Bis-benzimidazole STING agonist immunoconjugates and uses thereof

Abstract

The present invention provides an immunoconjugate of formula I, comprising an antibody linked by a bond to one or more STING agonist moieties. The present invention also provides a STING agonist-linker intermediate compound comprising a reactive functional group. Such intermediate compositions are suitable substrates for the formation of said immunoconjugates via a linker or linking moiety. The present invention further provides a method of treating cancer using said immunoconjugates.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 215,100, filed June 25, 2021, which is incorporated by reference in its entirety.

[0002] Technical Field The present invention relates generally to immunoconjugates comprising an antibody conjugated to one or more bis-benzimidazole molecules. [Background technology]

[0003] STING (stimulator of interferon genes), also known as transmembrane protein 173 (TMEM173) and MPYS / MITA / ERIS, is a protein encoded by the STING1 gene in humans. STING is widely expressed, especially in immune cells, lungs, and ovaries. STING plays a role in innate immunity by inducing the production of type I interferons when cells are infected with intracellular pathogens such as viruses, mycobacteria, and intracellular parasites. STING-mediated type I interferons protect the same infected cells and neighboring cells from local infection by binding to cells that secrete them through autocrine signaling and to neighboring cells through paracrine signaling. STING functions as both a direct cytoplasmic DNA sensor (CDS) and an adaptor protein in type I interferon signaling through different molecular mechanisms. STING has been shown to activate downstream transcription factors STAT6 and IRF3 through TBK1, which are involved in antiviral and innate immune responses against intracellular pathogens. Compounds that bind to and act as agonists of STING have been shown to induce the secretion of proinflammatory cytokines, including type 1 interferon, when incubated with human PBMCs (WO2017 / 175147). STING modulators may be useful in the treatment of various disorders, such as allergic diseases, neurodegenerative diseases, precancerous syndromes, and cancer, and may also be useful in immunogenic compositions or vaccine adjuvants.

[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. Summary of the Invention

[0005] The invention generally comprises an antibody covalently linked to one or more STING agonist moieties by a linker, the antibody having 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; D is a STING agonist moiety having the formula: [ka] The substituents are defined herein.

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

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

[0008] Another aspect of the invention is a method of treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an antibody linked by a bond to one or more bis-benzimidazole moieties.

[0009] Another aspect of the invention is the use of immunoconjugates comprising antibodies linked by bonds to one or more bis-benzimidazole moieties to treat cancer.

[0010] Another aspect of the invention is a method for preparing an immunoconjugate by conjugating one or more bis-benzimidazole moieties to an antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the appended claims.

[0012] 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.

[0013] definition The term "immunoconjugate" refers to an antibody construct that is covalently attached to an adjuvant moiety via a linker. The term "adjuvant" refers to a substance that can induce an immune response in a subject exposed to the adjuvant. The term "adjuvant moiety" refers to an adjuvant that is covalently attached to an antibody construct, for example, via a linker, as described herein. The adjuvant moiety can induce an immune response while attached to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject.

[0014] The terms "immunostimulant" and "immunostimulatory" are used interchangeably and refer to moieties, substances, or adjuvants capable of eliciting an immune response in a subject exposed to the immunostimulatory moiety or immunostimulatory compound after in vivo cleavage of the linker. The term "adjuvant moiety" or "immunostimulatory moiety" refers to an adjuvant that is covalently attached to a cell binding agent, such as an elastase substrate, an antibody construct via a peptide linker, or referred to as a "payload," as described herein. The adjuvant moiety can elicit an immune response while bound to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct after administration of the immunoconjugate to a subject. The immunoconjugate allows for targeted delivery of the active adjuvant moiety while the target antigen is bound.

[0015] The term "pattern recognition receptors" (PRRs) refers to germline-encoded host sensors that detect pathogen-typical molecules and regulate the function of the innate immune system (Mahla, R.S. et al, (2013), Frontiers in Immunology, 4:248; Kumar, H. et al, (2011), Intl. Rev. of Immun. 30:16-34; Schroder K. et al, (2010), Cell, 140(6):821-832). PRRs are proteins expressed primarily by cells of the innate immune system, such as dendritic cells, macrophages, monocytes, neutrophils and epithelial cells, and identify pathogen-associated molecular patterns (PAMPs) associated with microbial pathogens and damage-associated molecular patterns (DAMPs) associated with components of host cells that are released during cellular damage or death. PRRs are also called early pattern recognition receptors, as they arise before other parts of the immune system, especially before adaptive immunity. PRRs also mediate the initiation of antigen-specific adaptive immune responses and the release of inflammatory cytokines. PRRs include, but are not limited to, Toll-like receptors (TLRs), STING-like receptors, RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin-like receptors (CLRs), and DNA sensors.

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

[0017] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. As used herein, an "antibody fragment" and all grammatical variations thereof are defined as a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, wherein said portion does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, (1) single chain Fv (scFv) molecules; (2) single chain polypeptides containing only one light chain variable domain without the relevant heavy chain portion, or fragments thereof containing the three CDRs of the light chain variable domain; (3) single chain polypeptides containing only one heavy chain variable region without the relevant light chain portion, or fragments thereof containing the three CDRs of the heavy chain variable region; (4) nanobodies or other specific single domain binding modules containing a single Ig domain from a non-human species; and (5) multispecific or multivalent structures formed from antibody fragments, including Fab, Fab', Fab'-SH, F(ab') and / or F(ab') fragments. 2 and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (herein referred to as a "single-chain antibody fragment" or "single-chain polypeptide"). In antibody fragments comprising one or more heavy chains, the heavy chain(s) can contain any of the constant domain sequences found in the non-Fc region of an intact antibody (e.g., CH1 of IgG isotypes) and / or can contain any hinge region sequence found in an intact antibody and / or can contain a leucine zipper sequence fused or located to the hinge region sequence or constant domain sequence of the heavy chain(s).

[0018] "Antibody" refers to a polypeptide comprising an antigen-binding region (including complementarity determining regions (CDRs)) derived from an immunoglobulin gene or 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 include, for example, the variable domains or regions of the light and heavy chains (V and V, respectively). L and V H ), light and heavy chain constant domains or regions (C L and C H ) and are classified into various categories according to size and function. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids, called the paratope, which is primarily responsible for antigen recognition, i.e., the antigen-binding domain. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. IgG antibodies are large molecules of about 150 kDa composed of four peptide chains. IgG antibodies contain two identical class gamma heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus comprising a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain by a disulfide bond, respectively. The resulting tetramer has two identical halves that together form a Y-shaped configuration. Both ends of the branch contain the same antigen-binding domain. In humans, there are four IgG subclasses (IgG1, IgG2, IgG3, and IgG4), named in order of abundance in serum (i.e., IgG1 is the most abundant). The antigen-binding domain of an antibody is usually the most important for the specificity and affinity of binding to cancer cells.

[0019] 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-GM1, CTLA-4, and CD44 (WO2017 / 196598).

[0020] "Antibody construct" refers to an antibody or a fusion protein comprising (i) an antigen-binding domain and (ii) an Fc domain.

[0021] In some embodiments, the binding agent is an antigen-binding antibody "fragment," which is a construct that includes at least the antigen-binding region of an antibody, either alone or together with other components that together form an antigen-binding construct. Many different types of antibody "fragments" are known in the art, including, for example: (i) V L , V H , C L and C.H. 1 (ii) a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region, called F(ab') 2 (iii) a V fragment of a single arm of an antibody L and V H (iv) F(ab') fragments consisting of Fv domains under mild reducing conditions; 2 (v) disulfide-stabilized Fv fragments (dsFv), which result from cleavage of the disulfide bridges of the Fv fragment; and (vi) the two domains of the Fv fragment linked by a synthetic linker that allows the two domains to be synthesized as one polypeptide chain (i.e., V L and VH ) and single-chain Fv (scFv).

[0022] An antibody or antibody fragment may be part of a larger construct, e.g., a conjugation 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 optionally to an 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 TROP2. 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.

[0023] By "epitope" is meant any antigenic or epitopic determinant of an antigen to which an antigen-binding domain binds (i.e., at the paratope of the antigen-binding domain). Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0024] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. There are three major classes of Fc receptors: (1) FcγR, which binds IgG; (2) FcαR, which binds IgA; and (3) FcεR, which binds IgE. The FcγR family includes several members, such as FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). Fcγ receptors have different affinities for IgG and for IgG subclasses (e.g., IgG1, IgG2, IgG3, IgG4).

[0025] As used herein, the phrase "immune checkpoint inhibitor" refers to any modulator that inhibits the activity of an immune checkpoint molecule. Immune checkpoint inhibitors may include, but are not limited to, immune checkpoint molecule binding proteins, small molecule inhibitors, antibodies (including bispecific and multispecific antibodies having at least one antigen-binding region that targets an immune checkpoint protein, e.g., bispecific or multispecific antibodies that do not target only an immune checkpoint protein, as well as antibodies that are dual immunomodulators (that target two immunomodulatory targets simultaneously) (resulting in blockade of inhibitory targets, depletion of suppressor cells, and / or activation of effector cells); tumor-targeted immunomodulators (that induce potent costimulation in tumor-infiltrating immune cells by targeting tumor antigens and costimulatory molecules such as CD40 or 4-1BB); NK cell redirecting agents (that redirect NK cells to malignant cells by targeting tumor antigens and CD16A); or T cell redirecting agents (that redirect T cells to malignant cells by targeting tumor antigens and CD3)), antibody derivatives (including Fc fusions, Fab fragments, and scFv), antibody drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides, and peptidomimetics.

[0026] 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-Coffe, and ALIGN (for nucleic acid and amino acid sequence alignment), BLAST programs (e.g., BLAST2.1, BL2SEQ, BLASTp, BLASTn, etc.), and FASTA programs (e.g., FASTA3x, FASTM, and SRESEARCH) (for sequence alignment and sequence similarity search). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7):951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17):3389-3402 1997) and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997). Percent sequence identity (%) is calculated, for example, as 100×1[(identical positions) / min(TG A ,T.G. B)], and TG A and T.G. B T.G. A and T.G. B is the sum of the number of residues and internal gap positions of peptide sequences A and B in the alignment that minimizes (see, e.g., Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0027] The binding agent comprises Ig heavy and light chain variable region polypeptides that together form an antigen-binding site. Each of the heavy and light chain variable regions is a polypeptide that comprises three complementarity determining regions (CDR1, CDR2, and CDR3) linked by framework regions. The binding agent can be any of a variety of types of binding agents known in the art, including Ig heavy and light chains. For example, the binding agent can be an antibody, an antigen-binding antibody "fragment," or a T-cell receptor.

[0028] "Biosimilar" refers to an antibody construct that has activity profiles similar to sacituzumab, a previously approved antibody construct targeting Trop2, e.g., sacituzumab govitecan (TRODELVY®, Immunomedics, IMMU-132).

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

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

[0031] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

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

[0033] Non-natural (non-naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in the L- or D-configuration that function similarly to natural amino acids. An "amino acid analog" can be a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., a carbon bonded to a hydrogen, a carboxyl group, and an amino group), but has a modified side group or peptide backbone, such as homoserine, norleucine, methionine sulfoxide, 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 similarly to a natural amino acid.

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

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

[0036] "Divalent" refers to a chemical moiety that includes two points of attachment for linking two functional groups. A multivalent 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.

[0037] Wavy line [ka] represents the point of attachment of a particular chemical moiety. [ka] When present, it is understood that the chemical moiety can be used in both ways, i.e., read from left to right or right to left. In some embodiments, the two wavy lines present [ka] A particular site having a suffix suffix is ​​considered to be used as read from left to right.

[0038] "Alkyl" refers to a straight chain (linear) or branched saturated aliphatic radical having the number of carbon atoms indicated. Alkyl can contain any number of carbons, for example, from 1 to 12. Examples of alkyl groups include methyl (Me, -CH3 ), ethyl (Et, -CH 2 CH 3 ), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1-Butyl (n-Bu, n-Butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 ) 2 ), 2-Butyl (s-Bu, s-Butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl-2-butyl (-C(CH 3 ) 2 CH 2 CH 3 ), 3-methyl-2-butyl (-CH(CH 3 )CH(CH 3 ) 2 ), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH 3 ) 2 ), 2-methyl-1-butyl (-CH 2 CH(CH 3 )CH 2 CH 3 ), 1-hexyl (-CH 2 CH2 CH 2 CH 2 CH 2 CH 3 ), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 )), 2-methyl-2-pentyl (-C(CH 3 ) 2 CH 2 CH 2 CH 3 ), 3-methyl-2-pentyl (-CH(CH 3 )CH(CH 3 )CH 2 CH 3 ), 4-methyl-2-pentyl (-CH(CH 3 )CH 2 CH(CH 3 ) 2 ), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 ) 2 ), 2-methyl-3-pentyl (-CH(CH 2 CH 3 )CH(CH 3 ) 2 ), 2,3-dimethyl-2-butyl (-C(CH 3 ) 2 CH(CH 3 ) 2 ), 3,3-dimethyl-2-butyl (-CH(CH 3 )C(CH 3 ) 3Examples of alkyl groups include, but are not limited to, 1-heptyl, 1-octyl, and the like. Alkyl groups can be substituted or unsubstituted. Substituted alkyl groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy. Substituted alkyl groups can be geminally substituted, where a carbon atom of the alkyl forms a spiro, cycloalkyl group, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0039] The term "alkyldiyl" refers to a divalent alkyl radical. Examples of alkyldiyl groups include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene (-CH 2 CH 2 CH 2 -), and the like. An alkyldiyl group may also be referred to as an "alkylene" group. An alkyldiyl group can be substituted or unsubstituted. A substituted alkyldiyl group can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy. A substituted alkyldiyl group can be geminally substituted, where a carbon atom of the alkyl forms a spiro, cycloalkyl group, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0040] "Alkenyl" refers to a straight chain (linear) or branched unsaturated aliphatic radical having the indicated number of carbon atoms and at least one carbon-carbon double bond, sp2. Alkenyl can contain from 2 to about 12 or more carbon atoms. Alkenyl groups are radicals that have "cis" and "trans" orientations, alternatively "E" and "Z" orientations. Examples include ethylenyl or vinyl (-CH=CH 2 ), allyl (-CH 2 CH=CH 2Alkenyl groups include, but are not limited to, butenyl, butenyl, pentenyl, and isomers thereof. Alkenyl groups can be unsubstituted or substituted. "Substituted alkenyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0041] The term "alkenylene" or "alkenyldiyl" refers to a straight-chain or branched divalent hydrocarbon radical. Examples include ethylenylene or vinylene (-CH=CH-), allyl (-CH 2 CH=CH-), but are not limited to these.

[0042] "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, C 2 -C 6 Alkynyl includes ethynyl (-C≡CH), propynyl (propargyl, -CH 2 Alkynyl groups include, but are not limited to, C≡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.

[0043] The terms "alkynylene" or "alkynyldiyl" refer to a divalent alkynyl radical.

[0044] The terms "carbocycle", "carbocyclyl", "carbocycle" and "cycloalkyl" refer to saturated or partially unsaturated monocyclic, fused bicyclic, spiro, or bridged polycyclic assemblies containing 3 to 12 ring atoms or the number of atoms indicated. Saturated monocyclic carbocycles include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocycles include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. Carbocyclic groups are partially unsaturated and may have one or more double or triple bonds in the ring. Representative partially unsaturated carbocyclic groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene.

[0045] The term "cycloalkyldiyl" refers to a divalent cycloalkyl radical.

[0046] "Aryl" means an aromatic ring system having 6 to 20 carbon atoms (C 6 -C 20 ) monovalent aromatic hydrocarbon radical. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or joined by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. Other aryl groups include benzyl, which has a methylene linking group. Some aryl groups have 6 to 12 ring members, such as phenyl, naphthalene, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl.

[0047] "Arylene" or "aryldiyl" refers to an aryl group of 6 to 20 carbon atoms (C ) derived by the removal of two hydrogen atoms from two carbon atoms of a parent aromatic ring system. 6-C 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 described herein.

[0048] The terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic radical of 3 to about 20 ring atoms, in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atoms are C, and one or more ring atoms are optionally substituted independently by one or more substituents described below. The heterocycle may be a monocycle having 3-7 ring members (2-6 carbon atoms and 1-4 heteroatoms selected from N, O, P, and S) or a bicycle having 7-10 ring members (4-9 carbon atoms and 1-6 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.

[0049] 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 where one or more ring atoms are optionally substituted independently with one or more of the substituents described. Five- and six-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S-dioxothiomorpholinyldiyl.

[0050] The term "heteroaryl" refers to a monovalent aromatic radical of 5, 6, or 7 rings, including fused ring systems of 5 to 20 atoms, at least one of which is aromatic, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0051] 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.

[0052] The heterocycle or heteroaryl group may be carbon (carbon-linked) or nitrogen (nitrogen-linked) linked, where possible. By way of example and not limitation, a carbon-linked heterocycle or heteroaryl is linked at the 2-, 3-, 4-, 5-, or 6-position of a pyridine, the 3-, 4-, 5-, or 6-position of a pyridazine, the 2-, 4-, 5-, or 6-position of a pyrimidine, the 2-, 3-, 5-, or 6-position of a pyrazine, the 2-, 3-, 4-, or 5-position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of an oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of an isoxazole, pyrazole, or isothiazole, the 2-, or 3-position of an aziridine, the 2-, 3-, or 4-position of an azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of a quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of an isoquinoline.

[0053] 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.

[0054] The terms "halo" or "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0055] The term "carbonyl" by itself or as part of another substituent refers to C(=O) or -C(=O)-, i.e., a carbon atom double-bonded to an oxygen and bonded to two other groups in the carbonyl-containing moiety.

