reverse phase HPLC, 8SO2BzL-9 (0.03 g, 43%) was obtained after evaporation. LC / MS [M+H] 985.47 (calculated); LC / MS [M+H] = 985.88 (observed).

[0218] Example L-10 Synthesis of 2-amino-8-(N-(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)-N-methylsulfamoyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-10 [ka] Preparation of tert-butyl (2-((2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepin-8-yl)sulfonyl)-5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34-yl)carbamate, 8SO2BzL-10b A solution of 2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-sulfonyl chloride, 8SO2BzL-10a (1.00 mL, 0.15 mmol) in acetonitrile was added dropwise to a stirred mixture of tert-butyl (5,8,11,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34-yl)carbamate (0.11 g, 0.18 mmol) and triethylamine (0.08 mL, 0.60 mmol) in acetonitrile (3 mL). After 15 min, the reaction was concentrated and purified by reverse phase chromatography using a gradient of 10-90% ACN / water (+0.1% TFA) over 10 min to give 8SO2BzL-10b (0.05 g, 37%).

[0219] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-N-methylsulfamoyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-10c To a solution of 8SO2BzL-10b (0.05 g, 0.05 mmol) in acetonitrile (3 mL) was added aqueous HCl (6 M, 3 mL) and the mixture was stirred at room temperature for 45 min. The solvent was removed and the isolated syrup was azeotroped with ACN (3 mL) to give 8SO2BzL-10c (0.04 g, 91%) as the HCl salt as an opaque white film.

[0220] Preparation of 8SO2BzL-10 To a solution of 8SO2BzL-10c HCl (0.04 g, 0.05 mmol) in DMF (3 mL) was added triethylamine (0.03 mL, 0.20 mmol). 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (0.02 g, 0.06 mmol) was added in small portions. After the addition was complete, acetic acid (9 uL) was added and the solvent was removed in vacuo. After purification by reverse phase HPLC, 8SO2BzL-10 (0.02 g, 42%) was obtained after evaporation. LC / MS [M+H] 999.49 (calculated); LC / MS [M+H] 999.92 (observed).

[0221] Example L-12 Synthesis of 2-amino-6-[5-[3-[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]propanoylamino]pentyl]-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-12 [ka] [ka] Preparation of 2-amino-8-bromo-N-ethoxy-6-iodo-N-propyl-3H-1-benzazepine-4-carboxamide, 8SO2BzL-12b To a solution of 2-amino-8-bromo-6-iodo-3H-1-benzazepine-4-carboxylic acid, 8SO2BzL-12a (2.0 g, 4.91 mmol, 1.0 equiv.) in DCM (20 mL) and DMA (10 mL) was added methanesulfonic acid, CH3SO3H (472 mg, 4.91 mmol, 350 uL, 1.0 equiv.), N-ethoxypropan-1-amine (823 mg, 5.90 mmol, 1.2 equiv., HCl), and EDCI (3.77 g, 19.7 mmol, 4 equiv.). The mixture was stirred at 25 °C for 2 h. The pH of the reaction mixture was adjusted to about 9 with saturated Na2CO3. The aqueous phase was extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed with brine (20 mL x 2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was triturated with EtOAc at 25 °C for 10 min to give 8SO2BzL-12b (1.24 g, 2.52 mmol, 51.3% yield) as a yellow solid. LC / MS [M+H] 491.97 (calculated); LC / MS [M+H] 491.9 (observed).

[0222] Preparation of tert-butyl N-[5-[2-amino-8-bromo-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-6-yl]pent-4-ynyl]carbamate, 8SO2BzL-12c A mixture of 8SO2BzL-12b (800 mg, 1.63 mmol, 1.0 equiv), tert-butyl N-pent-4-ynylcarbamate (328 mg, 1.79 mmol, 1.1 equiv), Pd(PPh3)2Cl2 (114 mg, 163 umol, 0.1 equiv), copper iodide, CuI (61.9 mg, 325 umol, 0.2 equiv) in DMF (16 mL) and Et3N (6 mL) was degassed and purged with N2 three times, then stirred at 80° C. under N2 atmosphere for 2 h. The mixture was poured into ice water (w / w=1 / 1) (20 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (30 mL×3). The combined organic phase was washed with brine (20 mL×2), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate=1 / 0, 2 / 1) to give 8SO2BzL-12c (690 mg, 1.26 mmol, 77.5% yield) as a yellow oil. 1 H NMR(MeOD,400MHz)δ 7.53(s,1H),7.27(s,2H),3.93(q,J = 7.2 Hz,2H),3.74(t,J = 7.2 Hz,2H),3.19(t,J = 7.2 Hz,2H),2.90-2.83(m,2H),2.51(t,J = 7.2 Hz,2H),1.82-1.70(m,4H),1.43(s,9H),1.17(t,J = 7.2 Hz,3H),0.98(t,J = 7.2 Hz,3H).LC / MS [M+H]547.2 (calculated value); LC / MS[M+H]547.2 (observed value).

[0223] Preparation of tert-butyl N-[5-[2-amino-8-benzylsulfanyl-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-6-yl]pent-4-ynyl]carbamate, 8SO2BzL-12d A mixture of 8SO2BzL-12c (350 mg, 639 umol, 1.0 equiv), phenylmethanethiol, BnSH (0.25 g, 2.01 mmol, 236 uL, 3.15 equiv), DIEA (165 mg, 1.28 mmol, 223 uL, 2.0 equiv), Xantphos (74.0 mg, 128 umol, 0.2 equiv), and Pd2(dba)3 (117 mg, 128 umol, 0.2 equiv) in dioxane (10 mL) was degassed and purged with N2 three times, then stirred at 110 °C under N2 atmosphere for 1 h. The residue was poured into ice water (w / w=1 / 1) (10 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with brine (10 mL x 1), dried over anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1) to give 8SO2BzL-12d (300 mg, 508 umol, 79.4% yield) as a yellow solid. 1 H NMR(MeOD,400MHz)δ 7.56(s,1H),7.37-7.34(m,2H),7.30-7.19(m,3H),7.09(d,J = 1.6 Hz,1H),7.03(d,J = 1.6 Hz,1H),4.20(s,2H),3.93(q,J = 7.2 Hz,2H),3.73(t,J = 7.2 Hz,2H),3.30(s,2H),3.19(t,J = 6.8 Hz,2H),2.49(t,J = 7.2 Hz,2H),1.81-1.72(m,4H),1.43(s,9H),1.16(t,J = 7.2 Hz,3H),0.98(t,J = 7.2 Hz, 3H).LC / MS [M+H] 591.3 (calculated);LC / MS [M+H] 591.3 (observed).