[0056] As used herein, the phrase "quaternary ammonium salt" refers to an ammonium salt having an alkyl substituent (e.g., a C 1 -C 2 -O 4 -O 6 -O 8 -O 9 -O 10 -O 11 -O 12 -O 13 -O 14 -O 15 -O 16 -O 17 -O 18 -O 19 -O 20 -O 21 -O 22 -O 23 -O 34 -O 35 -O 36 -O 37 -O 38 -O 40 -O 41 -O 42 -O 43 -O 44 -O 45 -O 46 -O 47 -O 48 -O 49 -O 50 -O 51 -O 52 -O 53 -O 54 -O 55 -O 56 -O 57 -O 58 -O 59 -O 60 - 1 -C 4 It refers to a tertiary amine that has been quaternized with an alkyl group.

[0057] The terms "treat," "treatment," and "treating" refer to any indicia of success in treating or ameliorating an injury, condition, state (e.g., cancer) or symptom (e.g., cognitive impairment), and include any objective or subjective parameter, such as remission, remission, relief of symptoms, or making the symptom, injury, condition, or condition more tolerable to the patient, reducing the rate of progression of a symptom, reducing the frequency or duration of a symptom or condition, or, in some circumstances, preventing the onset of a symptom. Treatment or amelioration of a symptom can be based on any objective or subjective parameter, including the results of a physical examination.

[0058] 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, and circulating cancers, such as leukemias.

[0059] 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), including minimal residual disease, and including both primary and metastatic tumors; and liquid cancers (e.g., blood cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain tumors.

[0060] The "pathology" of cancer includes any phenomenon that compromises the well-being of a patient, including, but not limited to, abnormal or uncontrolled cell proliferation, metastasis, inhibition of normal function of neighboring cells, release of abnormal levels of cytokines or other secretions, suppressed or exacerbated inflammatory or immune responses, neoplasms, pre-malignant or malignant tumors, and invasion of surrounding or distant tissues or organs such as lymph nodes.

[0061] 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.

[0062] As used herein, the term "metastasis" refers to the growth of a cancer tumor in an organ or body part that is not directly connected to the organ in which the cancer tumor resides. Metastasis will be understood to include micrometastasis, which is the presence of undetectable amounts of cancer cells in an organ or body part that is not directly connected to the organ in which the cancer tumor resides. Metastasis can also be defined as a several-step process, including the departure of cancer cells from the original tumor site, the migration and / or infiltration of cancer cells to other parts of the body.

[0063] The phrases "effective amount" and "therapeutically effective amount" refer to the dosage or amount of a substance, such as an immunoconjugate, that produces the therapeutic effect for which it is administered. The actual amount administered will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22nd Edition, (Pharmaceutical Press, London, 2012)). In the case of cancer, a therapeutically effective amount of the immunoconjugate may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with cancer. To the extent that the immunoconjugate may inhibit the growth of and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. With respect to cancer therapy, efficacy may be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0064] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably and refer to any mammalian subject (e.g., human) for whom diagnosis, treatment, or therapy is desired. For purposes of therapy, "mammal" refers to any animal classified as a mammal, including humans, domestic and farm animals, zoo, sport, and pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, and the like. In certain embodiments, the mammal is a human.

[0065] 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.

[0066] As used herein, the term "administering" means parenterally, intravenously, intraperitoneally, intramuscularly, intratumorally, intralesionally, intranasally or subcutaneously, orally, as a suppository, topically, intrathecally, or by implantation of a sustained release device, e.g., a mini-osmotic pump, into the subject.

[0067] The terms "about" and "approximately" used herein to modify a numerical value indicate a close range surrounding that numerical value. Thus, when "X" is a value, "about X" or "approximately X" indicates a value between 0.9X and 1.1X, such as between 0.95X and 1.05X, or between 0.99X and 1.01X. Reference to "about X" or "approximately X" specifically indicates at least the values ​​X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" and "approximately X" are intended to teach and provide support for the present specification for a claim limitation of, for example, "0.98X."

[0068] antibody The immune complexes of the present invention include antibodies. Included within the scope of the embodiments of the present invention are functional variants of the antibody constructs or antigen-binding domains described herein. The term "functional variant" as used herein refers to an antibody construct having an antigen-binding domain with substantial or significant sequence identity or similarity with a parent antibody construct or antigen-binding domain, and this functional variant retains the biological activity of the antibody construct or antigen-binding domain it is a variant of. Functional variants include, for example, variants of the antibody constructs or antigen-binding domains described herein (parent antibody constructs or antigen-binding domains) that retain a similar, equal or greater ability to recognize target cells expressing Trop2 than the parent antibody construct or antigen-binding domain.

[0069] With respect to an antibody construct or antigen-binding domain, a functional variant can be, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the antibody construct or antigen-binding domain.

[0070] A functional variant may, for example, comprise an amino acid sequence of a parent antibody construct or antigen-binding domain with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise an amino acid sequence of a parent antibody construct or antigen-binding domain with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the parent antibody construct or antigen-binding domain.

[0071] The antibodies comprising the immunoconjugates of the present invention comprise Fc engineered variants. In some embodiments, the mutations in the Fc region that result in modulated binding to one or more Fc receptors include the following mutations: SD(S239D), SDIE(S239D / I332E), SE(S267E), SELF(S267E / L328F), SDIE(S239D / I332E), SDIEAL(S239D / I332E / A330L), GA(G236A), ALIE(A330L / I332E ... ), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R) and V11 (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328 and A330. Further Fc region modifications to modulate Fc receptor binding are described, for example, in US2016 / 0145350, US7416726 and US5624821, which are incorporated by reference in their entireties.

[0072] The antibodies comprising the immunoconjugates of the invention include glycan variants, such as defucosylation. In some embodiments, the Fc region of the binding agent is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0073] The amino acid substitutions in the antibody construct or antigen-binding domain of the invention are preferably conservative amino acid substitutions, which are known in the art and include amino acid substitutions that replace one amino acid having particular physical and / or chemical properties with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution can be an acidic / negatively charged polar amino acid (e.g., Asp or Glu) substituted for another acidic / negatively charged polar amino acid, a positively charged amino acid substituted for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side chain substituted for another amino acid with a beta-branched side chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0074] The antibody construct or antigen-binding domain may consist essentially of the specific amino acid sequence(s) described herein, such that other components, e.g., other amino acids, do not substantially alter the biological activity of the antibody construct or antigen-binding domain functional variant.

[0075] In some embodiments, the antibody in the immune complex comprises a modified Fc region, where the modification modulates binding of the Fc region to one or more Fc receptors.

[0076] In some embodiments, an antibody in an immune complex (e.g., an antibody conjugated to at least two adjuvant moieties) comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in an Fc region that result in modulated binding (e.g., increased or decreased binding) to one or more Fc receptors (e.g., FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) and / or FcγRIIIB (CD16b)) compared to a native antibody lacking the Fc region mutations. In some embodiments, an antibody in an immune complex comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in an Fc region that reduce binding of the Fc region of the antibody to FcγRIIB. In some embodiments, the antibody in the immune complex comprises one or more modifications (e.g., amino acid insertions, deletions, and / or substitutions) in the Fc region of the antibody that decrease binding of the antibody to FcγRIIB while maintaining the same or increased binding to FcγRI (CD64), FcγRIIA (CD32A) and / or FcγRIIIA (CD16a) compared to a native antibody lacking the Fc region mutation. In some embodiments, the antibody in the immune complex comprises one or more modifications in the Fc region of the antibody that increase binding of the Fc region of the antibody to FcγRIIB.

[0077] In some embodiments, the modulated binding is provided by mutations in the Fc region of the antibody compared to the native Fc region of the antibody. The mutations can be in the CH2 domain, the CH3 domain, or a combination thereof. A "native Fc region" is synonymous with a "wild-type Fc region" and comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature or identical to the amino acid sequence of an Fc region found in a native antibody (e.g., cetuximab). Native sequence human Fc regions include native sequence human IgG1 Fc regions, native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fc (Jefferis et al., (2009) mAbs, 1(4):332-338).

[0078] In some embodiments, the Fc region of the antibody of the immunoconjugate is modified to have an altered glycosylation pattern of the Fc region compared to the native, unmodified Fc region.

[0079] Human immunoglobulins are glycosylated at the Asn297 residue in the Cγ2 domain of each heavy chain. This N-linked oligosaccharide consists of the core heptasaccharide N-acetylglucosamine4mannose3 (GlcNAc4Man3). Removal of the heptasaccharide by endoglycosidases or PNGaseF is known to cause conformational changes in the antibody Fc region, which can significantly reduce antibody binding affinity for activating FcγRs and reduce effector functions. The core heptasaccharide is often modified with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially affect Fc binding to activating and inhibitory FcγRs. Furthermore, it has been demonstrated that α2,6-sialylation enhances anti-inflammatory activity in vivo, while defucosylation improves FcγRIIIa binding and leads to a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Thus, specific glycosylation patterns can be used to control inflammatory effector functions.

[0080] In some embodiments, the modification to alter the glycosylation pattern is a mutation, e.g., a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods of regulating immune responses by antibodies that modulate FcγR regulatory signaling are described, for example, in US7416726, US2007 / 0014795, and US2008 / 0286819, which are incorporated herein by reference in their entireties.

[0081] In some embodiments, the antibody of the immunoconjugate is modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern. For example, hybridomas can be engineered to secrete defucosylated mAbs, desialylated mAbs or deglycosylated Fc with specific mutations that allow for increased FcRγIIIa binding and effector function. In some embodiments, the antibody of the immunoconjugate is engineered to be defucosylated.

[0082] In some embodiments, the entire Fc region of the antibody in the immune complex is replaced with a different Fc region, so that the Fab region of the antibody is bound to a non-natural Fc region. For example, the Fab region of cetuximab, which normally contains an IgG1 Fc region, can bind to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which normally contains an IgG4 Fc region, can bind to IgG1, IgG2, IgG3, IgA1, or IgG2. In some embodiments, the Fc-modified antibody with a non-natural Fc domain also contains one or more amino acid modifications, such as the S228P mutation in IgG4 Fc, that modulate the stability of the Fc domain as described. In some embodiments, the Fc-modified antibody with a non-natural Fc domain also contains one or more amino acid modifications, as described herein, that modulate Fc binding to FcR.

[0083] In some embodiments, modifications that modulate binding of the Fc region to an FcR do not alter binding of the Fab region of the antibody to its antigen when compared to the native, unmodified antibody, hi other embodiments, modifications that modulate binding of the Fc region to an FcR also increase binding of the Fab region of the antibody to its antigen when compared to the native, unmodified antibody.

[0084] 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.

[0085] 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.

[0086] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0087] In some embodiments, the antibody in the immunoconjugate is a cysteine ​​engineered antibody that provides for site-specific attachment of adjuvant, label or drug moieties to the antibody via replacement of cysteines at sites where the engineered cysteines are available for attachment but do not interfere with immunoglobulin folding and assembly or alter antigen binding and effector function (Junutula, et al., (2008), Nature Biotech., 26(8):925-932; Dornan et al. (2009), Blood, 114(13):2721-2729; US7521541; US7723485; US2012 / 0121615; WO2009 / 052249). A "cysteine ​​engineered antibody" or "cysteine ​​engineered antibody variant" is an antibody in which one or more residues of an antibody are replaced with a cysteine ​​residue. Cysteine ​​engineered antibodies can be attached to thienoazepine adjuvant moieties as thienoazepine linker compounds with uniform stoichiometry (e.g., up to two thienoazepine moieties per antibody in an antibody with a single engineered cysteine ​​site).

[0088] In some embodiments, cysteine ​​engineered antibodies are used to prepare immunoconjugates. The immunoconjugates may have a reactive cysteine ​​thiol residue introduced at a site on the light chain, e.g., at the 149-lysine site (LC K149C), or at a site on the heavy chain, e.g., at the 122-serine site (HC S122C), as numbered by Kabat numbering. In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced at the 118-alanine site (EU numbering) of the heavy chain (HC A118C). This site is numbered 121 in the SEQ ID NO: system and 114 in the Kabat numbering. In other embodiments, the cysteine ​​engineered antibody has a cysteine ​​residue introduced into (i) the light chain at G64C, R142C, K188C, L201C, T129C, S114C or E105C according to the Kabat numbering, (ii) the heavy chain at D101C, V184C, T205C or S122C according to the Kabat numbering, or (iii) other cysteine ​​mutated antibodies, and as described in Bhakta, S. et al, (2013), "Engineering THIOMABs for Site-Specific Conjugation of Thiol-Reactive Linkers", Laurent Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular Biology, vol. 1045, pages 189-203; WO 2011 / 156328; US9000130.

[0089] Immune checkpoint inhibitors In some embodiments, the antibody of the immune complex is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins. In another embodiment, the immune checkpoint inhibitor reduces the interaction between one or more immune checkpoint proteins and their ligands. Inhibitory nucleic acids that reduce the expression and / or activity of immune checkpoint molecules can also be used in the methods disclosed herein.

[0090] The immune checkpoint inhibitors nivolumab and atezolizumab may be modified to contain an IgG1 Fc and subsequently converted into the immunoconjugates of the invention.

[0091] Most checkpoint antibodies are designed to block signal transduction rather than have effector functions that kill cells. The immunoconjugates of the present invention can restore the "effector functionality" required to activate myeloid immunity.

[0092] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152), T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-inducible TNFR-related protein (GITR, also known as TNFRSF18), inducible T cell costimulatory (ICOS, also known as CD278), CD96, poliovirus receptor-related 2 (PVRL2, also known as CD112R), programmed cell death protein 1 (PD-1, also known as CD279), programmed cell death 1 ligand 1 (PD-L1, also known as B7-H3 and CD274), programmed cell death ligand 2 (PD-L2, also known as B7-DC and CD273). Also known as CD40, CD134, lymphocyte activation gene 3 (LAG-3, also known as CD223), B7-H4, killer immunoglobulin receptor (KIR), tumor necrosis factor receptor superfamily member 4 (TNFRST4, also known as OX40 and CD134) and its ligand OX40L (CD252), indoleamine 2,3-dioxygenase 1 (IDO-1), indoleamine 2,3-dioxygenase 2 (IDO-2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), B- and T-lymphocyte attenuator (BTLA, also known as CD272), T-cell membrane protein 3 (TIM3), adenosine A2A receptor (A2Ar), and V-domain Ig suppressor of T-cell activation (VISTA protein). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-L1.

[0093] In some embodiments, the antibody is selected from ipilimumab (also known as YERVOY®), pembrolizumab (also known as KEYTRUDA®), nivolumab (also known as OPDIVO®), atezolizumab (also known as TECENTRIQ®), avelumab (also known as BAVENCIO®), and durvalumab (also known as IMFINZI®).

[0094] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4. In some embodiments, the immune checkpoint inhibitor is an antibody to CTLA4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to CTLA4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to CTLA4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as CTLA4.

[0095] In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody to PD-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to PD-1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to PD-1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-1.

[0096] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody to PD-L1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to PD-L1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to PD-L1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L1. In some embodiments, the immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L1.

[0097] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody to PD-L2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to PD-L2. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to PD-L2. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L2. In some embodiments, the immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L2.

[0098] In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG-3. In some embodiments, the immune checkpoint inhibitor is an antibody to LAG-3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to LAG-3. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to LAG-3. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as LAG-3.

[0099] In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7-H4. In some embodiments, the immune checkpoint inhibitor is an antibody to B7-H4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to B7-H4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to B7-H4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as B7-H4.

[0100] In one embodiment, the immune checkpoint inhibitor is an inhibitor of KIR. In some embodiments, the immune checkpoint inhibitor is an antibody against KIR. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against KIR. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against KIR. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as KIR.

[0101] In some embodiments, the immune checkpoint inhibitor is an inhibitor of TNFRSF4. In some embodiments, the immune checkpoint inhibitor is an antibody against TNFRSF4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against TNFRSF4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against TNFRSF4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as TNFRSF4.

[0102] In some embodiments, the immune checkpoint inhibitor is an inhibitor of OX40L. In some embodiments, the immune checkpoint inhibitor is an antibody to OX40L. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to OX40L. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to OX40L. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as OX40L. In some embodiments, the immune checkpoint inhibitor reduces the interaction between TNFRSF4 and OX40L. In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO-1. In some embodiments, the immune checkpoint inhibitor is an antibody to IDO-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to IDO-1, and in some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to IDO-1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as IDO-1.

[0103] In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO-2. In some embodiments, the immune checkpoint inhibitor is an antibody to IDO-2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to IDO-2. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to IDO-2. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as IDO-2.

[0104] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CEACAM1. In some embodiments, the immune checkpoint inhibitor is an antibody to CEACAM1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to CEACAM1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to CEACAM1. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as CEACAM1.

[0105] In some embodiments, the immune checkpoint inhibitor is an inhibitor of BTLA. In some embodiments, the immune checkpoint inhibitor is an antibody against BTLA. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against BTLA. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against BMA. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as BTLA.

[0106] In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the immune checkpoint inhibitor is an antibody to TIM3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody to TIM3. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody to TIM3. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as TIM3.

[0107] In some embodiments, the immune checkpoint inhibitor is an inhibitor of A2Ar. In some embodiments, the immune checkpoint inhibitor is an antibody against A2Ar. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against A2Ar. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against A2Ar. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as A2Ar.

[0108] In some embodiments, the immune checkpoint inhibitor is an inhibitor of VISTA protein. In some embodiments, the immune checkpoint inhibitor is an antibody against VISTA protein. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against VISTA protein. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against VISTA protein. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as VISTA protein.