[0224] Preparation of tert-butyl N-[5-[2-amino-8-chlorosulfonyl-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepin-6-yl]pent-4-ynyl]carbamate, 8SO2BzL-12e To a solution of 8SO2BzL-12d (300 mg, 508 umol, 1.0 equiv) in MeCN (6 mL) and HO (0.6 mL) was added AcOH (305 mg, 5.08 mmol, 290 uL, 10 equiv) and NCS (271 mg, 2.03 mmol, 4.0 equiv), then stirred at 25° C. for 1 h. The reaction mixture was poured into ice water (w / w=1 / 1) (10 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (10 mL×3), and the combined organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give the crude product 8SO2BzL-12e (250 mg, 441 umol, 86.8% yield) as a yellow oil, which was used in the next step without further purification. LC / MS [M+H] 567.2 (calculated); LC / MS [M+H] 567.3 (observed).

[0225] Preparation of 2-amino-6-(5-aminopent-1-ynyl)-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-12f A solution of 8SO2BzL-12e (250 mg, 441 umol, 1.0 equiv) in MeCN (2.5 mL) and H2O (13 mL) was stirred at 100 °C for 1 h. The mixture was concentrated in vacuo. The crude product 8SO2BzL-12f (200 mg, 412 umol, 93.5% yield, HCl) was used in the next step without further purification as a yellow solid. LC / MS [M+H] 449.18 (calculated); LC / MS [M+H] 449.1 (observed).

[0226] Preparation of 2-amino-6-[5-(tert-butoxycarbonylamino)pent-1-ynyl]-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-12g To a solution of 8SO2BzL-12f (200 mg, 446 umol, 1.0 equiv) in THF (5 mL) and H2O (5 mL) was added NaHCO3 (112 mg, 1.34 mmol, 52 uL, 3.0 equiv) and Boc2O (146 mg, 669 umol, 154 uL, 1.5 equiv), then stirred at 25°C for 1 h. The mixture was concentrated under reduced pressure at 30°C. The residue was purified by preparative HPLC (column: Phenomenex Luna 80 x 30 mm x 3 um; mobile phase: [water (TFA)-ACN]; B%: 10%-40%, 8 min) to give 8SO2BzL-12g (100 mg, 182 umol, 40.9% yield) as a yellow solid. 1 H NMR(MeOD,400MHz)δ 7.84(d,J = 1.2 Hz,1H),7.71(d,J = 1.2 Hz,1H),7.63(s,1H),3.98(d,J = 7.2 Hz,2H),3.76(t,J = 6.8 Hz,2H),3.38(s,2H),3.18(t,J = 6.8 Hz,2H),2.54(t,J = 7.2 Hz,2H),1.81-1.76(m,4H),1.43(s,9H),1.20(t,J = 7.2 Hz,3H),1.00(t,J = 7.2 Hz,3H).LC / MS[M+H]549.2(calculated value);LC / MS[M+H]549.3(observed value).

[0227] Preparation of 2-amino-6-[5-(tert-butoxycarbonylamino)pentyl]-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-12h A mixture of 8SO2BzL-12g (100 mg, 182 umol, 1.0 equiv.), Pd(OH)2 / C (64.0 mg, 91.1 umol, 20% purity, 0.5 equiv.) in MeOH (10 mL) was degassed and purged with H2 (367 ug, 182 umol, 1 equiv.) three times, then stirred at 25° C. for 1 h under H2 (30 psi). The mixture was filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 5%-55%, 8 min) to give 8SO2BzL-12h (72 mg, 130 umol, 71.5% yield) as a white solid. 1 H NMR(MeOD,400MHz)δ 7.70(d,J = 1.2 Hz,1H),7.65(d,J = 1.2 Hz,1H),7.46(s,1H),3.98(q,J = 7.2 Hz,2H),3.76(t,J = 6.8 Hz,2H),3.36(s,2H),3.02(t,J = 6.8 Hz,2H),2.84(t,J = 8.0 Hz,2H),1.83-1.74(m,2H),1.71-1.59(m,2H),1.52-1.34(m,13H),1.20(t,J = 7.2 Hz,3H),1.01(t,J = 7.2 Hz, 3H).LC / MS[M+H] 553.26 (calculated);LC / MS[M+H] 553.2 (observed).

[0228] Preparation of 2-amino-6-(5-aminopentyl)-4-[ethoxy(propyl)carbamoyl]-3H-1-benzazepine-8-sulfonic acid, 8SO2BzL-12i To a solution of 8SO2BzL-12h (60 mg, 108 umol, 1 equiv) in EtOAc (2 mL) was added HCl / EtOAc (4M, 1 mL, 37 equiv) and then stirred at 20° C. for 1 h. The mixture was concentrated in vacuo to give 8SO2BzL-12i (50 mg, 102 umol, 94.2% yield, HCl) as a white solid. LC / MS [M+H] 453.2 (calculated); LC / MS [M+H] 453.2 (observed).