[0109] antibody target In some embodiments, the antibody of the immune complex is 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, aromatase, ATX, AX1, AZGP1 (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAG1, BAI1, BCR, BC L2, BCL6, BDNF, BLNK, BLR1 (MDR15), BIyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRTA, BMPR1B, BMPR2, BPAG1 (plectin), BRCA1, C19orflO (IL27) w), C3, C4A, C5, C5R1, CANT1, CAPRIN-1, CASP1, CASP4, CAV1, CCBP2(D6 / JAB61), CCLI(1-309), CCLI1(eotaxin), CCL13(MCP-4), CCL15(MIP-Id), CCL16( HCC-4), CCL17(TARC), CCL18(PARC), CCL19(MIP-3b), CCL2(MCP-1), MCAF, CCL20(MIP-3a), CCL21(MEP-2), SLC, Exodus-2, CCL22(MDC / STC-1), CCL23(M PIF-I), CCL24(MPIF-2 / eotaxin-2), CCL25(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CCL3(MIP-Ia), CCL4(MIPIb), CCL5(RANTES), CCL7(M CP-3), CCL8(mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-IRB / RA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13) , CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBI1), CCR8(CMKBR8 / TERI / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), CD164, CD19,CDIC, CD2, CD20, CD21, CD200, CD-22, CD24, CD27, CD28, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD38, CD40, CD40L, CD44, CD45RB, CD47, CD 52, CD69, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD137, CD152, CD274, CDH1 (Ecadherin), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH5 , CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A(p21Wap1 / Cip1), CDKN1B(p27Kip1), CDKN1C, CDKN2A(p16INK4a), CDKN2B, CDKN2C , CDKN3, CEBPB, CERI, CHGA, CHGB, titinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (clausin-7), CLDN18. 2 (claudin 18.2), CLN3, CLU (clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTL8, CTNNB1 (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO), CXCLI1 (1-TAC / IP-9), CXCL12 (SDF1 ), CXCL13, CXCL14, CXCL16, CXCL2(GRO2), CXCL3(GRO3), CXCL5(ENA-78 / LIX), CXCL6(GCP-2), CXCL9(MIG), CXCR3(GPR9 / CKR-L2), CXCR4, CXCR6(TYMSTR / STRL33 / Bonzo), CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, Enola,ENO2, ENO3, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPRA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN- A2, EPHRINA3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERBB2(Her-2), EREG, ERK8, estrogen receptor, Earl, ESR2, F3 (TF), FADD, Famesyltransferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR3, FIGF (VEGFD), FIL I (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GDF5, GFI1, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (gelsolin), GSTP1, H AVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, hedgehog, HGF, HIF1A, HIP1, histamine and histamine receptor, HLA-A, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFNγ, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, ILIO,ILIORA, ILIORB, IL-1, IL1R1 (CD121a), IL1R2 (CD121b), IL-IRA, IL-2, IL2RA (CD25), IL2RB (CD122), IL2RG (CD132), IL-4, IL-4R (CD123), IL-5, IL5RA (CD125), IL3RB (CD131), IL-6, IL6RA, (CD126), IR6RB (CD130), IL-7, IL7RA (CD127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R (CD129), IL-10, IL10RA (CD210), IL10RB (CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, ILIR2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, IL4, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), LAMA5, LEP (leptin), Lingo-p75, Lingo-Troy,LPS, LRRC15, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​MACMARCKS, MAG or OMgp, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionectin-UI), mTOR, MTSS1, MUC1 (mucin), MYC, MYD88, NCK2, neurocan, nectin-4, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOX5, NPPB, NROB1, NRO B2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3 , NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZI, OPRDI, P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, PEG-asparaginase, PF4(CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 kinase, PIK3CG, PLAU(uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP(CXCL7), P PID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PIN, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, RXR, S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPENA1, SERPIN3, SERPINB5(マスピI), SERPINEI(PAI-I), SERPINFI SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SLI T2, SPP1, SPRR1B(Spr1), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TB4R2, TB X21, TCP1O, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGEBR 1, TGFBR2, TGFBR3, THIL, THBS1(proliferase-1), THBS2, THBS4, THPO, TIE(T ie-1) TIMP3, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9; TLR10, TLR11, TNF, TNF-a, TNFAIP2(B94), TNFAIP3, TNFRSF11A, TNFRSF1A. TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8, TNFRSF9 TNFSF1O(TRAIL), TNFSF11(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFSF13B, TNSF14(WHO-L), TNFRSF14(WHO), TNFSF15(VEGI), TNFSF18, T NFSF4 (OX40 factor), TNFSF5 (CD40 factor), TNFSF6 (FasL), TNFSF7 (CD27 factor) TNFSF8(CD30 fragment), TNFSF9(4-1BB fragment), TOLLIP, TNFSF8(CD30 fragment), TOP2A (fragment). 1ia) TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TR AF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, uPAR, VEGF EGFB, VEGFC, glycoprotein, VHLC5, VLA-4, Wnt-1, XCL1(SCM-Ib), XCL2(SCM-Ib). XCRI(GPR5 / CCXCR1), YYI, ZFPM2, CLEC4C(BDCA-2, DLEC, CD303, CLECSF7);CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin-1), PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (C D85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD8 5b, ILT8), NCR1(CD335, LY94, NKp46), NCR3(CD335, LY94, NKp46), NCR3(CD337, NKp30), O It can bind to (e.g., specifically bind to) one or more targets selected from SCAR, TARM1, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354), TREM2, and KLRF1 (NKp80).

[0110] In some embodiments, the antibody binds to an FcR.gamma coupled receptor. In some embodiments, the FcR.gamma coupled receptor is selected from the group consisting of GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, and TARM1.

[0111] In some embodiments, the antibody binds to a DAP12 coupled receptor, hi some embodiments, the DAP12 coupled receptor is selected from the group consisting of CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354), and TREM2.

[0112] In some embodiments, the antibody binds to a heme ITAM-containing receptor. In some embodiments, the heme ITAM-containing receptor is KLRF1 (NKp80).

[0113] In some embodiments, the antibody can bind to one or more targets selected from CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), and CLEC7A (Dectin-1). In some embodiments, the antibody can bind to CLEC6A (Dectin-2) or CLEC5A. In some embodiments, the antibody can bind to CLEC6A (Dectin-2).

[0114] In some embodiments, the antibody is selected from the group consisting of ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2 (P47738), ATPB (P56480), AATM (P052 02), TMM93(Q9CQW0), ERGI3(Q9CQE7), RTN4(Q99P72), CL041(Q8BQR4), ERLN2(Q8BFZ9), TERA(Q01853), DAD1(P61804), CALX(P35564), CALU(O35887), VAPA(Q9WV55), MOGS(Q80UM7), GANAB(Q8BHN3), ERO1A(Q8R180), UGGG1(Q6P5E4), P4HA1(Q60715), HYEP(Q9D379), CALR (P14211), AT2A2(O55143), PDIA4(P08003), PDIA1(P09103), PDIA3(P27773), PDIA6(Q922R8), CLH(Q68FD5), PPIB(P24369), TCPG(P80 318), MOT4(P57787), NICA(P57716), BASI(P18572), VAPA(Q9WV55), ENV2(P11370), VAT1(Q62465), 4F2(P10852), ENOA(P17182), ILK( O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH, (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), and CH10 (Q64433). In the preceding list, accession numbers are given in parentheses.

[0115] In some embodiments, the antibody binds to an antigen selected from CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM, MUC1, MUC16, EGFR, Her2, SLAMF7, and gp75. In some embodiments, the antigen is selected from CD19, CD20, CD47, EpCAM, MUC1, MUC16, EGFR, and Her2. In some embodiments, the antibody binds to an antigen selected from the Tn antigen and the Thomsen-Friedenreich antigen.

[0116] In some embodiments, the antibody or Fc fusion protein is selected from the group consisting of abagovomab, abatacept (also known as ORENCIA®), abciximab (also known as REOPRO®), c7E3Fab), adalimumab (also known as HUMIRA®), adecatumumab, alentuzumab (also known as CAMPATH®), MabCampath or Campath-1H), altumomab, afelimomab, anatumomab, mafenatox, anetumumab, anrukizumab, , apolizumab, actilumomab, acelizumab, atlizumab, atolimumab, bapinezumab, basiliximab (also known as SIMULECT®), bavituximab, bectumomab (also known as LYMPHSCAN®), belimumab (also known as LYMPHO-STAT-B®), bertilimumab, besilisomab, bevacizumab (also known as AVASTIN®), biciromab, bralobarbital, bivatuzumab mertansine, Campath, canakinumab (ACZ88 5), cantuzumab mertansine, capromab (also known as PROSTASCINT®), catumaxomab (also known as REMOVAB®), cedelizumab (also known as CIMZIA®), certolizumab pegol, cetuximab (also known as ERBITUX®), clenoliximab, decatuzumab, dacliximab, daclizumab (also known as ZENAPAX®), denosumab (also known as AMG162), dexamethasone, dacliximab (also known as DA ... Tumomab, drimomab alitox, drixizumab, dantumab, durimulumab, durumumab, ecloneximab, eculizumab (also known as SOLIRIS®), edovacomab, edrecolomab (Mab17-1A, also known as PANOREX®), efalizumab (also known as RAPTIVA®), efungumab (also known as MYCOGRAB®), ersilimomab, enlimomab pegol, epitumomab situxetan, efalizumab, epitumomab,Epratuzumab, erlizumab, ertumaxomab (also known as REXOMUN®), etanercept (also known as ENBREL®), etaracizumab (also known as etaracizumab, VITAXIN®, ABEGRIN®), exbivirumab, fanolesomab (also known as NEUTROSPEC®), faralimomab, felizumab, fontolizumab (also known as HUZAF®), galactamib, valproate ... cimatum, gantenerumab, gavilimomab (also known as ABXCBL®), gemtuzumab ozogamicin (also known as MYLOTARG®), golimumab (also known as CNTO148), golimiximab, ibalizumab (also known as TNX-355), ibritumomab tiuxetan (also known as ZEVALIN®), igovomab, imciromab, infliximab (also known as REMICADE®), inotropic steroids (also known as EGFR), gliomab ... Rimomab, inotuzumab ozogamicin, ipilimumab (also known as MDX-010, MDX-101), iratumumab, keliximab, labetuzumab, remaresomab, lebrilizumab, lerdelimumab, lexatumumab (also known as HGS-ETR2, ETR2-ST01), lexitumumab, ribivirumab, lintuzumab, lucatumumab, rumiliximab, mapatumumab (also known as HGS-ETR1, TRM-1), masumolimomab, matuzumab (also known as EMD72000) (also known as BOSATRIA®), mepolizumab (also known as BOSATRIA®), metelimuamab, miratumumab, minretumomab, mitumomab, morolimumab, motavizumab (also known as NUMAX®), muromonab (also known as OKT3), nacolomab butafenatox, naptumomab estafenatox, natalizumab (also known as TYSABRI®, ANTEGREN®), nebacumab, nerelimomab, nimotumab (also known as THERACIM hR3®, THERA-CIM-hR3®, THERALOC®),Nofetumomab merpentane (also known as VERLUMA®), ocrelizumab, odulimomab, ofatumumab, omalizumab (also known as XOLAIR®), oregovomab (also known as OVAREX®), otelixizumab, pagibaximab, palivizumab (also known as SYNAGIS®), panitumumab (also known as ABX-EGF, VECTIBIX®), pascolizumab, pemtumomab (also known as THERAGYN®), (also known as MEDI-507), sontuzumab, stamlumab (also known as stamtuzumab, ... MYO-029), suresomab (also known as LEUKOSCAN®), tacatuzumab, tetraxetan, tadocizumab, talizumab, taplitumomab, paptox, tefibazumab (also known as AUREXIS®), terimomab-alitox, teneliximab, teplizumab, ticilimumab, tocilizumab (also known as ACTEMRA®), toralizumab, tositumomab, trastuzumab (also known as HERCEPTIN®), tremelimumab (also known as CP-675,206), tucotuzumab celmoleukin, tuvilumab, urtoxazumab, ustekinumab (also known as CNTO1275), bapaliximab, veltuzumab, bapalimomab, visilizumab (also known as NUVION®), volociximab (also known as M200), votumumab (also known as HUMASPECT®), zalutumumab, zanolimumab (also known as HuMAX-CD4), zialimumab, zolimomab alitox, daratumumab,Selected from elotuxumab, ovintuzumab, olaratumab, brentuximab vedotin, afibercept, abatacept, belatacept, afibercept, etanercept, romiplostim, SBT-040 (sequences listed in US2017 / 0158772). In some embodiments, the antibody is rituximab.

[0117] 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.

[0118] 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.

[0119] Several antibodies targeting PD-L1, such as atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™) and avelumab (BAVENCIO™), have been developed for the treatment of cancer. Nevertheless, there remains a need for new PD-L1-binding agents, including agents that bind with high affinity to PD-L1 and effectively inhibit PD-L1 / PD-1 signaling, and agents that can deliver therapeutic payloads to PD-L1-expressing cells. Additionally, there is a need for novel PD-L1-binding agents for the treatment of autoimmune and infectious diseases.

[0120] 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.

[0121] In a particular embodiment, the immunoconjugate of the present invention comprises an anti-HER2 antibody. In one embodiment of the present invention, the anti-HER2 antibody of the immunoconjugate of the present invention comprises a humanized anti-HER2 antibody, such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8, as described in Table 3 of 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 called trastuzumab and is commercially available under the trade name HERCEPTIN™ (Genentech, Inc.).

[0122] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived IgG1κ, monoclonal antibody that is a humanized version of the murine anti-HER2 antibody (4D5) that selectively binds with high affinity (Kd=5 nM) to the extracellular domain of HER2 in cell-based assays (U.S. Pat. Nos. 5,677,171, 5,821,337, 6,054,297, 6,165,464, 6,339,142, 6,407,213, 6,639,055, 6,719,971, 6,800,738, 7,074,404; Coussens et al (1985) Science, 230:1132-9; Slamon et al (1999)). et al (1989) Science, 244:707-12, Slamon et al (2001) New Engl. J. Med. 344:783-792).

[0123] 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.

[0124] In another embodiment of the invention, the anti-HER2 antibody of the immunoconjugate of the invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US Patent No. 7,862,817. An exemplary humanized 2C4 antibody is pertuzumab (CAS Registry Number 380610-27-5), PERJETA™ (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI), which functions by inhibiting the ability of 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™ has been approved for the treatment of breast cancer.

[0125] 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.

[0126] 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.

[0127] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct that comprises an antigen-binding domain that specifically recognizes and binds CEA.

[0128] 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 analogue (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).

[0129] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds Trop2. Tumor-associated calcium signal transduction 2 (TROP2) is a transmembrane glycoprotein encoded by the TACSTD2 gene (Linnenbach AJ, et al. (1993) Mol Cell Biol. 13(3):1507-15; Calabrese G, et al (2001) Cytogenet Cell Genet. 92(1-2):164-5). Trop2 is an intracellular calcium signal transduction 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 important for prognosis. Several ligands that interact with Trop2 have been proposed. Trop2 signals cells through different pathways, which are transcriptionally regulated by a complex network of several transcription factors.

[0130] Human Trop2 (TACSTD2: tumor-associated calcium signaling substance 2, GA733-1, EGP-1, M1S1; hereafter referred to as hTrop2) is a single transmembrane type 1 cell membrane protein consisting of 323 amino acid residues. The existence of a cell membrane protein common to human trophoblast cells and cancer cells that is involved in immune resistance has been suggested for some time (Faulk WP, et al., Proc. Natl. Acad. Sci. 75(4): 1947-1951 (1978)). Meanwhile, an antigen molecule recognized by a monoclonal antibody against a cell membrane protein in a human choriocarcinoma cell line was identified and named Trop2 as one of the molecules expressed in human trophoblast cells (Lipinski M, et al., Proc. Natl. Acad. Sci. 78(8), 5147-5150 (1981)). This molecule was also designated as tumor antigen GA733-1, recognized by mouse monoclonal antibody GA733 (Linnenbach AJ, et al., Proc. Natl. Acad. Sci. 86(1), 27-31 (1989)), 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 it was confirmed that all these molecules are the same molecule (Fornaro M, et al., Int. J. Cancer, 62(5), 610-618 (1995)). The DNA and amino acid sequences of hTrop2 are available in public databases and can be referenced, for example, under accession numbers NM_002353 and NP_002344 (NCBI).

[0131] In response to such information suggesting a link with cancer, several anti-hTrop2 antibodies have been established and their antitumor effects have been investigated. 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), as well as antibodies that show antitumor activity as ADCs 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 still an unmet medical need for hTrop2 as a therapeutic target.

[0132] TROP2 expression in cancer cells correlates with drug resistance. Several strategies have targeted TROP2 on cancer cells, including antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, etc. In vitro and preclinical studies using these various therapeutic treatments have significantly suppressed tumor cell growth both in vitro and in vivo in mice. Clinical studies have explored the potential application of Trop2 both as a prognostic biomarker and as a therapeutic target for reverse resistance.

[0133] 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, the active metabolite of irinotecan (US2016 / 0297890; WO2015 / 098099).

[0134] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds Caprin-1 (Ellis JA, Luzio JP. (1995), J. Biol. Chem. 270(35):20717-23; Wang B, et al. (2005), J. Immunol. 175(7):4274-82; Solomon S, et al. (2007), Mol. Cell Biol. 27(6):2324-42). Caprin-1 is also known as GPIAP1, GPIP137, GRIP137, M11S1, RNG105, p137GPI, and cell cycle-related protein 1.

[0135] Cytoplasmic activation / proliferation-associated protein-1 (Caprin-1) is an RNA-binding protein involved in the regulation of cell cycle control-related genes. Caprin-1 selectively binds to the mRNAs of c-Myc and cyclin D2 and mediates the G 1 It accelerates the progression of cells through the IL-1 and IL-2 phases to the S phase, enhances cell viability, and promotes cell proliferation, indicating that it may play an important role in tumorigenesis (Wang B,et al.(2005),J.Immunol.175:4274-4282). Caprin-1 acts alone or in combination with other RNA-binding proteins such as RasGAP SH3 domain binding protein 1 and fragile X mental retardation protein. In the tumorigenesis process, Caprin-1 functions primarily by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, Caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, which contributes to radiation and chemotherapy resistance. Given its role in various clinical malignancies, Caprin-1 may be used as a biomarker and as a target for the development of novel therapeutic agents. (Yang,ZS,et al,(2019),Oncology Letters,18:15-21).

[0136] Antibodies targeting caprin-1 for treatment and detection have been described (WO2011 / 096519; WO2013 / 125654; ​​WO2013 / 125636; WO2013 / 125640; WO2013 / 125630; WO2013 / 018889; WO2013 / 018891; WO2013 / 018883; WO2013 / 018892; WO2014 / 014082; WO2014 / 014086; WO2015 / 020212; WO2018 / 079740).

[0137] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to claudin-1.

[0138] Claudin 1 is a member of the claudin family of transmembrane proteins located in the tight junctions between cells and acts as a coreceptor for HCV entry into hepatocytes (Kniesel U,et al.(2000).Cell.Mol.Neurobiol.20(1):57-76; Furuse M,et al.(1998).J.Cell Biol.141(7):1539-50; Swisshelm K,et al.(2005),Adv.Drug Deliv.Rev.57(6):919-28). Claudin 1 is also known as senescence-associated epithelial membrane protein, senescence-associated epithelial membrane protein 1, CLDN1, CLD1, ILVASC, SEMP1.

[0139] Claudins are abundant in luminal epithelial sheets where they maintain epithelial cell polarity. Claudin 1 is expressed in most tissues, such as the bladder, fallopian tube, liver, pancreas, prostate, and skin.

[0140] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds to Nectin-4.

[0141] Nectins are a protein family of cell adhesion molecules involved in calcium-dependent cell adhesion (Takai Y. et al. (2003), Cancer Science, 94(8):655-67; ​​Fuchs, A. et al. (2006), Seminars in Cancer Biology, 16(5):359-366; Miyoshi J. et al. (2007), American journal of nephrology, 27(6):590-604). Nectins play an important role in cell-cell junctions in many different tissues, including intermediate junctions in epithelial cells or chemical synapses in nerve cells.

[0142] In an exemplary embodiment, the immunoconjugate of the invention comprises an antibody construct comprising an antigen-binding domain that specifically recognizes and binds LRRC15 (leucine-rich repeat containing 15).

[0143] LRRC15 is a cell membrane-expressed protein encoded by the LRRC15 gene in humans. LRRC15 is expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreas), as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 may be useful as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin (Purcell, JW. et al. (2018), Cancer Res., 78(14):4059-4072).

[0144] Bis-benzimidazole adjuvant compounds The immunoconjugates of the present invention comprise a bis-benzimidazole adjuvant moiety. The adjuvant moieties described herein are compounds that induce an immune response (i.e., immunostimulants). Generally, the adjuvant moieties described herein are STING agonists.

[0145] Certain amido-benzimidazole compounds have demonstrated systemically active STING receptor agonists (Ramanjulu, JMet al. (2018), Nature, 564:439-443; Barber, GN (2015), Nature Rev. Immunol. 15:760-770; US2019 / 0300513).

[0146] STING is a dimeric structure with a large, symmetric binding pocket. The bis-benzimidazole compounds in Table 1 conjugated to targeting antibodies were designed to target and bind to the open conformation of the binding pocket of STING. Binding to small molecule agonists usually induces a closed conformation of the STING protein. This introduces the risk that the linker, especially if it is "non-cleavable", will prevent binding and activation. Bis-benzimidazoles have been reported to bind and activate via the open conformation, which we expected to be more suitable for linker attachment (Ramanjulu, JMet al. (2018), Nature, 564: 439-443; Barber, GN (2015), Nature Rev. Immunol, 15: 760-770).

[0147] Bis-benzimidazole linker compounds The immunoconjugates of the present invention are prepared by conjugation of an antibody with a bis-benzimidazole-linker compound. The bis-benzimidazole-linker compound comprises a bis-benzimidazole (BBI) moiety covalently attached to a linker unit (L). The linker unit comprises functional groups and subunits that affect the stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugate. The linker unit comprises a reactive functional group that reacts with, i.e., binds to, a reactive functional group of an antibody. For example, a nucleophilic group such as a lysine side chain amino of an antibody reacts with an electrophilic reactive functional group of a BBI-linker compound to form an immunoconjugate. Also, for example, a cysteine ​​thiol of an antibody reacts with a maleimide group or a bromoacetamide group of a BBI linker compound to form an immunoconjugate.

[0148] Considerations for the design of the immunoconjugates of the present invention include (1) preventing premature release of the bis-benzimidazole (BBI) moiety during in vivo circulation and (2) ensuring that the biologically active form of the BBI moiety is released at the desired site of action at an appropriate rate. The complex structure of the immunoconjugates together with their functional properties require careful design and selection of every component of the molecule, including the antibody, the binding site, the linker structure, and the bis-benzimidazole compound. The linker determines the mechanism and rate of adjuvant release.

[0149] In general, the linker unit (L) may be cleavable or non-cleavable. A cleavable linker unit may comprise a peptide sequence that is a substrate for a specific protease, such as a cathepsin, that recognizes and cleaves the peptide linker unit, separating the STING agonist from the antibody (Caculitan NG, et al. (2017), Cancer Res. 77(24):7027-7037).

[0150] Cleavable linker units may contain labile functionalities such as acid-sensitive disulfide groups (Kellogg, BA. et al. (2011), Bioconjugate Chem. 22, 717-727; Ricart, AD et al. (2011), Clin. Cancer Res. 17, 6417-6427; Pillow, T., et al. (2017), Chem. Sci. 8, 366-370; Zhang D, et al. (2016), ACS Med Chem Lett. 7(11):988-993).

[0151] In some embodiments, the linker is not cleavable under physiological conditions. As used herein, the term "physiological conditions" refers to a temperature range of 20-40 degrees Celsius, atmospheric pressure (i.e., 1 atm), a pH of about 6 to about 8, and one or more physiological enzymes, proteases, acids, and bases. One advantage of a non-cleavable linker between the antibody and the STING agonist in an immunoconjugate is that it minimizes premature payload release and corresponding toxicity. STING is a widely expressed receptor and is therefore a particularly relevant consideration.

[0152] In one embodiment, the invention includes a peptide linking unit, i.e., L or linker, between the cell-binding agent and the immunostimulatory moiety, which comprises a peptide radical based on a linear sequence of specific amino acid residues that can be selectively cleaved by a protease, such as a cathepsin, a tumor-associated elastase enzyme, or an enzyme with protease-like or elastase-like activity. The peptide radical may be from about 2 to about 12 amino acids. Enzymatic cleavage of the bond in the peptide linker releases the active form of the immunostimulatory moiety. This results in increased tissue specificity of the conjugates of the invention, and thus further reduced toxicity of the conjugates of the invention in other tissue types.

[0153] The linker provides sufficient stability of the immunoconjugate in biological media, e.g., culture medium or serum, while at the same time providing the desired intracellular action within the tumor tissue as a result of its specific enzymatic or hydrolytic cleavability with release of the immunostimulatory moiety, i.e., the "payload."

[0154] The enzymatic activity of proteases, cathepsins or elastases can catalyze the cleavage of covalent bonds in immune complexes under physiological conditions. The enzymatic activity is an expression product of cells associated with tumor tissue. The enzymatic activity at the cleavage site of the targeting peptide converts the immune complex into an active immunostimulant free of the targeting peptide and the linking group. The cleavage site may be specifically recognized by the enzyme. The cathepsins or elastases may catalyze the cleavage of a specific peptide bond between the C-terminal amino acid residue of a specific peptide and the immunostimulatory portion of the immune complex.

[0155] In one embodiment, the invention includes a linking unit, i.e., L or linker, between the cell-binding agent and the immunostimulatory moiety that includes a substrate for cleavage by glucuronidase (Jeffrey SC, et al. (2006), Bioconjug Chem. 17(3):831-40), or sulfatase (Bargh JD, et al. (2020), Chem. Sci. 11(9):2375-2380). In particular, L includes a Gluc unit and includes a formula selected from the following: [ka]

[0156] The specific cleavage of the immune complexes of the present invention takes advantage of the presence of tumor-infiltrating cells of the immune system and leukocyte-secreted enzymes to facilitate the activation of anti-cancer drugs at the tumor site.

[0157] Suitable reactive electrophilic functional groups (Q in formula II) for BBI 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); arylazides (primary amine reactive); fluorinated arylazides (reactive via carbon-hydrogen (CH) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) and sulfotetrafluorophenyl (STP) esters (amine reactive); imide esters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Additional reagents include those described in Hermanson, Bioconjugate Techniques, 2001, 144:1311-1323. nd Edition, Academic Press, 2008.

[0158] The present invention provides solutions to limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers, such as those containing peptide units and substrates for proteases, 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 may adversely affect the efficacy of intracellular release. 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. Furthermore, in standard conjugation processes, the amount of adjuvant / drug moiety loaded onto the antibody (i.e., drug loading), the amount of aggregates formed in the conjugation reaction, and the yield of the final purified conjugate that can be obtained are correlated. For example, aggregate formation is generally positively correlated to the number of equivalents of adjuvant / drug moiety and its derivatives 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 the yield of immunoconjugates and make the process difficult to scale up.

[0159] An exemplary embodiment is a bis-benzimidazole-linker compound of formula II: [ka] wherein X a and X b are independent, R 5 5-membered heteroaryl optionally substituted by R 1 and R 4 are independently H, F, Cl, Br, I, -CN, -OH, -O-(C 1 -C 6 alkyl) and R 5 selected from the group consisting of; R 2a and R 2b are independently H, -C(=O)N(R 6 )2 , and R 5 Selected from; X a , R b , R 1 , R 4 , R 2a , R 2b One of them is R 5 Replaced by; R 3 are F, Cl, -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 C optionally substituted with one or more groups selected from 1 -C 6 Alkyldiyl, -(C 1 -C 3 Alkyldiyl)-O-(C 1 -C 3 Alkyldiyl)-, C 2 -C 6 Alkenyldiyl and C 2 -C 6 alkynyldiyl; R 5 teeth, -(C 1 -C 12 Alkyldiyl)-L; -(C 1 -C 12 Alkyldiyl)-N(R 6 )-L; -(C 1 -C 12 Alkyldiyl)-OL; -(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-L; -O-(C 1 -C 12 Alkyldiyl)-L; -O-(C 1 -C 12 Alkyldiyl)-N(R 6 )-L; -O-(C 1 -C 12 Alkyldiyl)-OL; -O-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-L; -O-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-N(R 6 )-L; -OC(=O)N(R 6 )-L; -OC(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 6 )-L; -N(R 6 )-L; -N(R 6 )-(C 1 -C 12 -alkyldiyl)-L; -N(R 6 )-(C 1 -C 12 -alkyldiyl)-N(R 6 )-L; -N(R 6 )-(C 1 -C 12 -alkyldiyl)-OL; -N(R 6 )-(C 1 -C 12 Alkyldiyl)(C 2 -C 20 Heterocyclyldiyl)-L; -C(=O)N(R 6 )-L; -C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-L; -C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 6 )-L; -C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-OL; -(C 2 -C 20 Heterocyclyldiyl)-L; -S(=O) 2 -(C 2 -C 20 heterocyclyldiyl)-L; and -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 6 )-L; R 6 are independently H or C 1 -C 6 is alkyl; L is QC(=O)-PEG-; QC(=O)-PEG-C(=O)N(R 6 )-(C 1 -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 + (R6 ) 2 -PEG-C(=O)-PEP-; QC(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-; QC(=O)-PEG-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-; QC(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-; QC(=O)-PEG-SS-(C 1 -C 12 Alkyldiyl)-OC(=O)- QC(=O)-PEG-SS-(C 1 -C 12 Alkyldiyl)-C(=O)-; QC(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-; QC(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-; QC(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 5 )-C(=O); QC(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C12 Alkyldiyl)-N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-; 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-OC(=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(R 5 )-C(=O)-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 Alkyldiyl)-OC(=O)-; Q-(CH 2 ) m-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-; Q-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-; Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-; and Q-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 is a linker selected from the group consisting of: PEG is a cyclic amine having the formula: -(CH 2 CH 2 O) n -(CH 2 ) m where m is an integer from 1 to 5 and n is an integer from 2 to 50; Gluc has the formula: [ka] having; PEP has the formula: [ka] having 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 five-membered proline amino acid ring, with the wavy line indicating the point of attachment; Cyc is F, Cl, NO 2 , -OH, -OCH3 and glucuronic acid having the structure: 6 -C 20 Aryldiyl and C 1 -C 20 heteroaryldiyl; [ka] R 7 -CH(R 8 )O-, -CH 2 -, -CH 2 N(R 8 )-, and -CH(R 8 )OC(═O)—, R 8 is H, C 1 -C 6 Alkyl, C(=O)-C 1 -C 6 Alkyl, and -C(=O)N(R 9 ) 2 Selected from R 9 are independently H, C 1 -C 12 Alkyl, and -(CH 2 CH 2 O) n -(CH 2 ) m -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R 9 the groups taken together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; Q is F, Cl, NO 2 and S.O. 3 - N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, each 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 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 2N(CH 3 ) 2 、-CO 2 H、-COCH 3 、-CO 2 CH 3 、-CO 2 C(CH 3 ) 3 、-COCH(OH)CH 3 、-CONH 2 、-CONHCH 3 、-CON(CH 3 ) 2 、-C(CH 3 ) 2 CONH 2 、-NH 2 -NHCH 3 、-N(CH 3 ) 2 、-NHCOCH 3 、-N(CH 3 )COCH 3 、-NHS(O) 2 CH 3 、-N(CH 3 )C(CH 3 ) 2 CONH 2 、-N(CH 3 )CH 2 CH 2 S(O) 2 CH 3 、-NHC(=NH)H、-NHC(=NH)CH 3 、-NHC(=NH)NH 2 、-NHC(=O)NH 2 、-NO 2 、=O、-OH、-OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 OCH 3 、-OCH 2 CH 2 OH、-OC H2 CH 2 N(CH 3 ) 2 、-O(CH 2 CH 2 O) n -(CH 2 ) m CO2 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.

[0160] In an exemplary embodiment, L comprises PEP, wherein PEP is a dipeptide and has the formula: [ka] has.

[0161] In an exemplary embodiment, L comprises PEP, wherein PEP is a tripeptide and has the formula: [ka] has.

[0162] In an exemplary embodiment, the PEP is a tripeptide, wherein AA 1 is methyl and AA 2 forms proline and AA 3 is isopropyl.

[0163] In an exemplary embodiment, L comprises PEP, wherein PEP is a tetrapeptide and has the formula: [ka] has.

[0164] In an exemplary embodiment, the PEP is a tetrapeptide, wherein: AA 1is selected from the group consisting of Abu, Ala, and Val; AA 2 is selected from the group consisting of Nle(O-Bzl), Oic and Pro; AA 3 Ala and Met(O) 2 selected from the group consisting of; AA 4 Oic, Arg(NO 2 ), Bpa, and Nle(O-Bzl).

[0165] In one exemplary embodiment, the PEP is [ka] [ka] The amino acid residues are selected from the group consisting of:

[0166] In an exemplary embodiment, the PEP is selected from the group consisting of Ala-Pro-Val, Asn-Pro-Val, Ala-Ala-Val and other peptide sequences as described in WO2021 / 226440.

[0167] In an exemplary embodiment, PEP has the formula: [ka] has.

[0168] In an exemplary embodiment, PEP has the formula: [ka] has.

[0169] An exemplary embodiment of the STING agonist-linker intermediate compound is wherein L has the formula: [ka] This includes including.

[0170] Exemplary embodiments of the STING agonist-linker intermediate compound include where Q is selected from the following: [ka]

[0171] Exemplary embodiments of the STING agonist-linker intermediate compound include those in which Q is F, Cl, NO 2 , and S.O. 3 - and R 1 is substituted with one or more groups independently selected from the group consisting of phenoxy,

[0172] Exemplary embodiments of the STING agonist-linker intermediate compound include where Q is 2,3,5,6-tetrafluorophenoxy.

[0173] Exemplary embodiments of the STING agonist-linker intermediate compound include where Q is 2,3,5,6-tetrafluoro-4-sulfonato-phenoxy.

[0174] Exemplary embodiments of the STING agonist-linker intermediate compound include where Q is a maleimide.

[0175] Exemplary embodiments of the STING agonist-linker intermediate compound include where L is selected from the following structures: [ka] In the formula, the wavy line represents R 5 The attachment points to the

[0176] Exemplary embodiments of bis-benzimidazole, STING agonist-linker intermediate compounds are shown in Tables 1a and 1b. Each STING agonist-linker intermediate compound was prepared, characterized by mass spectrometry, and shown to have the mass shown. The STING agonist-linker intermediate compounds of Tables 1a and 1b exhibit surprising and unexpected properties of STING agonist selectivity that, when conjugated to an antibody, may predict useful therapeutic activity for treating cancer and other disorders. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0177] immune complex The immunoconjugates of the invention induce targeted activation of STING-expressing tumor cells themselves as well as immune effector cells such as myeloid cells. Tumor targeting provides specificity to minimize off-target STING activation, and the immunoconjugates allow phagocytosis to not only enhance effector cell activation, but also increase uptake of the immunoconjugate and subsequent processing and presentation of tumor antigens.