[0229] Preparation of 8SO2BzL-12 To a solution of 3-[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]propanoic acid (68.2 mg, 102 umol, 1.0 equiv.) in DMF (1 mL) was added DIEA (66.1 mg, 511 umol, 89 uL, 5.0 equiv.), 8SO2BzL-12i (50 mg, 102 umol, 1 equiv., HCl), and HATU (38.9 mg, 102 umol, 1.0 equiv.). This was stirred at 25° C. for 0.5 h. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8 min) to give 8SO2BzL-12 (14 mg, 12.7 umol, 12.43% yield) as a yellow oil. 1 H NMR(MeOD,400MHz)δ 7.72(s,1H),7.68(s,1H),7.48(s,1H),6.91(s,2H),4.19(s,2H),4.01(q,J = 7.2 Hz,2H),3.80-3.55(m,42H),3.43-3.35(m,4H),3.19(t,J = 6.8 Hz,2H),2.88(t,J = 8.0 Hz,2H),2.42(t,J = 6.4 Hz,2H),1.80(q,J = 7.2 Hz,2H),1.74-1.64(m,2H),1.60-1.51(m,2H),1.45-1.43(m,2H),1.23(t,J = 7.2 Hz,3H),1.02(t,J = 7.2 Hz,3H).LC / MS [M+H]1101.5(calculated value);LC / MS[M+H]1101.9(observed value).

[0230] Example L-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 (2-((2-amino-8-(N-methylsulfamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-13 [ka] Preparation of tert-butyl (2-((2-amino-8-(N-methylsulfamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-13b To a solution of tert-butyl (2-((2-amino-8-(chlorosulfonyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, 8SO2BzL-13a (0.14 M, 2.00 mL, 0.28 mmol) in acetonitrile / water (9:1) was added methylamine solution (2 M in THF, 0.70 mL, 1.40 mmol) at 0 °C. After 10 min, the solvent was removed and the crude product was purified by reverse phase HPLC using a gradient of 10-90% acetonitrile / water to give 8SO2BzL-13b (0.09 g, 68%) as a yellow film after evaporation of the solvent.

[0231] Preparation of 2-amino-N-(2-aminoethoxy)-8-(N-methylsulfamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamide, 8SO2BzL-13c A solution of 8SO2BzL-13b (0.09 g, 0.19 mmol) in acetonitrile (2 mL) and 6 N HCl (2.00 mL, 12.00 mmol) was stirred at room temperature for 45 min. The solvent was removed in vacuo to give 8SO2BzL-13c (0.08 g, 99%) as the HCl salt.

[0232] Preparation of 8SO2BzL-13 8SO2BzL-13c To a solution of HCl (0.03 g, 0.06 mmol) in DMF (4 mL) was added triethylamine (0.04 mL, 0.26 mmol) at room temperature. To this mixture was added a solution 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(4-nitrophenyl)carbonate, PNPC-PEG10-mal (0.05 g, 0.06 mmol) in DMF (2 mL). After 20 min, acetic acid (6 uL) was added and the reaction was concentrated in vacuo and purified by reverse phase HPLC using a gradient of 10-90% ACN / water (+0.1% TFA) over 10 min to give 8SO2BzL-13 (0.04 g, 52%) after concentration of pure fractions. LC / MS [M+H] 1060.47 (calculated); LC / MS [M+H] 1060.89 (observed).

[0233] Example L-15 Synthesis of 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]ethyl N-[2-[[2-amino-8-(thiazol-2-ylsulfamoyl)-3H-1-benzazepine-4-carbonyl]-propyl-amino]oxyethyl]carbamate, 8SO2BzL-15 [ka] Preparation of ethyl 8-bromo-2-(tritylamino)-3H-1-benzazepine-4-carboxylate, 8SO2BzL-15b A mixture of ethyl 2-amino-8-bromo-3H-1-benzazepine-4-carboxylate, 8SO2BzL-15a (5 g, 16.1 mmol, 1 equiv), TrtCl (6.76 g, 24.2 mmol, 1.5 equiv), TEA (4.91 g, 48.5 mmol, 6.75 mL, 3 equiv), and DMAP (395 mg, 3.23 mmol, 0.2 equiv) in DCM (50 mL) was degassed and purged with N2 three times, then stirred under N2 atmosphere at 40 °C for 16 h. The reaction mixture was quenched by addition of H2O (50 mL) and extracted with EtOAc (100 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=100:1-0:1) to give 8SO2BzL-15b (7.8 g, 14.1 mmol, 87.4% yield) as a yellow oil. LC / MS [M+H] 551.1 (calculated); LC / MS [M+H] 551.1 (observed).

[0234] Preparation of ethyl 8-benzylsulfanyl-2-(tritylamino)-3H-1-benzazepine-4-carboxylate, 8SO2BzL-15c A mixture of 8SO2BzL-15b (3 g, 5.44 mmol, 1 equiv.), DIEA (1.41 g, 10.8 mmol, 1.90 mL, 2 equiv., (1E,4E)-1,5-diphenylpenta-1,4-dien-3-one; palladium, Pd2(dba)3, CAS Registry Number 51364-51-3 (996 mg, 1.09 mmol, 0.2 equiv.), and (5-diphenylphosphanyl-9,9-dimethyl-xanthen-4-yl)-diphenyl-phosphane, Xphos (629 mg, 1.09 mmol, 0.2 equiv.) in dioxane (30 mL) was degassed and purged with N2 three times, then the phenylmethylsulfonyl ether was added. Tanthiol, BnSH (1.35 g, 10.8 mmol, 1.27 mL, 2 equiv.) was added and the mixture was stirred at 110° C. for 1 h under N2 atmosphere. The reaction mixture was quenched by adding H2O (50 mL) and extracted with EtOAc (100 mL×2). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=100:1-0:18) to give 8SO2BzL-15c (2.3 g, 3.87 mmol, 71.1% yield) as a yellow oil.