[0178] An exemplary embodiment of the immunoconjugate comprises an antibody covalently attached to one or more STING agonists, bis-benzimidazole (BBI) 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; D is a STING agonist moiety having the formula: [ka] X a and X b are independent, R 55-membered heteroaryl optionally substituted by R 1 and R 4 are independently H, F, Cl, Br, I, -CN, -OH, -O-(C 1 -C 6 alkyl) and R 5 selected from the group consisting of; R 2a and R 2b are independently H, -C(=O)N(R 6 ) 2 , and R 5 Selected from; X a , X b , R 1 , R 4 , R 2a , R 2b One of them is R 5 Replaced by; R 3 are F, Cl, -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 C optionally substituted with one or more groups selected from 1 -C 6 Alkyldiyl, -(C 1 -C 3 Alkyldiyl)-O-(C 1 -C 3 Alkyldiyl)-, C 2 -C 6 Alkenyldiyl and C 2 -C 6 alkynyldiyl; R 5 teeth, -(C 1 -C 12 Alkyldiyl)-*; -(C 1 -C 12Alkyldiyl)-N(R 6 )-*; -(C 1 -C 12 Alkyldiyl)-O-*; -(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-*; -O-(C 1 -C 12 Alkyldiyl)-*; -O-(C 1 -C 12 Alkyldiyl)-N(R 6 )-*; -O-(C 1 -C 12 Alkyldiyl)-O-*; -O-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-*; -O-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-N(R 6 )-*; -OC(=O)N(R 6 )-*; -OC(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )*; -N(R 6 )-*; -N(R 6 )-(C 1 -C 12 Alkyldiyl)-*; -N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 6 )-*; -N(R 6 )-(C 1 -C 12 Alkyldiyl)-O-*; -N(R 6 )-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-*; -C(=O)N(R 6 )-*; -C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-*; -C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )-*; -C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-O-*; -(C 2 -C 20 Heterocyclyldiyl)-*; -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-*; and -S(=O) 2 -(C 2 -C 20 Heterocyclyldiyl)-(C 1 -C 12 Alkyldiyl)-N(R 6 -)* selected from the group consisting of; Asterisk * indicates the binding site of L; R 6 are each independently H or C 1 -C 6 is alkyl; L is -C(=O)-PEG-; -C(=O)-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-Gluc-; -C(=O)-PEG-O-; -C(=O)-PEG-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 )-(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-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-; -C(=O)-PEG-SS-(C 1 -C 12 Alkyldiyl)-OC(=O)-; -C(=O)-PEG-SS-(C 1 -C 12 Alkyldiyl)-C(=O)-; -C(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-; -C(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-; -C(=O)-(C1 -C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 5 )-C(=O); -C(=O)-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-N(R 6 )C(=O)-(C 2 -C 5 Monoheterocyclyldiyl)-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-Gluc-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-O-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-OC(=O)-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-N(R 5 )-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6)-PEG-N(R 5 )-C(=O)-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)-PEP-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-SS-(C 1 -C 12 Alkyldiyl)-OC(=O)-; -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)-; -Succinimidyl-(CH 2 ) m -C(=O)N(R 6 )-PEG-C(=O)N(R 6 )-(C 1 -C 12 Alkyldiyl)-C(=O)-PEP-; -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-; and -Succinimidyl-(CH 2 ) m -C(=O)-PEP-N(R 6 )-(C 1 -C 12 Alkyldiyl)N(R 6 )C(=O)-(C 2 -C 5 is a linker selected from the group consisting of: PEG has the formula: -(CH 2 CH 2 O) n -(CH 2 ) mwhere m is an integer from 1 to 5 and n is an integer from 2 to 50; Gluc [ka] having; PEP has the formula: [ka] where AA is 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 is F, Cl, NO 2 , -OH, -OCH 3 and C optionally substituted with one or more selected from glucuronic acid having the structure 6 -C 20 Aryldiyl and C 1 -C 20 heteroaryldiyl; [ka] R 7 -CH(R 8 )O-, -CH 2 -, -CH 2 N(R 8 )-, and -CH(R 8 )OC(═O)—, R 8 is H, C 1 -C 6 Alkyl, C(=O)-C 1 -C 6 Alkyl, and -C(=O)N(R 9 ) 2 Selected from R 9 are independently H, C 1 -C 12 Alkyl, and -(CH 2 CH 2 O) n -(CH 2 ) m -OH, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R 9the 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 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 , -CH2 NHSO 2 CH 3 、-CH 2 NHCH 3 、-CH 2 N(CH 3 ) 2 、-CO 2 H、-COCH 3 、-CO 2 CH 3 、-CO 2 C(CH 3 ) 3 、-COCH(OH)CH 3 、-CONH 2 、-CONHCH 3 、-CON(CH 3 ) 2 、-C(CH 3 ) 2 CONH 2 、-NH 2 -NHCH 3 、-N(CH 3 ) 2 、-NHCOCH 3 、-N(CH 3 )COCH 3 、-NHS(O) 2 CH 3 、-N(CH 3 )C(CH 3 ) 2 CONH 2 、-N(CH 3 )CH 2 CH 2 S(O) 2 CH 3 、-NHC(=NH)H、-NHC(=NH)CH 3 、-NHC(=NH)NH 2 、-NHC(=O)NH 2 、-NO 2 、=O、-OH、-OCH 3 、-OCH 2 CH 3 、-OCH 2 CH 2 OCH 3 、-OCH 2 CH 2 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.

[0179] Antibodies target tumor-specific and / or immune-specific (e.g., PD-L1) antigens, providing specificity in targeting of immune complexes and allowing safe, systemic delivery.

[0180] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is an antibody construct having an antigen-binding domain that binds to PD-L1, such as atezolizumab, durvalumab, and avelumab, or a biosimilar or biobetter thereof.

[0181] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is an antibody construct having an antigen binding domain that binds to HER2, such as trastuzumab and pertuzumab, or a biosimilar or biobetter thereof.

[0182] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is an antibody construct having an antigen binding domain that binds to CEA, such as labetuzumab, or a biosimilar or biobetter thereof.

[0183] Exemplary embodiments of the immunoconjugate of Formula I include where the antibody is an antibody construct having an antigen binding domain that binds to Trop2, such as sacituzumab, or a biosimilar or biobetter thereof.

[0184] Exemplary embodiments of the immunoconjugate of formula I include X a and X b is independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl.

[0185] Exemplary embodiments of the immunoconjugate of formula I include X a and X b are -CH 3 , -CH 2 CH 3 , -CH=CH 2 , -C≡CH, -C≡CCH 3 , -CH 2 CH 2 CH 3 , -CH(CH 3 ) 2 , and -CH 2 CH(CH 3 ) 2 and wherein the pyrazolyl is substituted by one or more groups selected from the group consisting of:

[0186] Exemplary embodiments of the immunoconjugate of formula I include X a and X b One of them is R 5 This includes being replaced with:

[0187] Exemplary embodiments of the immunoconjugate of formula I include R 1 -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OCH 3 , -OCH 2 CH 2OH and -OCH 2 CH 2 N(CH 3 ) 2 It is included that the compound is selected from the group consisting of:

[0188] Exemplary embodiments of the immunoconjugate of formula I include R 1 -OCH 3 This includes being.

[0189] Exemplary embodiments of the immunoconjugate of formula I include R 1 This includes the fact that F.

[0190] Exemplary embodiments of the immunoconjugate of formula I include R 2a and R 2b are -C(=O)NH 2 This includes being.

[0191] Exemplary embodiments of the immunoconjugate of formula I include R 2a and R 2b One of them is R 5 This includes being replaced with:

[0192] Exemplary embodiments of the immunoconjugate of formula I include R 3 Ga-CH 2 CH 2 -, -CH=CH-, and -C≡C-.

[0193] Exemplary embodiments of the immunoconjugate of formula I include R 3 F, -OH and -OCH 3 C substituted with one or more groups selected from 2 -C 4 It is contemplated that the alkenyl group may be alkenyldiyl.

[0194] Exemplary embodiments of the immunoconjugate of formula I include R 4 -O-(C 1 -C 12 Alkyldiyl)-(C 2 -C 20 Heterocyclyldiyl)-* is included.

[0195] Exemplary embodiments of the immunoconjugate of formula I include 1 -C 12 The alkyldiyl is propyldiyl, and C 2 -C 20 It is included that heterocyclyldiyl is piperidiyl.

[0196] Exemplary embodiments of the immunoconjugate of formula I include R 1 and R 4 One of them is R 5で This includes being replaced.

[0197] Exemplary embodiments of the immunoconjugate of Formula I include where L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0198] Exemplary embodiments of the immunoconjugate of Formula I include those in which L is attached to a cysteine ​​thiol of the antibody.

[0199] Exemplary embodiments of the immunoconjugates of Formula I include, for PEG, m is 1 or 2 and n is an integer from 2 to 10.

[0200] Exemplary embodiments of the immunoconjugate of Formula I include where n is 10 for PEG.

[0201] Exemplary embodiments of the immunoconjugate of formula I include those in which L comprises PEP, where PEP is a dipeptide and has the formula: [ka]

[0202] Exemplary embodiments of the immunoconjugate of formula I include those in which L comprises PEP, where PEP is a tripeptide and has the formula: [ka]

[0203] Exemplary embodiments of the immunoconjugate of formula I include those in which L comprises PEP, where PEP is a tetrapeptide and has the formula: [ka]

[0204] Exemplary embodiments of the immunoconjugate of formula I include where L comprises a Glu selected from the following formulas: [ka]

[0205] Exemplary embodiments of the immunoconjugate of Formula I include where L is selected from the following structures: [ka] In the formula, the wavy line represents R 5 The attachment points to the

[0206] The present invention includes all rational combinations and permutations of features of the embodiments of Formula I.

[0207] In certain embodiments, the immunoconjugate compounds of the invention include those with immunostimulatory activity. The antibody-drug conjugates of the invention selectively deliver effective doses of bis-benzimidazole drugs to tumor tissue, thereby achieving greater selectivity (i.e., lower effective doses) while increasing the therapeutic index ("therapeutic window") compared to unconjugated bis-benzimidazole.

[0208] Drug loading is represented by p, the number of BBI moieties per antibody in the immunoconjugate of Formula I. Drug (BBI) loading may range from 1 to about 8 drug moieties (D) per antibody. The immunoconjugate of Formula I comprises a mixture or population of antibodies conjugated to a range of 1 to about 8 drug moieties. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of reactive or available amino acid side chain residues, such as lysine and cysteine. In some embodiments, a free cysteine ​​residue is introduced into the antibody amino acid sequence by the methods described herein. In such embodiments, p may be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges therein, e.g., 1-8 or 2-5. In such embodiments, p and n are equal (i.e., p=n=1, 2, 3, 4, 5, 6, 7, or 8, or some range therebetween). Exemplary immunoconjugate compounds 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.

[0209] For some immunoconjugates, p may be limited by the number of binding sites on the antibody. For example, as in certain exemplary embodiments described herein, where 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 on the antibody may be available and reactive for conjugation with the BBI linker compound of formula II. In certain embodiments, even higher drug loading, e.g., p greater than 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading of the immunoconjugate ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

[0210] The loading of the immunoconjugate (drug / antibody ratio) may be controlled in different ways, as well as by, for example, (i) limiting the molar excess of the BBI linker intermediate compound compared to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing denaturation conditions for optimized antibody reactivity.

[0211] 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 may be identified in the mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, KJ, et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004, Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American (See, Association for Cancer Research, 2004, Annual Meeting, March, 27-31, 2004, Proceedings of the AACR, 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.

[0212] Exemplary embodiments of the immunoconjugates of formula I are selected from the immunoconjugates of Tables 2a and 2b. Assessment of immunoconjugate activity in vitro may be performed according to the method of Example 202.

[0213] STING activation is typically associated with induction of type I / III IFN (interferon) via IRF3 (interferon regulatory factor 3) signaling, but can also induce proinflammatory cytokines such as TNFα (tumor necrosis factor alpha) via the NF-κB (nuclear factor kappa light-chain enhancer of activated B cells) pathway. Certain immune complexes show the ability to induce TNFα as well as IFNλ1 (interferon lambda 1), consistent with STING activation. As a comparison factor, trastuzumab did not substantially induce TNFα or IFNλ1 in PBMC tumor co-culture assays (Example 202). [Table 3] [Table 4-1] [Table 4-2]

[0214] Composition of the immunoconjugate The invention provides compositions, e.g., pharmaceutical or pharmacologically acceptable compositions or formulations, comprising a plurality of immunoconjugates as described herein and optionally a carrier therefor, e.g., a pharma- ceutically or pharmacologically acceptable carrier. The immunoconjugates may be the same or different in the composition, i.e., the composition may include immunoconjugates with the same number of BBI adjuvants linked to the same position on the antibody construct, and / or may include immunoconjugates with the same number of BBI adjuvants linked to different positions on the antibody construct, with different numbers of adjuvants linked to the same position on the antibody construct, or with different numbers of adjuvants linked to different positions on the antibody construct.

[0215] In an exemplary embodiment, the composition comprising immunoconjugate compounds comprises a mixture of immunoconjugate compounds, wherein the average drug per antibody (BBI) loading in the mixture of immunoconjugate compounds is about 2 to about 5.

[0216] The immunoconjugate compositions of the invention can have an average adjuvant to antibody construct ratio (DAR) of about 0.4 to about 10. One of skill in the art will recognize that because the number of thienoazepine adjuvants attached to an antibody construct may vary from one immunoconjugate to another in a composition comprising multiple immunoconjugates of the invention, the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average, sometimes referred to as the drug / 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.

[0217] The average number of adjuvant moieties per antibody (DAR) in preparing immune complexes from conjugation reactions can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of immune complexes in the composition in terms of p may also be determined. In certain cases, separation, purification, and characterization of homogeneous immune complexes with a particular value of p from immune complexes with other drug loads may be achieved by means such as reverse-phase HPLC or electrophoresis.

[0218] 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 intratumorally (intratumorally). A composition for injection will generally comprise a solution of the immunoconjugate dissolved in a pharma- ceutically acceptable carrier. Among the acceptable vehicles and solvents that may be used are water and isotonic solutions of one or more salts, such as sodium chloride, for example, Ringer's solution. In addition, sterile fixed oils can conventionally be used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, can be used in the preparation of injectables as well. These compositions are desirably sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharma- ceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., as required to approximate physiological conditions.

[0219] The composition may contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition may vary widely and is selected primarily based on fluid volume, viscosity, weight, etc., according to the particular mode of administration selected and the needs of the patient. In certain embodiments, the concentration of the immunoconjugate in the solution formulation for injection ranges from about 0.1% (w / w) to about 10% (w / w).

[0220] Immunoconjugate therapy for cancer The present invention provides a method for treating cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate described herein (e.g., a composition described herein) to a subject in need thereof, e.g., a subject having cancer and in need of cancer treatment. The method comprises administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 2.

[0221] It is contemplated that the immunoconjugates of the invention may be used to treat a variety of hyperproliferative diseases or disorders, such as those characterized by overexpression of tumor antigens. Exemplary hyperproliferative disorders include benign or malignant solid tumors, as well as hematological disorders, such as leukemia and lymphoid malignancies.

[0222] In another aspect, the immunoconjugate is provided for use as a drug.In certain embodiments, the present invention provides an immunoconjugate for use in a method for treating an individual, comprising administering to the individual an effective amount of the immunoconjugate.In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0223] In a further aspect, the present invention provides the use of the immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer, and the method comprises administering an effective amount of the medicament to an individual having cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0224] 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 secondary metastases), lung, breast, and skin.

[0225] Soft tissue tumors are a highly diverse group of rare tumors derived from connective tissue. Examples of soft tissue tumors include alveolar soft part sarcoma, hemangioid fibrous histiocytoma, chondromyxoid fibroma, skeletal chondrosarcoma, extraskeletal myxoid chondrosarcoma, clear cell sarcoma, desmoplastic small round cell tumor, dermatofibrosarcoma protuberans, endometrial stromal tumor, Ewing's sarcoma, fibromatosis (desmoid), infantile fibromatosis, gastrointestinal stromal tumor, giant cell tumor of bone, giant cell tumor of tendon sheath, inflammatory myofibroblastic tumor, uterine fibroid, leiocytoma, lipoblastoma, typical lipoma, spindle cell or pleomorphic lipoma, atypical lipoma, chondroid lipoma, well-differentiated liposarcoma, myxoid / round cell liposarcoma, pleomorphic liposarcoma, myxoid malignant fibrous histiocytoma, high grade malignant fibrous histiocytoma, myxofibrosarcoma, malignant peripheral nerve tumor, These include, but are not limited to, schwannoma, mesothelioma, neuroblastoma, osteochondroma, osteosarcoma, primitive neuroectodermal tumor, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, benign or malignant schwannoma, synovial sarcoma, Evans tumor, nodular fasciitis, desmoid-type fibromatosis, solitary fibrous tumor, dermatofibrosarcoma protuberans (DFSP), angiosarcoma, epithelioid hemangioendothelioma, giant cell tumor of tendon sheath (TGCT), pigmented villonodular synovitis (PVNS), fibrous dysplasia, myxofibrosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, neurofibroma, pleomorphic adenoma of soft tissue, and tumors derived from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and nerve sheath cells.

[0226] 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, Askin tumor; botryoid sarcoma; chondrosarcoma; Ewing sarcoma; malignant hemangioendothelioma; malignant nerve sheath tumor; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodes; dermatofibrosarcoma protuberans (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; angiosarcoma (more commonly called "angiosarcoma"); Kaposi's sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neuroblastoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma).

[0227] 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.

[0228] Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma may begin in a mole (cutaneous melanoma) but may also begin in other pigmented tissues, such as in the eye or in the intestine.