[0235] Preparation of ethyl 8-chlorosulfonyl-2-(tritylamino)-3H-1-benzazepine-4-carboxylate, 8SO2BzL-15d A mixture of 8SO2BzL-15c (3 g, 5.04 mmol, 1 equiv), NCS (2.69 g, 20.2 mmol, 4 equiv), AcOH (3.03 g, 50.4 mmol, 2.88 mL, 10 equiv) in MeCN (30 mL) and H2O (3 mL) was stirred at 25 °C for 1 h. The reaction mixture was quenched by adding H2O (50 mL) and extracted with EtOAc (50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 99:1-0:1) to give 8SO2BzL-15d (2 g, 3.50 mmol, 69.4% yield) as a yellow solid. LC / MS [M+H] 571.1 (calculated); LC / MS [M+H] 571.2 (observed).

[0236] Preparation of ethyl 8-(thiazol-2-ylsulfamoyl)-2-(tritylamino)-3H-1-benzazepine-4-carboxylate, 8SO2BzL-15e A mixture of 8SO2BzL-15d (1.5 g, 2.63 mmol, 1 equiv) and 1-methylimidazole (258 mg, 3.15 mmol, 251 uL, 1.2 equiv) in MeCN (30 mL) was degassed and purged with N2 three times, then stirred at 25 °C under N2 atmosphere for 2 h. Then 4,5-dihydrothiazol-2-amine (1.07 g, 10.5 mmol, 4 equiv) was added and the resulting mixture was stirred at 25 °C under N2 atmosphere for an additional 16 h. The reaction mixture was quenched by the addition of H2O (50 mL) and extracted with EtOAc (50 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=100:1-0:1) to give 8SO2BzL-15e (0.5 g, 787 umol, 29.9% yield) as a yellow solid. 1H NMR(MeOD,400 MHz)δ7.71(s,1H),7.23(m,20H),6.75(d,J = 4.8 Hz,1H),4.36(q,J = 7.2 Hz,2H),2.98(s,2H),1.38(t, J = 7.2Hz,3H).LC / MS[M+H]635.17(calculated value);LC / MS[M+H]635.1(observed value).

[0237] Preparation of 8-(N-(thiazol-2-yl)sulfamoyl)-2-(tritylamino)-3H-benzo[b]azepine-4-carboxylic acid, 8SO2BzL-15f A mixture of 8SO2BzL-15e (0.5 g, 787.69 umol, 1 equiv), LiOH.HO (264 mg, 6.30 mmol, 8 equiv) in HO (4 mL) and THF (4 mL) was stirred at 25 °C for 3 h. The reaction was quenched by the addition of 2 M HCl to adjust the pH to about 6, and then filtered to give 8SO2BzL-15f (0.45 g, 699.66 umol, 88.82% yield, HCl) as a white solid. LC / MS [M+H] 607.1 (calculated); LC / MS [M+H] 607.2 (observed).

[0238] Preparation of tert-butyl N-[2-[propyl-[8-(thiazol-2-ylsulfamoyl)-2-(tritylamino)-3H-1-benzazepine-4-carbonyl]amino]oxyethyl]carbamate, 8SO2BzL-15g A mixture of 8SO2BzL-15f (0.42 g, 692 umol, 1 equiv), tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (181 mg, 830 umol, 1.2 equiv), methanesulfonic acid (133 mg, 1.38 mmol, 98.5 uL, 2 equiv), EDCI (663 mg, 3.46 mmol, 5 equiv) in DMA (5 mL) and DCM (5 mL) was degassed and purged with N2 three times, then stirred under N2 atmosphere at 25 °C for 2 h. The reaction mixture was quenched by addition of Na2HCO3 (3 mL) until pH was about 7 and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate=99:1-20:80) to give 8SO2BzL-15g (0.5 g, 619 umol, 89.5% yield) as a yellow solid. LC / MS [M+H] 807.3 (calculated); LC / MS [M+H] 807.3 (observed).

[0239] Preparation of 2-amino-N-(2-aminoethoxy)-N-propyl-8-(thiazol-2-ylsulfamoyl)-3H-1-benzazepine-4-carboxamide, 8SO2BzL-15h A mixture of 8SO2BzL-15g (0.5 g, 619 umol, 1 equiv), TFA (5.65 g, 49.5 mmol, 3.67 mL, 80 equiv) in DCM (10 mL) was stirred at 25 °C for 16 h. The reaction mixture was quenched by addition of HO (5 mL) and extracted with MTBE (10 mL) (5 mL × 2) to remove excess TFA. The combined aqueous layers were concentrated under reduced pressure to give 8SO2BzL-15h (0.25 g, 432 umol, 69.7% yield, TFA) as a white solid. LC / MS [M+H] 465.1 (calculated); LC / MS [M+H] 465.1 (observed).

[0240] Preparation of 8SO2BzL-15 A mixture of 8SO2BzL-15h (0.2 g, 288 umol, 1 equiv, 2 TFA), 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 (232 mg, 288 umol, 1 equiv), DIEA (111 mg, 866 umol, 150 uL, 3 equiv) in DMF (0.5 mL) was degassed and purged with N2 three times, then the mixture was stirred under N2 atmosphere at 0° C. for 1 h. The reaction solution was quenched with TFA until the pH was about 6. The residue was purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 15%-40%, 8 min) to give 8SO2BzL-15 (15.0 mg, 13.2 umol, 4.6% yield) as a white solid. 1 H NMR(MeOD-d4,400 MHz) δ7.97(s,1H),7.88(d,J = 2.0 Hz,1H),7.86(d,J = 2.0 Hz,1H),7.39(s,1H),7.18(d,J = 4.8 Hz,1H),6.90(s,2H),6.81(d,J = 4.8 Hz,1H),4.17(s,2H),3.74(m,2H),3.67(m,2H),3.59(m,42H),3.38(m,6H),1.80-1.72(m,2H) 1.00(t,J = 7.2 Hz,3H).LC / MS [M+H] 1129.4 (calculated); LC / MS [M+H] 1129.5 (observed).