[0229] Merkel cell carcinoma is a rare type of skin cancer that usually appears as flesh-colored or bluish-red nodules on the face, head, or neck. Merkel cell carcinoma is also called a neuroendocrine carcinoma of the skin. In some embodiments, a method of treating Merkel cell carcinoma includes administering an immunoconjugate containing an antibody construct capable of binding to Trop2 (e.g., sacituzumab, sacituzumab govitecan, biosimilars thereof, or biobetters thereof). In some embodiments, the Merkel cell carcinoma has metastasized at the time the administration is performed.

[0230] Leukemias are cancerous cells that originate in blood-forming tissues, such as 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 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 myeloid leukemia or myeloblastic leukemia. Lymphoid leukemia is also called lymphoblastic leukemia or lymphocytic leukemia. Lymphoid leukemia cells may collect in lymph nodes, causing them to swell. Examples of leukemias include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0231] Lymphoma is a cancer that begins in the cells of the immune system. For example, lymphoma can arise in bone marrow-derived cells that would normally mature in the lymphatic system. There are two basic categories of lymphoma. One category of lymphoma is Hodgkin lymphoma (HL), which is characterized by the presence of a type of cell called Reed-Sternberg cells. There are currently six recognized types of HL. Examples of Hodgkin lymphoma include nodular sclerosing classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocytopenic CHL, lymphocyte-rich CHL, and nodular lymphocyte-predominant HL.

[0232] Another category of lymphoma is non-Hodgkin's lymphoma (NHL), which includes a large and diverse group of cancers of immune system cells. Non-Hodgkin's lymphomas can be further divided into indolent (slow-growing) and aggressive (fast-growing) cancers. There are currently 61 recognized types of NHL. Examples of non-Hodgkin's lymphomas include, but are not limited to, AIDS-related lymphoma, anaplastic large cell lymphoma, hematologic immunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small noncleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathy-type T-cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T-cell lymphoma, T-cell leukemia, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, treatment-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0233] Brain tumors include cancers of brain tissue. Examples of brain tumors include, but are not limited to, gliomas (e.g., glioblastoma, astrocytoma, oligodendroglioma, ependymoma, etc.), meningiomas, pituitary adenomas, and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas).

[0234] 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.

[0235] The immunoconjugates of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration, if desired for localized therapy. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, 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.

[0236] The immunoconjugates described herein can be used to treat the same types of cancer as sacituzumab, sacituzumab govitecan, their biogenerics, and their biobetters, particularly breast cancer, especially triple negative (tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma.

[0237] In some embodiments, the immunoconjugates described herein may be effective in treating bladder cancer, salivary gland cancer, endometrial 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.

[0238] The immunoconjugate is administered to a subject in need thereof in a therapeutically effective amount using any suitable dosing regimen, such as those utilized for sacituzumab, sacituzumab govitecan, their biosimilars, and their biobetters. For example, the method can include administering the immunoconjugate to provide a dose of about 100 ng / kg to about 50 mg / kg to the subject. The dose of the immunoconjugate can range from about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of the immunoconjugate can be outside of these ranges depending on the particular conjugate and the type and severity of the cancer being treated. The frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once a month to about five times a week. In some embodiments, the immunoconjugate is administered once a week.

[0239] In another aspect, the invention provides a method for preventing cancer. The method includes administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to the particular cancer to be prevented. For example, the method can include administering the immunoconjugate to provide the subject with a dose of about 100 ng / kg to about 50 mg / kg. The dose of the immunoconjugate can range from about 5 mg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, or about 100 μg / kg to about 1 mg / kg. The dose of the immunoconjugate can be about 100, 200, 300, 400, or 500 μg / kg. The dose of the immunoconjugate can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The dose of the immunoconjugate can be outside of these ranges depending on the particular conjugate and the type and severity of the cancer being treated. The frequency of administration can range from a single dose to multiple doses per week, or more frequently. In some embodiments, the immunoconjugate is administered from about once a month to about five times a week. In some embodiments, the immunoconjugate is administered once a week.

[0240] Some embodiments of the present invention provide a method for treating cancer as described above, where the cancer is breast cancer.Breast cancer can originate from various regions of the breast, and various types of breast cancer have been characterized.For example, the immunoconjugates of the present invention can be used to treat other forms of breast cancer, such as ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular, medullary, mucinous, papillary, or cribriform carcinoma of the breast); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; triple negative (tests negative for estrogen receptor, progesterone receptor, and excess HER2 protein) breast cancer.

[0241] In some embodiments, the cancer is susceptible to a STING-induced pro-inflammatory response. EXAMPLES

[0242] Bis-benzimidazole-linker (BBI-L) Preparation of compounds of formula II and intermediates Example 3: Synthesis of (2E)-1-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-(3-piperazin-1-ylpropoxy)-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazole-5-carboxamide, 3 [ka] Preparation of 4-chloro-3-methoxy-5-nitro-benzamide, 3b A solution of methyl 4-chloro-3-methoxy-5-nitro-benzoate was added with NH 3 H 2 3a (15 g, 61.0 mmol, 1 equiv.) in 0 (136.5 g, 973.7 mmol, 150 mL, 25% purity, 16.0 equiv.) was added and stirred at 50° C. for 24 h. The mixture was filtered and the filter cake was washed with water (300 mL). The cake was dried under reduced pressure to give 3b (11 g, 47.70 mmol, 78.11% yield) as an off-white solid. 1 H NMR (DMSO-d 6 ,400MHz)δ8.29(s,1H),8.05(d,J=1.6Hz,1H),7.88(d,J=1.6Hz,1H),7.78(s,1H),4.02(s,3H).

[0243] Preparation of 4-chloro-3-hydroxy-5-nitro-benzamide, 3c To a solution of 3b (6 g, 26.0 mmol, 1 equiv) in DCM (100 mL) was added AlCl 3 (21.0 g, 156 mmol, 8.50 mL, 6 equiv) was added and stirred at 40° C. for 12 h. The reaction mixture was cooled to 0° C. and quenched by the addition of 50 mL of 2 M aqueous HCl at 0° C., then stirred at 15° C. for 30 min and extracted with EtOAc (50 mL×3). The combined organic layers were washed with brine (50 mL) and diluted with Na 2 SO 4 It was dried over ice, filtered and concentrated under reduced pressure to give 3c (4 g, crude) as a pale yellow solid.1 H NMR (DMSO-d 6 ,400MHz)δ11.52(s,1H),8.17(s,1H),7.92(d,J=2.0Hz,1H),7.71(d,J=2.0Hz,1H),7.66(s,1H).

[0244] Preparation of 3-[3-[tert-butyl(dimethyl)silyl]oxypropoxy]-4-chloro-5-nitro-benzamide, 3d To a mixture of 3-bromopropan-1-ol (25 g, 180.0 mmol, 16.0 mL, 1 equiv.) in THF (250 mL), imidazole (25.0 g, 360 mmol, 2 equiv.) and TBSCl (32.6 g, 216 mmol, 26.50 mL, 1.2 equiv.) were added and stirred at 15° C. for 2 h. The mixture was diluted with water (300 mL) and extracted with EtOAc (100 mL×3). The organic layer was washed with brine (100 mL) and diluted with Na 2 SO 4 The residue was dried over ice, filtered and concentrated, and purified by flash silica gel chromatography (ISCO®; 70 g SepaFlash® silica flash column, eluent of 0-10% ethyl acetate / petroleum ether gradient, 100 mL / min) to give 3-bromopropoxy-tert-butyl-dimethyl-silane (43 g, 170 mmol, 94.40% yield) as a colorless oil. 1 H NMR (CDCl 3 ,400MHz)δ3.75(t,J=5.6Hz,2H),3.53(t,J=6.4Hz,2H),2.11-1.99(m,2H),0.91(s,9H),0.08(s,6H).

[0245] A solution of 3-bromopropoxy-tert-butyl-dimethyl-silane (19.8 g, 78.0 mmol, 1.3 equiv.) in DMF (200 mL) was treated with 3c (13 g, 60.0 mmol, 1 equiv.) and K 2 CO 3(16.60 g, 120.0 mmol, 2 equiv.) was added at 15° C. and stirred at 100° C. for 2 h. The mixture was diluted with water (600 mL) and extracted with EtOAc (200 mL×3). The organic layer was washed with brine (200 mL×3) and diluted with Na 2 SO 4 After drying over low heat, filtration and concentration, the residue was purified by flash silica gel chromatography (ISCO®; 35 g SepaFlash® silica flash column, eluent of 0-50% ethyl acetate / petroleum ether gradient, 65 mL / min) to give 3d (24 g, crude) as a pale yellow solid. 1 H NMR (DMSO-d 6 ,400MHz)δ8.28(s,1H),8.04(d,J=1.6Hz,1H),7.88(d,J=1.6Hz,1H),7.76(s,1H),4.2 9(t,J=6.0Hz,2H),3.79(t,J=6.0Hz,2H),2.01-1.93(m,2H),0.83(s,9H),0.01(s,6H).

[0246] Preparation of 4-chloro-3-(3-hydroxypropoxy)-5-nitro-benzamide, 3e Acetyl chloride (48.4 g, 617 mmol, 44.0 mL, 10 equiv) was added to a solution of 3d (24 g, 61.7 mmol, 1 equiv) in MeOH (300 mL) and stirred at 15 °C for 2 h. Solid NaHCO 3 The pH of the mixture was adjusted to about 8 by slow addition of, then filtered and concentrated to give 3e (15 g, 54.61 mmol, 88.50% yield) as a pale yellow solid. 1 H NMR (DMSO-d 6 ,400MHz)δ8.35(br s,1H),8.04(d,J=1.6Hz,1H),7.91(d,J=1.6Hz,1H),7.76(s,1H),4.64(t, J=5.2Hz,1H),4.30(t,J=6.4Hz,2H),3.64-3.52(m,2H),1.97-1.89(m,2H).

[0247] Preparation of 3-(5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl 4-methylbenzenesulfonate, 3f To a solution of 3e (15 g, 54.6 mmol, 1 equiv) in THF (150 mL) was added Et 3 N (27.6 g, 273 mmol, 38.0 mL, 5 equiv), DMAP (667 mg, 5.5 mmol, 0.1 equiv) and TsCl (11.60 g, 164 mmol, 3 equiv) were added and stirred at 15° C. for 12 h. The mixture was diluted with water (300 mL) and extracted with EtOAc (100 mL×3). The organic layer was washed with brine (100 mL) and diluted with Na 2 SO 4 The residue was dried over ice, filtered and concentrated, and purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent of 0-80% ethyl acetate / petroleum ether gradient at 45 mL / min) to give 3f (20 g, 46.6 mmol, 85.39% yield) as a pale yellow solid. 1 H NMR (DMSO-d 6 ,400MHz)δ8.28(s,1H),8.04(d,J=1.6Hz,1H),7.79(s,1H),7.75-7.69(m,3H),7.27(d,J= 8.0Hz,2H),4.22(t,J=5.6Hz,2H),4.13(t,J=5.6Hz,2H),2.27(s,3H),2.15-2.07(m,2H).

[0248] Preparation of tert-butyl 4-[3-(5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl]piperazine-1-carboxylate, 3 g To a solution of 3f (13.5 g, 31.5 mmol, 1 equiv) in THF (150 mL) was added Et 3 N (6.40 g, 63.0 mmol, 8.80 mL, 2 eq.) and tert-butyl piperazine-1-carboxylate; hydrochloride (9.80 g, 44.0 mmol, 1.4 eq.) were added and stirred at 80° C. for 12 h. The mixture was diluted with water (300 mL) and extracted with EtOAc (100 mL×3). The organic layer was washed with brine (100 mL) and diluted with Na 2 SO 4After drying over low heat, filtering and concentrating, the residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® silica flash column, eluent of 0-80% ethyl acetate / petroleum ether gradient at 45 mL / min) to give 3g (10 g, 22.58 mmol, 71.72% yield) as a pale yellow solid. 1 H NMR (DMSO-d 6 ,400MHz)δ8.28(br s,1H),8.04(d,J=1.6Hz,1H),7.87(d,J=1.6Hz,1H),7.77(br s,1H),4.27(t,J=6.4Hz,2H),3.30(br t,J=4.8Hz,4H),2.48-2.44(m,2H),2.32(br t,J=4.8Hz,4H),1.95(br t,J=6.4Hz,2H),1.39(s,9H).

[0249] [ka] [ka] Preparation of 2-[(E)-4-bromobut-2-enyl]isoindoline-1,3-dione, 3h To a solution of (1,3-dioxoisoindolin-2-yl)potassium (33.6 g, 181 mmol, 0.8 equiv.) in DMF (300 mL) was added (E)-1,4-dibromobut-2-ene (48.5 g, 227 mmol, 1 equiv.) and stirred at 15° C. for 12 h. The mixture was diluted with water (1000 mL) and extracted with EtOAc (500 mL×3). The organic layer was washed with brine (300 mL×3) and diluted with Na 2 SO 4 The residue was dried over 100 ml of ethyl acetate, filtered, and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 100 g SepaFlash® silica flash column, eluent of 0-60% ethyl acetate / petroleum ether gradient, 65 mL / min) to give 2-[(E)-4-bromobut-2-enyl]isoindoline-1,3-dione (50 g, 178 mmol, 78.72% yield) as a white solid.1 H NMR (CDCl 3 ,400MHz)δ7.89-7.83(m,2H),7.77-7.69(m,2H),6.00-5.90(m,1H),5.88-5.79(m,1H),4.35-4.28(m,2H),3.93-3.88(m,2H).

[0250] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-(1,3-dioxoisoindolin-2-yl)but-2-enyl]carbamate, 3i A solution of 3h (50 g, 178 mmol, 1 equiv.) in DMF (500 mL) was added to Cs 2 CO 3 (87.2 g, 268 mmol, 1.5 equiv.) and tert-butyl N-tert-butoxycarbonylcarbamate (50.0 g, 232 mmol, 1.3 equiv.) were added and stirred at 15° C. for 12 h. The mixture was diluted with water (2000 mL) and extracted with EtOAc (500 mL×3). The organic layer was washed with brine (500 mL×3) and diluted with Na 2 SO 4 The residue was dried over ice, filtered and concentrated, and purified by flash silica gel chromatography (ISCO®; 100 g SepaFlash® silica flash column, eluent of a gradient of 0-70% ethyl acetate / petroleum ether at 65 mL / min) to give 3i (46.5 g, 112 mmol, 62.55% yield) as a white solid. 1 H NMR (CDCl 3 , 400MHz) δ7.87-7.82(m,2H),7.75-7.69(m,2H),5.82-5.63(m,2H),4.27(d,J=5.6Hz,2H),4.14(d,J=5.6Hz,2H),1.46(s,18H).

[0251] Preparation of tert-butyl N-[(E)-4-aminobut-2-enyl]-N-tert-butoxycarbonyl-carbamate, 3j To a solution of 3i (46.5 g, 112 mmol, 1 equiv) in MeOH (500 mL) was added hydrazine; hydrate (19.7 g, 335 mmol, 19.0 mL, 85% purity, 3 equiv) at 20° C. and stirred at 70° C. for 3 h. The mixture was filtered and the filtrate was concentrated. The crude product was triturated with MTBE at 15° C. for 20 min and the filtrate was concentrated to give 3j (33 g, crude) as a pale yellow oil. 1 H NMR (CDCl 3 ,400MHz)δ5.79-5.69(m,1H),5.66-5.55(m,1H),4.15(dd,J=6.0,1.2Hz,2H),3.29(dd,J=5.6,1.2,Hz,2H),1.50(s,18H)

[0252] Preparation of tert-butyl N-[(E)-4-(4-carbamoyl-2-methoxy-6-nitro-anilino)but-2-enyl]carbamate, 3k A mixture of 4-chloro-3-methoxy-5-nitro-benzamide, 3b (5 g, 21.7 mmol, 1.0 equiv.) and 3j (18.6 g, 65.1 mmol, 3.0 equiv.) in DIEA (11.21 g, 86.73 mmol, 15.11 mL, 4.0 equiv.) (neat reaction) was stirred for 5.5 h at 110° C. The reaction mixture was then cooled to 25° C. and ethyl acetate (100 mL) was added and stirred for 10 min before being poured into ice water (100 mL) and stirred for 10 min to allow the desired solid to precipitate from the mixture, which was then filtered to give 3k (10 g, crude) as an orange solid.

[0253] Preparation of 4-[[(E)-4-aminobut-2-enyl]amino]-3-methoxy-5-nitro-benzamide, 3l To a mixture of 3k (10 g, 26.29 mmol, 1 equiv) in EtOAc (80 mL) at 15° C. was added HCl / EtOAc (4 M, 197 mL, 30 equiv) and then stirred for 1 h at 15° C. The mixture was concentrated to give 3l (8.5 g, 24.07 mmol, 91.54% yield, 2HCl) as a red solid. 1H NMR(MeOD,400MHz)δ8.21(d,J=2.0Hz,1H),8.05(s,4H),7.60(d,J=2.0Hz,1H),7.33(s,1H),5. 91-5.84(m,1H),5.66-5.61(m,1H),4.17(d,J=5.2Hz,2H),3.89(s,3H),3.39(t,J=5.6Hz,2H).

[0254] Preparation of tert-butyl 4-[3-[5-carbamoyl-2-[[(E)-4-(4-carbamoyl-2-methoxy-6-nitro-anilino)but-2-enyl]amino]-3-nitro-phenoxy]propyl]piperazine-1-carboxylate, 3m A solution of 3l (5.31 g, 15.0 mmol, 1.26 equiv, 2HCl) in butan-1-ol (106.00 mL) was added to NaHCO 3 (5.03 g, 59.8 mmol, 2.33 mL, 5 equiv.) and DIEA (7.73 g, 59.8 mmol, 10.4 mL, 5 equiv.) were added. The mixture was stirred at 20° C. for 30 min and then diluted with N 2 tert-Butyl 4-[3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl]piperazine-1-carboxylate, 3g (5.3 g, 11.97 mmol, 1 equiv.) was added under 10° C. at 20° C., and the mixture was stirred at 120° C. for 12 h. The reaction mixture was concentrated in vacuo to give a residue. The residue was diluted with EtOAc (200 mL) and ice water (200 mL), which was stirred for 10 min. The precipitate was filtered to give 3m (14 g, crude) as a red solid. 1 H NMR (DMSO-d 6 ,400MHz)δ8.15(dd,J=5.6,1.6Hz,2H),8.03(s,2H),7.81-7.65(m,3H),7.53-7.47(m,2H),7.33(s,2H),5.65-5.55(m,2H),4.12-4.10 (m,2H),4.08-3.97(m,4H),3.81(s,3H),3.30-3.27(m,4H),2.40(t,J=7.2Hz,2H),2.31-2.24(m,4H),1.93-1.82(m,2H),1.38(s,9H).

[0255] Preparation of tert-butyl 4-[3-[3-amino-2-[[(E)-4-(2-amino-4-carbamoyl-6-methoxy-anilinobut-2-enyl]amino]-5-carbamoyl-phenoxy]propyl]piperazine-1-carboxylate, 3n Dissolve 3m (10.5 g, 15.0 mmol, 1 equiv.) in MeOH (50 mL), THF (50 mL) and H 2 To a solution of 100 (10 mL) NaHCO 3 (6.5 g, 76.0 mmol, 3.0 mL, 5 eq.) and disodium dithionite Na 2 S 2 O 4 (40.0 g, 229 mmol, 15 equiv.) was added and stirred at 20° C. for 5 h. The mixture was diluted with water (200 mL) and extracted with DCM:i-PrOH=3:1 (100 mL×6). The organic layer was extracted with Na 2 SO 4 It was dried over, filtered and concentrated to give 3n (4.6 g, 7.34 mmol, 48.00% yield) as a yellow solid. 1 H NMR(MeOD,400MHz)δ6.94-6.91(m,2H),6.88-6.83(m,2H),5.77-5.64(m,2H),4.00(t,J=6.0Hz,2H),3.78(s,3H),3.59(br d,J=4.4Hz,2H),3.56(br d,J=4.4Hz,2H),3.51(br s,4H),2.89-2.64(m,6H),2.11-2.02(m,2H),1.46(s,9H)

[0256] Preparation of 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate [ka] A solution of 2-ethyl-5-methyl-pyrazole-3-carboxylic acid (6 g, 39.0 mmol, 1 equiv.) in DCE (60 mL) was added at 0° C. with N 2 Thionyl chloride, SOCl 2(23.0 g, 195 mmol, 14.0 mL, 5 equiv) was added and stirred for 12 h at 80° C. The mixture was concentrated to give 2-ethyl-5-methyl-pyrazole-3-carbonyl chloride (8 g, crude) as a colorless oil. 1 H NMR (CDCl 3 , 400MHz) δ6.91(s,1H),4.45(q,J=7.2Hz,2H),2.31(s,3H),1.41(t,J=7.2Hz,3H).

[0257] A solution of potassium thiocyanate (4.0 g, 41.7 mmol, 4.0 mL, 1.2 equiv.) in acetone (20 mL) was 2 2-Ethyl-5-methyl-pyrazole-3-carbonyl chloride (6 g, 34.8 mmol, 1 equiv) was added under 0° C. and stirred for 1 h at 0° C. The mixture was filtered and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 10 g SepaFlash® silica flash column, eluent of 0-40% ethyl acetate / petroleum ether gradient, 45 mL / min) to give 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate (5.5 g, 28.17 mmol, 81.04% yield) as a pale yellow oil. 1 H NMR (CDCl 3 , 400MHz) δ6.73(s,1H),4.50(q,J=7.2Hz,2H),2.30(s,3H),1.41(t,J=7.2Hz,3H).

[0258] Preparation of tert-butyl 4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, 3o A solution of 3n (500 mg, 798 umol, 1 eq.) and 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate (327 mg, 1.68 mmol, 2.1 eq.) in DMF (5 mL) was stirred at 0° C. for 30 min, then EtN (565 mg, 5.60 mmol, 777 uL, 7 eq.) and EDCI (765.0 mg, 4.0 mmol, 5 eq.) were added and the mixture was stirred at 15° C. for an additional 12 h. The residue was dissolved in NaHCO 3 (30 mL) of saturated aqueous solution, filtered, and the cake was 2 The crude product was washed with 20 mL of CH40 (10 mL × 3) and dried to obtain a crude product. The crude product was stirred at 20 °C for 20 min. 3 Trituration with CN gave 3o (500 mg, 527 umol, 66.04% yield) as a pale yellow solid. 1 H NMR(MeOD,400MHz)δ7.58(d,J=14.0Hz,2H),7.29-7.17(m,2H),6.63-6.52(m,2H),5.89-5.76(m,2H),5.00(br dd,J=4.4,8.8Hz,4H),4.64-4.55(m,4H),3.89(br t,J=5.2Hz,2H),3.71(s,3H),3.37-3.33(m,4H),2.39-2.33(m,2H),2.29-2 .26(m,4H),2.03(s,6H),1.78-1.67(m,2H),1.46(s,9H),1.38-1.32(m,6H).

[0259] Preparation of 3. To a solution of 3o (50 mg, 47.0 umol, 1 equiv, TFA) in EtOAc (10 mL) was added HCl / EtOAc (4 M, 11.0 mL), which was stirred for 1 h at 15° C. The mixture was concentrated and then lyophilized to give 3 (39 mg, 42.3 umol, 89.95% yield, 2HCl) as a pale yellow solid. 1H NMR(MeOD,400MHz)δ7.66(d,J=2.8Hz,2H),7.37(d,J=6.4Hz,2H),6.66(d,J=11.2Hz,2H),6.00(br d,J=14.8Hz,1H),5.88-5.78(m,1H),5.13(br d,J=6.4Hz,4H),4.69-4.59(m,4H),4.19-4.07(m,2H),3.84(s,3H),3.69(br s, 8H), 3.43-3.37 (m, 2H), 2.23 (s, 6H), 2.22-2.15 (m, 2H), 1.44-1.35 (m, 6H). HPLC: 95.099% (220nm). LCMS(ESI):C 42 H 52 N 14 O 6 Calculated mass 848.42, observed m / z 849.4 [M+H] + .

[0260] Example L-1: Synthesis of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (2,3,5,6-tetrafluorophenyl), BBI-L-1 [ka] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate To a mixture of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (5 g, 8.52 mmol, 1 equiv.) and (4-nitrophenyl)carbonochloridate (1.89 mg, 9.37 mmol, 1.1 equiv.) in DCM (100 mL) was added pyridine (1.01 mg, 12.8 mmol, 1.03 mL, 1.5 equiv.) at 0° C., then stirred for 2 h at 25° C. The mixture was poured into ice water (w / w=1 / 1) (100 mL), stirred for 10 min, and the pH of the mixture was adjusted to about 4 with HCl (1 M). The aqueous phase was extracted with DCM (50 mL x 2) and anhydrous Na 2 SO 4 The residue was purified by silica gel chromatography (column height 250 mm, diameter 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 1 / 0, 0 / 1, ethyl acetate / methanol = 1 / 0, 2 / 1) to give tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (3 g, 3.99 mmol, 46.82% yield) as a colorless oil. 1 H NMR(400MHz,MeOD)δ8.36-8.28(m,2H),7.54-7.46(m,2H),4.46-4.40(m,2H),3.83-3.77(m,2H),3.72-3.57(m,40H),1.45(s,9H)

[0261] [ka] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, L-1a (2E)-1-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-(3-piperazin-1-ylpropoxy)-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazole-5-carboxamide in MeOH (5 mL) A mixture of 3 (70 mg, 79.1 umol, 1 equiv, HCl) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (55.5 mg, 94.8 umol, 1.2 equiv) was added to the mixture of 3 (70 mg, 79.1 umol, 1 equiv, HCl) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (55.5 mg, 94.8 umol, 1.2 equiv) at 15°C with NaBH 3 CN (14.9 mg, 237 umol, 3 equiv) was added in one portion. The mixture was stirred at 15°C for 16 h. It was then concentrated under reduced pressure at 35°C. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-40%, 10 min) to give L-1a (80 mg, 56.43 umol, 71.38% yield) as a yellow solid.

[0262] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazin-1-yl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, L-1b H 2 To a mixture of L-1a (80 mg, 52.2 umol, 1 equiv, TFA) in O (2 mL) was added HCl (12 M, 65.3 uL, 15 equiv) at 15° C. The mixture was stirred at 80° C. for 1 h and then concentrated under reduced pressure at 50° C. to give L-1b (80 mg, crude, HCl) as a pale yellow oil.

[0263] Preparation of BBI-L-1 To a mixture of L-1b (68.1 mg, 50.07 umol, 1 eq, HCl) in DCM (0.3 mL) and DMA (0.03 mL) was added 2,3,5,6-tetrafluorophenol (66.5 mg, 400 umol, 8 eq) and EDCI (96.0 mg, 501 umol, 10 eq) in one portion at 15°C. The mixture was stirred at 15°C for 30 min. It was then concentrated under reduced pressure at 35°C. The residue was purified by preparative HPLC (column: Phenomenex Synergi C18 150*25*10 μm (micron); mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-50%, 8 min) to give BBI-L-1 (17.4 mg, 10.72 umol, 21.40% yield, TFA) as a pale yellow oil. 1H NMR(MeOD,400MHz)δ7.63(s,1H),7.58(s,1H),7.48-7.37(m,1H),7.33(s,1H),7.30(s,1H),6.65( s,1H),6.59(s,1H),5.87(s,2H),5.10-5.00(m,4H),4.74-4.55(m,4H),4.00(t,J=6.4Hz,2H),3.89 -3.82(m,4H),3.78(s,3H),3.73-3.56(m,42H),3.28-3.21(m,2H),3.15(,2H),2.97(t,J=6.0Hz,2 H),2.79(s,2H),2.63-2.55(m,2H),2.24(s,3H),2.22(s,3H),1.79-1.77(m2H),1.44-1.32(m,6H). HPLC: 96.08% (220nm). LCMS(ESI):C 71 H 96 F 4 N 14 O 16 Calculated mass 1508.70, observed m / z 1509.5 [M+H] + .

[0264] Example L-3: 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5 Synthesis of -methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, BBI-L-3 [ka] [ka] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, L-3a To a solution of (2E)-1-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-(3-piperazin-1-ylpropoxy)-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazole-5-carboxamide, 3 (180 mg, 203 umol, 1 equiv, HCl) in DMF (3.00 mL), was added Et 3 N (82.0 mg, 813 umol, 113 uL, 4 eq) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(3-tert-butoxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (2,3,5,6-tetrafluorophenyl) (171 mg, 223 umol, 1.1 eq) were added and then stirred at 0° C. for 1 hour. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-50%, 8 min) to give L-3a (280mg, 194umol, 95.3% yield) as a pale yellow solid.

[0265] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazin-1-yl]-3-oxo-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, L-3b H 2 To a solution of L-3a (280 mg, 194 umol, 1 equiv) in O (3.00 mL), HCl (12 M, 0.20 mL, 12.4 equiv) was added and then stirred at 80 °C for 1 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%-30%, 8 min) to give L-3b (100 mg, 66.51 umol, 34.34% yield, TFA) as a pale yellow solid.

[0266] Preparation of BBI-L-3 To a solution of L-3b (100 mg, 71.9 umol, 1 eq.) and (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxysodium (77.0 mg, 288 umol, 4 eq.) in DCM (2.00 mL) and DMA (0.10 mL), EDCI (55.0 mg, 288 umol, 4 eq.) was added and then stirred at 20°C for 1 h. The mixture was concentrated to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-40%, 8 min) to give BBI-L-3 (28 mg, 16.17 umol, 22.47% yield, TFA) as a pale yellow solid. 1H NMR(MeOD,400MHz)δ7.66-7.58(m,2H),7.28(br d,J=14.0Hz,2H),6.60(br d,J=12.0Hz,2H),5.85-5.78(m,2H),5.06-4.97(m,4H),4.70-4.51(m,4H),4.34-4.23(m,2H),4.05-3.98(m, 2H),3.86-3.80(m,2H),3.78-3.66(m,5H),3.64-3.47(m,38H),3.38-3.32(m,2H),3.18-3.07(m,2H),2.94(br t,J=5.6Hz,6H),2.57-2.45(m,2H),2.28-2.14(m,6H),2.10-2.03(m,2H),1.43-1.30(m,6H). HPLC: 96.21% (220nm), 97.95% (254nm). LCMS(ESI):C 72 H 96 F 4 N 14 O 22 Calculated mass of S: 1616.65, observed m / z 1617.5 [M+H] +

[0267] Example L-9: Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazin-1-yl]ethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyloxy]-2,3,5,6-tetrafluoro-benzenesulfonic acid, BBI-L-9 [ka] Preparation of N-[2-[4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazin-1-yl]ethyl]carbamate tert-butyl, L-9a To a solution of 1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-(3-piperazin-1-ylpropoxy)benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, 3 (530 mg, 496 umol, 1.0 equiv, 2HCl), and tert-butyl N-(2-oxoethyl)carbamate (395 mg, 2.48 mmol, 5.0 equiv) in MeOH (20 mL) was added acetic acid, AcOH (2.98 mg, 49.6 umol, 2.84 uL, 0.1 equiv), which was stirred at 20 °C for 0.5 h. Sodium cyanoborohydride, NaBH 3 CN (62.4 mg, 993 umol, 2.0 equiv) was added and the mixture was stirred for an additional hour at 20° C. The reaction mixture was diluted with H 2 The reaction was quenched by adding 20O (1 mL) and then saturated NaHCO 3 The pH of the mixture was adjusted to 8 by HCl. The aqueous phase was extracted with ethyl acetate (10 mL x 5). The combined organic phase was washed with brine (20 mL x 1) and anhydrous Na 2 SO 4 The crude product was dried over hexane, filtered and concentrated in vacuo. The crude product was triturated with MTBE at 20° C. for 10 min to give L-9a (600 mg, 450 umol, 90.60% yield) as a yellow solid.

[0268] Preparation of 1-[(E)-4-[7-[3-[4-(2-aminoethyl)piperazin-1-yl]propoxy]-5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, L-9b To a solution of L-9a (20 mg, 20.2 umol, 1.0 equiv) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 1 mL), then stirred at 20° C. for 1 h. The mixture was concentrated in vacuum. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 5%-35%, 8 min) to give L-9b (5 mg, 4.05 umol, 20.10% yield, 3 TFA) as a yellow solid. 1 H NMR(400MHz,MeOD)δ7.60(d,J=8.4Hz,2H),7.27(d,J=8.4Hz,2H),6.59(d,J=16.8Hz,2H),5.80(s,2H),5.06-5.00(m,4H),4.67-4.53(m,4H),4.05 -3.95(m,4H),3.75(s,3H),3.33-3.80(m,10H),2.75-2.70(m,2H),2.52- 2.45(m,2H),2.21(d,J=3.6Hz,6H),2.10-1.91(m,2H),1.40-1.32(m,6H). HPLC: 100.00% (220nm). LCMS(ESI):C 44 H 57 N 15 O 6 Calculated mass 891.46, observed m / z 892.4 [M+H] +

[0269] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazin-1-yl]ethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, L-9c A solution of L-9b (150 mg, 145 umol, 1 equiv, 4HCl) in DMF (2 mL) was added with Et 3 N (58.5 mg, 578 mmol, 80.5 uL, 4.0 equiv.) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4-nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (120 mg, 159 umol, 1.1 equiv.) were added. The mixture was stirred at 20° C. for 1 h. Water (5 mL) was added to the mixture and stirred for 10 min. The aqueous phase was extracted with DCM / i-PrOH=3 / 1 (5 mL×3). The combined organic phase was washed with anhydrous Na 2 SO 4 It was dried over ice, filtered and concentrated in vacuo to give L-9c (150 mg, 81.2 umol, 56.20% yield) as a yellow oil.

[0270] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazin-1-yl]ethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, L-9d MeCN (0.5 mL) and H 2 To a solution of L-9c (150mg, 81.2umol, 1.0eq) in 2mL of O was added TFA (92.6mg, 812umol, 60.1uL, 10eq), then stirred at 80℃ for 1h. The mixture was filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1%TFA)-ACN]; B%: 15%~40%, 8min) to give L-9d (100mg, 55.8umol, 68.7% yield, 3TFA) as a yellow oil. 1 H NMR(400MHz,MeOD)δ7.59(d,J=15.2Hz,2H),7.29(d,J=19.2Hz,2H),6.65-6.56(m,2H),5 .83(s,2H),5.02(s,4H),4.69-4.49(m,4H),4.21(s,2H),4.01-3.91(m,2H),3.75(s,3H) ,3.73-3.67(m,6H),3.64-3.57(m,36H),3.43-3.35(m,2H),2.98-2.92(m,4H),2.78-2.7 0(m,4H),2.53(t,J=6.4Hz,2H),2.26-2.16(m,6H),1.83-1.79(m,2H),1.39-1.32(m,6H).