[0241] Example 201 Preparation of immune complexes (IC) To prepare ricin-conjugated immunoconjugates, the antibody is buffer exchanged into conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3 using a G-25 SEPHADEX™ desalting column (Sigma-Aldrich, St. Louis, MO) or a Zeba™ spin desalting column (Thermo Fisher Scientific). The eluate is then adjusted to a concentration of about 1-10 mg / ml each using buffer, and then sterile filtered. The antibody is pre-warmed to 20-30° C. and rapidly mixed with 2-20 (e.g., 7-10) molar equivalents of tetrafluorophenyl (TFP) or sulfonic acid tetrafluorophenyl (sulfoTFP) ester, 8-sulfonyl-2-aminobenzazepine-linker (8SO2Bz-L) compound of formula II dissolved in dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5-20 mM. The reaction is allowed to proceed for approximately 16 hours at 30° C., and the immune complexes (IC) are separated from the reactants by passing 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 complexes (IC) in Tables 3a and 3b. The adjuvant-antibody ratios (DAR) are determined by liquid chromatography mass spectrometry using a C4 reversed-phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) coupled to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0242] To prepare cysteine-conjugated immunoconjugates, antibodies are buffer-exchanged into conjugation buffer containing PBS, pH 7.2, with 2 mM EDTA using Zeba™ spin desalting columns (Thermo Fisher Scientific). Interchain disulfides are reduced using a 2-4 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. Maleimide-8SO2Bz-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-8SO2Bz-L. In some cases, up to 20% (v / v) additional DMA or DMSO was added to improve the solubility of maleimide-8SO2Bz-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-8SO2Bz-L using two consecutive Zeba™ spin desalting columns. The columns are pre-equilibrated with phosphate buffered saline (PBS), pH 7.2. The adjuvant-to-antibody ratio (DAR) is estimated by liquid chromatography mass spectrometry using a C4 reversed-phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0243] 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 8SO2Bz-L compound is dissolved in a solvent system that includes at least one polar aprotic solvent, as described elsewhere herein. In some such embodiments, the 8SO2Bz-L is dissolved in a pH 8 Tris buffer (e.g., 50 mM Tris) to a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM, and ranges thereof, such as about 5 mM to about 50 mM, or about 10 mM to about 30 mM. In some embodiments, the 8-sulfonyl-2-aminobenzazepine-linker intermediate is dissolved in DMSO (dimethyl sulfoxide), DMA (dimethylacetamide), acetonitrile, or another suitable dipolar aprotic solvent.

[0244] Alternatively, an equivalent excess of the 8SO2Bz-L solution may be diluted and combined with the antibody solution in the conjugation reaction. The 8SO2Bz-L solution may be suitably diluted with at least one polar aprotic solvent and at least one polar protic solvent, examples of which include water, methanol, ethanol, n-propanol, and acetic acid. The molar equivalent of the 8SO2Bz-L intermediate to the antibody may range from about 1.5:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1, and from about 1.5:1 to about 20:1, from about 1.5:1 to about 15:1, from about 1.5:1 to about 10:1, from about 3:1 to about 15:1, from about 3:1 to about 10:1, from about 5:1 to about 15:1, or from about 5:1 to about 10:1. The reaction may be suitably monitored for completion by methods known in the art, such as LC-MS. The conjugation reaction is usually complete within a range of about 1 hour to about 16 hours. After the reaction is complete, a reagent may be added to the reaction mixture to quench the reaction. If the antibody thiol group has reacted with a thiol-reactive group, such as maleimide, of the 8SO2Bz-L linker intermediate, the unreacted antibody thiol group may be reacted with a capping reagent. An example of a suitable capping reagent is ethylmaleimide.

[0245] After binding, the immune complex may be purified and separated from unbound reactants and / or bound 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 may be diluted with 20 mM sodium succinate, pH 5, etc. The diluted solution is applied to a cation exchange column, followed by washing with, for example, at least 10 column volumes of 20 mM sodium succinate, pH 5. The complex may be suitably eluted with a buffer such as PBS.

[0246] 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. Then, cells were cultured at 4 × 10 4 Cells were seeded in 96-well plates at 100 cells / well. Activity was measured at wavelengths of 620–655 nm using a plate reader.

[0247] Example 203 Evaluation of immune complex activity in vivo This example demonstrates that the immunoconjugates of the invention are effective in inducing immune activation and are therefore useful in the treatment of cancer.

[0248] a) Isolation of human antigen-presenting cells: Human bone marrow 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. Negative selection was then performed using the EASYSEP™ human monocyte enrichment kit (Stem Cell Technologies) containing monoclonal antibodies against CD14, CD16, CD40, CD86, CD123, and HLA-DR, without CD16 deletion, to purify immature APCs with a purity of >90%.

[0249] b) Bone marrow 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). Cell-free supernatants are analyzed 18 hours later via ELISA to measure TNFα secretion as a readout of the proinflammatory response.

[0250] 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 96-well plates (Corning, Corning, NY) co-cultured with CEA-expressing tumor cells (e.g., MKN-45, HPAF-II) at an effector-to-target cell ratio of 10:1. Cells were stimulated with various concentrations of unconjugated (naked) antibodies and immunoconjugates of the invention (prepared according to the above examples). Cell-free supernatants were analyzed by cytokine bead array using the LegendPlex™ kit according to the manufacturer's guidelines (BioLegend®, San Diego, CA).

[0251] 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 EASYSEP™ human myeloid DC enrichment kit (Stem Cell Technologies).

[0252] 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.

[0253] 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).

[0254] 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) may bind via the CDR region of the antibody. After 18-48 h of incubation, activation may be assessed by upregulation of cell surface costimulatory molecules using flow cytometry or by measurement of secreted pro-inflammatory cytokines. For cytokine measurements, cell-free supernatants are harvested and analyzed by cytokine bead arrays (e.g., LegendPlex from Biolegend) using flow cytometry.

[0255] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

Claims

1. Formula II: 【change】 8-sulfonyl-2-aminobenzazepine-linker compounds (In the formula, R １ , R ２ , R ３ , and R ４ are independent, H, C １ -C １２ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryl, C ２ -C ９ Heterocyclyl, and C １ -C ２０ heteroaryl, wherein alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl are independently and optionally selected from the group consisting of: -(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C １ -C １２ alkyldiyl)-OR ５ , -(C ３ -C １２ carbocyclyl), -(C ３ -C １２ carbocyclyl)-*, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-NR ５ - *, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C ３ -C １２ carbocyclyl)-NR ５ -C(=NR ５ ) NR ５ - *, -(C ６ -C ２０ aryl), -(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 ) ２ , -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-NR ５ -C(=NR ５ａ ) N (R ５ )-*, -(C ２ -C ２０ heterocyclyl), -(C ２ -C ２０ heterocyclyl)-*, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-NR ５ - *, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C ２ -C ９ heterocyclyl)-C(═O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C ２ -C ９ Heterocyclyl)-NR ５ -C(=NR ５ａ ) NR ５ - *, -(C ２ -C ９ Heterocyclyl)-NR ５ -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C ２ -C ９ heterocyclyl)-(C ６ -C ２０ Aryldiyl)-*, -(C １ -C ２０ heteroaryl), -(C １ -C ２０ heteroaryldiyl)-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C １ -C ２０ Heteroaryl)-NR ５ -C(=NR ５ａ ) N (R ５ )-*, -(C １ -C ２０ heteroaryl)-N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -C(=O)-*, -C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -C(=O)-(C ２ -C ２０ heterocyclyldiyl)-*, -C(=O)N(R ５ ) ２ 、 -C(=O)N(R ５ )-*、 -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ ) C(=O)R ５ , -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)N(R ５ ) ２ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-N(R ５ ) CO ２ R ５ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-N(R ５ ) C(=NR ５ａ ) N (R ５ ) ２ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５ C (=NR ５ａ ) R ５ , -C(=O)NR ５ -(C １ -C ８ alkyldiyl)-NR ５ (C ２ -C ５ heteroaryl), -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-N(R ５ )-*, -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-*, -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-(C ２ -C ２０ heterocyclyldiyl)-C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５ - *, -N(R ５ ) ２ 、 -N(R ５ )-*、 -N(R ５ )C(=O)R ５ 、 -N(R ５ )C(=O)-*、 -N(R ５ )C(=O)N(R ５ ) ２ 、 -N(R ５ )C(=O)N(R ５ )-*、 -N(R ５ )CO ２ R ５ 、 -NR ５ C(=NR) ５ａ )N(R ５ ) ２ 、 -NR ５ C(=NR) ５ａ )N(R ５ )-*、 -NR ５ C(=NR) ５ａ )R ５ 、 -N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -N(R ５ )-(C ２ -C ５ heteroaryl), -N(R ５ ) -S(=O) ２ -(C １ -C １２ alkyl), -O-(C １ -C １２ alkyl), -O-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -O-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -O-C(=O)N(R ５ ) ２ 、 -O-C(=O)N(R ５ )-*、 -O-(R ５ )-*、 -OR ５ 、 -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-*, -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-NR ５ -*, and -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ or substituted with one or more groups selected from: or R ２ and R ３ together form a 5- or 6-membered heterocyclyl ring, X １ , X ２ , X ３ , and X ４ are independently a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N (R ５ ) selected from the group consisting of R ５ are independent, H, C ６ -C ２０ Aryl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups together form a 5- or 6-membered heterocyclyl ring; R ５ａ But C ６ -C ２０ Aryl and C １ -C ２０ heteroaryl; where the asterisk * indicates the binding site of L, and R １ , R ２ , R ３ , and R ４ is attached to L; L, Q-C(=O)-PEG-, Q-C(=O)-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, Q-C(=O)-PEG-O-, Q-C(=O)-PEG-O-C(=O)-, Q-C(=O)-PEG-C(=O)-, Q-C(=O)-PEG-C(=O)-PEP-, Q-C(=O)-PEG-N(R ６ )-、 Q-C(=O)-PEG-N(R ６ )-C(=O)-、 Q-C(=O)-PEG-N(R ６ )-PEG-C(=O)-PEP-、 Q-C(=O)-PEG-N ＋ (R) ６ ) ２ -PEG-C(=O)-PEP- Q-C(=O)-PEG-C(=O)-PEP-N(R ６ )-(C １ -C １２ alkyldiyl)-, Q-C(=O)-PEG-C(=O)-PEP-N(R ６ )-(C １ -C １２ alkyldiyl)N(R ６ ) C(=O)-(C ２ -C ５ monoheterocyclyldiyl)-, Q-C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-, Q-C(=O)-PEG-SS-(C １ -C １２ alkyldiyl)-C(=O)-, Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(PEG-CO ２ H)-PEG-N(R ５ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-PEG-N(R ５ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(PEG-CO ２ H)-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(═O)-PEP-, and Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ the linker is selected from the group consisting of: (alkyldiyl)-OC(=O)-; R ６ are independently H or C １ -C ６ is alkyl, PEG is a group represented by the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -, m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc is a compound of the formula: 【Transformation 6】 and PEP has the formula: 【Transformation 7】 and wherein AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is optionally F, Cl, NO ２ , —OH, —OCH ３ , and structure: 【Transformation 8】 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 )OC(=O)-(wherein, R ８ But H, C １ -C ６ Alkyl, C(=O)-C １ -C ６ Alkyl, and —C(═O)N(R ９ ) ２ wherein R ９ are independent, H, C １ -C １２ Alkyl, and -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -OH, wherein m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R ９ the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12, z is 0 or 1; Q is F, Cl, NO ２ , and SO ３ － is selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, substituted with one or more groups independently selected from Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH) ３ ) ２ 、-CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-C(CH ３ ) ２ CONG 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 CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-O(CH ２ CH ２ O) ｎ -(CH) ２ ) ｍ CO ２ H, —O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H).

2. Q is, 2. The 8-sulfonyl-2-aminobenzazepine-linker compound of claim 1, selected from:

3. 3. The 8-sulfonyl-2-aminobenzazepine-linker compound of claim 2, wherein Q is maleimide.

4. L, Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(PEG-CO ２ H)-PEG-N(R ５ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-PEG-N(R ５ )-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(PEG-CO ２ H)-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-PEG-C(=O)-、 Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(═O)-PEP-, and Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-; 2. The 8-sulfonyl-2-aminobenzazepine-linker compound of claim 1, wherein Q is maleimide.

5. L, Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)- 5. The 8-sulfonyl-2-aminobenzazepine-linker compound of claim 4, wherein

6. structure:

2. The 8-sulfonyl-2-aminobenzazepine-linker compound of claim 1, having the formula:

7. 1. An antibody comprising an antibody covalently linked by a linker to one or more 8-sulfonyl-2-aminobenzazepine moieties, and having formula I: Ab-[L-D] ｐ I an immunoconjugate comprising During the ceremony, Ab is the antibody; p is an integer from 1 to 8; L is the linker; D is a compound of the formula: 【Chemistry 1】 the 8-sulfonyl-2-aminobenzazepine moiety having the formula R １ , R ２ , R ３ , and R ４ are independent, 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, each of which is independently and optionally selected from the group consisting of: -(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C １ -C １２ alkyldiyl)-OR ５ , -(C ３ -C １２ carbocyclyl), -(C ３ -C １２ carbocyclyl)-*, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-NR ５ - *, -(C ３ -C １２ carbocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C ３ -C １２ carbocyclyl)-NR ５ -C(=NR ５ ) NR ５ - *, -(C ６ -C ２０ aryl), -(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 ) ２ , -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-NR ５ -C(=NR ５ａ ) N (R ５ )-*, -(C ２ -C ２０ heterocyclyl), -(C ２ -C ２０ heterocyclyl)-*, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-NR ５ - *, -(C ２ -C ９ heterocyclyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C ２ -C ９ heterocyclyl)-C(═O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C ２ -C ９ Heterocyclyl)-NR ５ -C(=NR ５ａ ) NR ５ - *, -(C ２ -C ９ Heterocyclyl)-NR ５ -(C ６ -C ２０ aryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C ２ -C ９ heterocyclyl)-(C ６ -C ２０ Aryldiyl)-*, -(C １ -C ２０ heteroaryl), -(C １ -C ２０ heteroaryl)-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -(C １ -C ２０ heteroaryl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -(C １ -C ２０ Heteroaryl)-NR ５ -C(=NR ５ａ ) N (R ５ )-*, -(C １ -C ２０ heteroaryl)-N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -C(=O)-*, -C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -C(=O)-(C ２ -C ２０ heterocyclyldiyl)-*, -C(=O)N(R ５ ) ２ 、 -C(=O)N(R ５ )-*、 -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ ) C(=O)R ５ , -C(=O)N(R ５ )-(C １ -C １２ alkyldiyl)-N(R ５ )C(=O)N(R ５ ) ２ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-N(R ５ ) CO ２ R ５ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-N(R ５ ) C(=NR ５ａ ) N (R ５ ) ２ , -C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５ C (=NR ５ａ ) R ５ , -C(=O)NR ５ -(C １ -C ８ alkyldiyl)-NR ５ (C ２ -C ５ heteroaryl), -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-N(R ５ )-*, -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-*, -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -C(=O)NR ５ -(C １ -C ２０ heteroaryldiyl)-(C ２ -C ２０ heterocyclyldiyl)-C(=O)NR ５ -(C １ -C １２ alkyldiyl)-NR ５ - *, -N(R ５ ) ２ 、 -N(R ５ )-*、 -N(R ５ )C(=O)R ５ 、 -N(R ５ )C(=O)-*、 -N(R ５ )C(=O)N(R ５ ) ２ 、 -N(R ５ )C(=O)N(R ５ )-*、 -N(R ５ )CO ２ R ５ 、 -NR ５ C(=NR) ５ａ )N(R ５ ) ２ 、 -NR ５ C(=NR) ５ａ )N(R ５ )-*、 -NR ５ C(=NR) ５ａ )R ５ 、 -N(R ５ ) C(=O)-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -N(R ５ )-(C ２ -C ５ heteroaryl), -N(R ５ ) -S(=O) ２ -(C １ -C １２ alkyl), -O-(C １ -C １２ alkyl), -O-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -O-(C １ -C １２ alkyldiyl)-N(R ５ )-*, -O-C(=O)N(R ５ ) ２ 、 -O-C(=O)N(R ５ )-*、 -O-(R ５ )-*、 -OR ５ 、 -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-*, -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ５ ) ２ , -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-NR ５ -*, and -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ or substituted with one or more groups selected from: or R ２ and R ３ together form a 5- or 6-membered heterocyclyl ring, X １ , X ２ , X ３ , and X ４ are independently a bond, C(=O), C(=O)N(R ５ ), O, N(R ５ ), S, S(O) ２ , and S(O) ２ N (R ５ ) selected from the group consisting of R ５ are independent, H, C ６ -C ２０ Aryl, C ３ -C １２ Carbocyclyl, C ６ -C ２０ Aryldiyl, C １ -C １２ Alkyl, and C １ -C １２ alkyldiyl, or two R ５ the groups together form a 5- or 6-membered heterocyclyl ring; R ５ａ But C ６ -C ２０ Aryl and C １ -C ２０ heteroaryl; where the asterisk * indicates the binding site of L, and R １ , R ２ , R ３ , and R ４ is attached to L; L, -C(=O)-PEG-, -C(=O)-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(═O)-Gluc-, -C(=O)-PEG-O-, -C(=O)-PEG-O-C(=O)-, -C(=O)-PEG-C(=O)-, -C(=O)-PEG-C(=O)-PEP-, - C ( = O ) - PEG - N ( R ) ６ )-、 - C ( = O ) - PEG - N ( R ) ６ )-C(=O)-、 - C ( = O ) - PEG - N ( R ) ６ )-PEG-C(=O)-PEP-、 - C ( = O ) - PEG - N ＋ (R) ６ ) ２ -PEG-C(=O)-PEP- -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)-, -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(PEG-CO ２ H)-PEG-N(R ５ ) -, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-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-N(PEG-CO ２ H)-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(═O)-PEP-, and -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ the linker is selected from the group consisting of (alkyldiyl)-OC(=O)-, R ６ are independently H or C １ -C ６ is alkyl, PEG is a group represented by the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -, m is an integer from 1 to 5, and n is an integer from 2 to 50; Gluc is a compound of the formula: 【Chemistry 2】 and PEP has the formula: 【Transformation 3】 and wherein AA are independently selected from natural or unnatural amino acid side chains, or one or more of AA and the adjacent nitrogen atom form a 5-membered ring proline amino acid, and the wavy line indicates the point of attachment; Cyc is optionally F, Cl, NO ２ , —OH, —OCH ３ , and structure: 【Chemistry 4】 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 )OC(=O)-(wherein, R ８ But H, C １ -C ６ Alkyl, C(=O)-C １ -C ６ Alkyl, and —C(═O)N(R ９ ) ２ wherein R ９ are independent, H, C １ -C １２ Alkyl, and -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ -OH, wherein m is an integer from 1 to 5 and n is an integer from 2 to 50, or two R ９ the groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12, z is 0 or 1; Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl are independently and optionally selected from F, Cl, Br, I, —CN, —CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH (CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH) ３ ) ２ 、-CO ２ H, -COCH ３ 、-CO ２ CH ３ 、-CO ２ C(CH) ３ ) ３ 、-COCH(OH)CH ３ 、-CONH ２ 、-CONHCH ３ 、-CON(CH) ３ ) ２ 、-C(CH ３ ) ２ CONG 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 CONG ２ 、-N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、-NHCC(=NH)H、-NHCC(=NH)CH ３ 、-NHCC(=NH)NH ２ 、-NHCC(=O)NH ２ 、-NO ２ 、=O、-OH、-OCH ３ 、-OCH ２ CH ３ 、-OCH ２ CH ２ OCH ３ 、-OCH ２ CH ２ OH,-OCH ２ CH ２ N(CH) ３ ) ２ 、-O(CH ２ CH ２ O) ｎ -(CH) ２ ) ｍ CO ２ H, —O(CH ２ CH ２ O) ｎ H, -OCH ２ F, -OCHF ２ , -OCF ３ , -OP(O)(OH) ２ , -S(O) ２ N (CH ３ ) ２ , -SCH ３ , -S(O) ２ CH ３ , and -S(O) ３ H, or a pharmaceutically acceptable salt thereof.

8. X １ is a bond, and R １ The immune complex of claim 7, wherein is H.

9. X ２ is a bond, and R ２ is C １ -C ８ The immunoconjugate of claim 7, wherein the carboxyl group is alkyl.

10. X ２ and X ３ are each a bond, and R ２ and R ３ became independent and C １ -C ８ Alkyl, —O—(C １ -C １２ alkyl), -(C １ -C １２ alkyldiyl)-OR ５ , -(C １ -C ８ alkyldiyl)-N(R ５ ) CO ２ R ５ , -(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ , —O—(C １ -C １２ alkyl)-N(R ５ ) CO ２ R ５ , and —O—(C １ -C １２ alkyl)-OC(O)N(R ５ ) ２ The immune complex of claim 7, wherein the immune complex is selected from the group consisting of:

11. R ２ and R ３ are each independently —CH ２ CH ２ CH ３ , -OCH ２ CH ３ , -OCH ２ CF ３ , -CH ２ CH ２ CF ３ , -OCH ２ CH ２ OH, and -CH ２ CH ２ CH ２ 11. The immune complex of claim 10, wherein the amino acid is selected from the group consisting of OH.

12. R ４ is C １ -C １２ The immunoconjugate of claim 7, wherein the carboxyl group is alkyl.

13. R ４ But-(C １ -C １２ alkyldiyl)-N(R ５ 8. The immune complex of claim 7, wherein the asterisk * indicates the binding site of L.

14. The immune complex of claim 7 , wherein L is attached to a cysteine ​​thiol of the antibody.

15. 8. The immune conjugate of claim 7, wherein for the PEG, m is 1 or 2 and n is an integer from 2 to 10.

16. 16. The immune complex of claim 15, wherein n is 10.

17. An 8-sulfonyl-2-aminobenzazepine-linker compound selected from the group consisting of:

18. A pharmaceutical composition comprising a therapeutically effective amount of the immunoconjugate of claim 7 and one or more pharmaceutically acceptable diluents, vehicles, carriers, or excipients.

19. 8. The immunoconjugate of claim 7 for use in the treatment of cancer, wherein the cancer is selected from cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial cancer, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

20. A method for preparing an immunoconjugate of formula I according to claim 7, wherein the 8-sulfonyl-2-aminobenzazepine-linker compound according to claim 1 is conjugated to an antibody.

21. 8-sulfonyl-2-aminobenzazepine-linker compound 21. The method of claim 20, wherein:

22. 8. The immunoconjugate of claim 7, wherein the antibody binds to a target selected from the group consisting of PD-L1, HER2, TROP2, and CEA.

23. 23. The immunoconjugate of claim 22, wherein the antibody is selected from the group consisting of trastuzumab, pertuzumab, labetuzumab and sacituzumab.

24. L, -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(PEG-CO ２ H)-PEG-N(R ５ ) -, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-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-N(PEG-CO ２ H)-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(PEG-CO ２ H)-PEG-C(=O)-, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(═O)-PEP-, and -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)- The immune complex of claim 7, selected from the group consisting of:

25. L, -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)- 25. The immune complex of claim 24, wherein:

26. An antibody, 8. The immunoconjugate of claim 7, prepared by conjugation with an 8-sulfonyl-2-aminobenzazepine-linker compound selected from the group consisting of:

27. An antibody, 8-Sulfonyl-2-aminobenzazepine-linker compounds:

27. The immunoconjugate of claim 26, prepared by conjugation with

Images