[0271] Preparation of BBI-L-9 To a solution of L-9d (100 mg, 55.8 umol, 1.0 equiv, 3 TFA) in DCM (2 mL) and DMA (1 mL) was added (2,3,5,6-tetrafluoro-4-hydroxy-phenyl)sulfonyloxysodium (59.9 mg, 223 umol, 4.0 equiv) and EDCI (42.8 mg, 223 umol, 4.0 equiv). The mixture was stirred at 20°C for 1 h. The mixture was concentrated in vacuum. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-35%, 8 min) to give BBI-L-9 (43 mg, 22.6 umol, 40.42% yield, 2 TFA) as a yellow solid. 1 H NMR(400MHz,MeOD)δ7.59(d,J=18.8Hz,2H),7.26(d,J=22.8Hz,2H),6.57(d,J=22.4Hz,2H ),5.86-5.72(m,2H),5.02-4.95(m,4H),4.65-4.50(m,4H),4.19(s,2H),4.00-3.94(m,2H) ,3.84(t,J=5.6Hz,2H),3.75(s,3H),3.69-3.54(m,40H),3.48-3.42(m,4H),3.24-3.07(m ,6H),2.95(t,J=5.6Hz,2H),2.19(d,J=10.4Hz,6H),1.95-1.90(m,2H),1.39-1.30(m,6H). HPLC:100.00%(220nm), LCMS(ESI):C 74 H 101 F 4 N 15 O 23 Calculated mass for S 1675.69, observed m / z 1676.6 [M+H] +

[0272] Example L-13: 4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazine-1-carboxylic acid [4-[[(2S)-2 Synthesis of -[[(2S)-1-[(2S)-2-[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]ethoxy]ethoxy]propanoylamino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methyl-butanoyl]amino]phenyl]methyl, BBI-L-13 [ka] [ka]

[0273] Preparation of (S)-1-((9H-fluoren-9-yl)methyl) 2-(2,5-dioxopyrrolidin-1-yl)pyrrolidine-1,2-dicarboxylate, 13b To a solution of (2S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid, 13a (15 g, 44.5 mmol, 1.0 equiv.) in DCM (200 mL) was added 1-hydroxypyrrolidine-2,5-dione (5.12 g, 44.5 mmol, 1.0 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDCI, EDAC, EDC, CAS Registry Number 25952-53-8 (10.2 g, 53.44 mmol, 1.2 equiv.) and stirred at 20° C. for 12 h. The mixture was cooled to 30° C. with NaHCO 3 The organic layer was washed with a saturated aqueous solution of Na 2 SO 4Drying over and concentration gave 13b (17.5 g, 40.28 mmol, 90.60% yield) as a white solid.

[0274] Preparation of (2S)-2-[[(2S)-1-(9H-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carbonyl]amino]-3-methyl-butanoic acid, 13c A solution of (2S)-2-amino-3-methyl-butanoic acid, L-valine (4.95 g, 42.3 mmol, 1.05 equiv.) in THF (200 mL) was 2 NaHCO in O (50 mL) 3 (3.55 g, 42.3 mmol, 1.64 mL, 1.05 equiv) and 13b (17.5 g, 40.28 mmol, 1.0 equiv) were added and stirred at 20 °C for 12 h. The mixture was extracted with MTBE (2 × 100 mL) (discarded). The pH of the aqueous layer was adjusted to 5-6 with HCl (6 M) and extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with Na 2 SO 4 Drying over and concentration gave 13c (15 g, 34.36 mmol, 85.31% yield) as a white solid. 1 H NMR(MeOD,400MHz)δ7.80(d,J=7.2Hz,2H),7.70-7.54(m,2H),7.43-7.28(m,4H),4.49-4 .15(m,5H),3.69-3.38(m,2H),2.42-2.01(m,3H),2.00-1.82(m,2H),1.01-0.86(m,6H).

[0275] Preparation of 9H-fluoren-9-ylmethyl(2S)-2-[[(1S)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]-2-methyl-propyl]carbamoyl]pyrrolidine-1-carboxylate, 13d To a solution of 13c (10 g, 22.9 mmol, 1.0 equiv) and (4-aminophenyl)methanol (4.23 g, 34.4 mmol, 1.5 equiv) in MeOH (80 mL) and DCM (80 mL) was added 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, EEDQ, CAS Registry Number 16357-59-8 (8.50 g, 34.36 mmol, 1.5 equiv) and then stirred for 12 h at 20° C. The mixture was concentrated in vacuo to give a residue which was purified by flash silica gel chromatography (silica flash column, eluent of 0-40% ethyl acetate / MeOH, 65 mL / min) to give 13d (13 g, 24.0 mmol, 52.38% yield) as a yellow solid. 1 H NMR(MeOD,400MHz)δ7.85-7.71(m,2H),7.68-7.48(m,3H),7.47-7.16(m,7H),4.53(d,J=15.2Hz,2H),4.49-4.41(m,1H),4.40-4.33(m,2) H),4.32-4.27(m,1H),4.26-4.17(m,1H),4.16-4.07(m,1H),3.69-3.38(m,2H),2.40-2.05(m,2H),1.99-1.82(m,2H),1.08-0.88(m,6H).

[0276] Preparation of (2S)-N-[(1S)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]-2-methyl-propyl]pyrrolidine-2-carboxamide, 13e To a solution of 13d (13 g, 24.0 mmol, 1.0 equiv) in DCM (130 mL) was added piperidine (10.22 g, 120 mmol, 11.85 mL, 5.0 equiv) and then stirred for 2 h at 20° C. The mixture was concentrated to give a residue, which was triturated with EtOAc at 20° C. for 20 min to give 7e (8 g, crude) as a white solid.

[0277] Preparation of 9H-fluoren-9-ylmethyl N-[(1S)-2-[(2S)-2-[[(1S)-1-[[4-(hydroxymethyl)phenyl]carbamoyl]-2-methyl-propyl]carbamoyl]pyrrolidin-1-yl]-1-methyl-2-oxo-ethyl]carbamate, 13f To a solution of (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (3.95 g, 12.68 mmol, 1.5 equiv.) in DMF (30 mL), 1-bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, azabenzotriazole tetramethyluronium hexafluorophosphate, HATU, CAS Registry Number 148893-10-1 (4.82 g, 12.68 mmol, 1.5 equiv.) and DIPEA (3.28 g, 25.36 mmol, 4.42 mL, 3 equiv.) were added at 0° C. After addition, the mixture was stirred at this temperature for 5 min, then 13e (2.7 g, 8.45 mmol, 1 equiv.) was added at 0° C., and the resulting mixture was stirred at 0° C. for 25 min. The reaction mixture was 2 The reaction was quenched by adding O (150 mL) and then extracted with EtOAc (70 mL x 3). The combined organic layers were washed with brine (50 mL x 3) and Na 2 SO 4 The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:0 to 0:1), then (SiO 2 , EtOAc:MeOH=1:0-10:1) to give 13f (2.94 g, 4.80 mmol, 56.76% yield) as an off-white solid. 1 H NMR (MeOD-d 4, 400MHz) δ7.79(d,J=7.6Hz,2H),7.66(t,J=6.4Hz,2H),7.54(d,J=8.4Hz,2H),7.3 9(t,J=7.2Hz,2H),7.35-7.26(m,4H),4.59-4.51(m,3H),4.50-4.40(m,1H),4.39-4 .30(m,2H),4.29-4.18(m,2H),3.83-3.71(m,1H),3.68-3.63(m,1H),2.31-2.09(m ,2H),2.07-1.91(m,3H),1.36(dd,J=6.4,9.6,Hz,4H),1.03(dd,J=4.0,6.8Hz,6H).

[0278] Preparation of [4-[[(2S)-2-[[(2S)-1-[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoyl]pyrrolidine-2-carbonyl]amino]-3-methyl-butanoyl]amino]phenyl]methyl(4-nitrophenyl)carbonate, 13 g To a solution of 13f (2.4 g, 3.92 mmol, 1 equiv.) in DMF (20 mL) was added bis(4-nitrophenyl)carbonate (2.38 g, 7.83 mmol, 2 equiv.) and DIPEA (1.01 g, 7.83 mmol, 1.36 mL, 2 equiv.), then stirred at 20° C. for 1 h. The reaction mixture was cooled to 0° C. and cooled to 10° C. 2 The reaction was quenched by adding O (100 mL) and then extracted with EtOAc (80 mL x 3). The combined organic layers were washed with brine (50 mL x 3) and Na 2 SO 4 The residue was purified by column chromatography (SiO 2 The residue was purified by elution with 2,4-dichloromethane (petroleum ether:ethyl acetate) = 1:0 to 0:1 to give 13g (2.7 g, 3.47 mmol, 88.62% yield) as a white solid. 1 H NMR (CDCl 3,400MHz)δ8.32-8.22(m,3H),7.78(d,J=7.6Hz,2H),7.67(br d,J=8.4Hz,2H),7.60(br d,J=7.6Hz,2H),7.46-7.29(m,8H),7.18(br d,J=8.4Hz,1H),5.60(br d,J=7.6Hz,1H),5.25(s,2H),4.70-4.66(m,1H),4.62-4.52(m,1H),4.46-4.28(m,3H),4.28-4.19(m,1H),3.81- 3.68(m,1H),3.62-3.58(m,1H),2.48-2.29(m,2H),2.19-1.98(m,3H),1.41(d,J=7.2Hz,3H),1.10-0.94(m,6H). LC / MS[M+H]778.3 (calculated value); LC / MS[M+H]778.2 (observed value).

[0279] Preparation of tert-butyl 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]propanoate, 13i To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 13h (11.3 g, 19.3 mmol, 1 equiv), 2-(2,5-dioxopyrrol-1-yl)acetic acid (3 g, 19.3 mmol, 1 equiv) and DIPEA (10.0 g, 77.4 mmol, 13.5 mL, 4 equiv) in DCM (100 mL) was added HATU (8.09 g, 21.3 mmol, 1.1 equiv) at 0° C., and then the mixture was stirred at 0° C. for 30 min. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column: Phenomenex luna c18 250mm*100mm*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%~55%, 25 min) to give 13i (4.5g, 6.23mmol, 32.2% yield) as a yellow oil. 1 H NMR (CDCl 3,400MHz)δ6.88-6.80(m,1H),6.78(s,2H),4.22(s,2H),3.77-3.54(m,40H),3.47(q,J=5.2Hz,2H),2.51(t,J=6.4Hz,2H),1.46(s,9H).

[0280] Preparation of 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]propanoic acid, 13j CH 3 CN (25 mL) and H 2 To a solution of 13i (4.5 g, 6.23 mmol, 1 equiv) in 2O (25 mL) was added TFA (5.68 g, 49.8 mmol, 3.69 mL, 8 equiv) and then stirred at 80° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give CH 3 The CN was removed. The residue was extracted with MTBE (10 mL × 3) and disposed of. The aqueous phase was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (TFA condition; column: Phenomenex Luna c18 250 mm × 100 mm × 10 μm; mobile phase: [water (0.1% TFA)-ACN]; B%: 0%~25%, 24 min) to give 13j (1.6 g, 2.40 mmol, 38.6% yield) as a pale yellow oil. 1 H NMR (CDCl 3 ,400MHz)δ6.95(br s,1H),6.78(s,2H),4.22(s,2H),3.78(t,J=6.4Hz,2H),3.70-3.63(m,36H),3.60-3.54(m,2H),3.46(q,J=5.2Hz,2H),2.61(t,J=6.0Hz,2H). LCMS(ESI):C 42 H 43 N 5 O 10 Calculated mass 667.3, observed m / z 667.2 [M+H] +

[0281] Preparation of 4-[3-[[(2E)-6-carbamoyl-3-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-1H-benzimidazol-4-yl]oxy]propyl]piperazine-1-carboxylate [4-[[(2S)-2-[[(2S)-1-[(2S)-2-aminopropanoyl]pyrrolidine-2-carbonyl]amino]-3-methyl-butanoyl]amino]phenyl]methyl, 13l (2E)-1-[(E)-4-[(2E)-5-carbamoyl-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-(3-piperazin-1-ylpropoxy)-3H-benzimidazol-1-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-7-methoxy-3H-benzimidazole-5-carb To a solution of oxamide 13k (150 mg, 151 umol, 1 equiv, 4HCl) and 13g (129 mg, 166 umol, 1.1 equiv) in DMF (3.00 mL) was added DIPEA (97.0 mg, 754 umol, 131 uL, 5 equiv) at 20° C., the mixture was stirred at this temperature for 2 hours, and then piperidine (39.0 mg, 452.35 umol, 45.0 uL, 3 equiv) was added. The mixture was stirred at 20° C. for another 2 hours. The mixture was filtered and the residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%~40%, 8 min) to give 13l (90mg, 60.26umol, yield 39.97%, 2TFA) as a pale yellow solid. 1H NMR(MeOD,400MHz)δ7.67-7.53(m,4H),7.37(d,J=8.4Hz,2H),7.25(dd,J=1.2 ,14.0Hz,2H),6.60(d,J=7.6Hz,2H),5.85-5.68(m,2H),5.14(s,2H),5.00(br s,5H),4.64-4.54(m,4H),4.33-4.18(m,2H),3.95(br t,J=6.0Hz,2H),3.74-3.58(m,5H),3.25-3.13(m,4H),2.21(s,3H),2.19(s,3H) ,2.16-1.92(m,6H),1.52(d,J=7.2Hz,3H),1.40-1.29(m,6H),1.08-0.98(m,6H).

[0282] Preparation of BBI-L-13 To a solution of 13l (50 mg, 33.5 umol, 1 equiv, 2 TFA) and 7j (22.0 mg, 33.5 umol, 1 equiv) in DMF (1.00 mL) was added Et 3 N (7.00 mg, 66.9 umol, 9.00 uL, 2 eq.) and 1-propanephosphonic anhydride, T 3 P, CAS Registry Number 68957-94-8 (32.0mg, 50.2umol, 30.0uL, 50% purity, 1.5eq) was added and then stirred at 20°C for 2h. The mixture was filtered and the residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1%TFA)-ACN]; B%: 15%~40%, 8min) to give BBI-L-13 (16mg, 7.47umol, 22.31% yield, 2TFA) as a white solid. 1H NMR(MeOD,400MHz)δ7.68-7.53(m,4H),7.37(d,J=8.4Hz,2H),7.30-7.20(m,2H),6.88(s,2H),6.60(d,J=12Hz,2H),5.78(br s,2H),5.14(s,2H),4.99(br s,4H),4.68-4.47(m,8H),4.29-4.25(m,1H),4.16(s,3H),3.96(br t,J=5.6Hz,2H),3.80(td,J=6.8,9.2Hz,2H),3.74-3.71(m,2H),3.70(s,4H),3.67-3.56(m,36H),3.53(br t,J=5.6Hz,2H),3.48(td,J=1.6,3.2Hz,1H),3.36(br t,J=5.2Hz,2H),3.23-3.13(m,4H),2.54-2.43(m,2H),2.20(d,J=10.4Hz,6H),2.1 8-2.10(m,2H),2.06-1.94(m,5H),1.40-1.28(m,9H),1.02(dd,J=5.2,6.4Hz,6H). LCMS(ESI):C 42 H 43 N 5 O 10 Calculated mass 1913.9, observed m / z 1914.0 [M+H] + .

[0283] Example L-18: Synthesis of 1-[(E)-4-[5-carbamoyl-7-[3-[4-[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]piperazin-1-yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-3a,7a-dihydrobenzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, BBI-L-18 [ka] 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (68.9 mg, 108 umol, 2 eq.) and 1-[(E)-4-[5-carbamoyl-2-[(2 -ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-(3-piperazin-1-ylpropoxy)-3a,7a-dihydrobenzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, 3 (50 mg, 54.1 umol, 1 equiv, 2HCl) to a mixture of N 2 Sodium cyanoborohydride, NaBH 3 CN (6.8mg, 108umol, 2eq) was added and then stirred at 25℃ for 13h. The mixture was concentrated in vacuum to give a residue. The residue was purified by preparative HPLC column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (0.1%TFA)-ACN]; B%: 15%~40%, 8min to give BBI-L-18 (15mg, 9.46umol, 17.48% yield, TFA) as a pale yellow oil. 1H NMR(400MHz,MeOD)δ7.61(d,J=1.2Hz,1H),7.57(d,J=1.2Hz,1H),7.31(d,J=1.2Hz,1H) ,7.28(d,J=1.2Hz,1H),6.87(s,2H),6.63(s,1H),6.57(s,1H),5.88-5.83(m,2H),5.08 -5.01(m,4H),4.54-4.69(m,4H),4.16(s,2H),4,01-3.95(m,2H),3.80-3.88(m,2H),3. 55-3.72(m,38H),3.52(t,J=5.5Hz,2H),3.38-3.30(m,6H),2.69-2.93(m,4H),2.60(br t,J=7.2Hz,2H),2.21(d,J=8.8Hz,6H),1.71-1.82(m,2H),1.40-1.32(m,6H). HPLC: 99.67% (220nm). LC / MS [M+H] 1469.7 (calculated), LC / MS [M+H] 1469.8 (observed).

[0284] Example L-24: Synthesis of 1-[4-[5-carbamoyl-7-[3-[4-[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]piperazin-1-yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]butyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, BBI-L-24 [ka] [ka] Preparation of tert-butyl 4-[3-[6-carbamoyl-3-[4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]butyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, L-24a A solution of tert-butyl 4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, 3o (0.3 g, 316.10 umol, 1 equiv.) in EtOAc (20 mL) was added with N 2 Under the condition of Pd(OH) 2 10% by weight, 0.1 g of 1000 ml ... 2 Purge several times with H 2 (50 psi) at 20° C. for 12 h. The reaction mixture was filtered and concentrated under reduced pressure to give L-24a (0.2 g, 210.29 umol, 66.53% yield) as a black solid. LC / MS [M+H] 951.5 (calculated); LC / MS [M+H] 951.4 (observed).

[0285] Preparation of 1-[4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-(3-piperazin-1-ylpropoxy)benzimidazol-1-yl]butyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, L-24b To a solution of L-24a (0.2 g, 210.29 umol, 1 equiv) in EtOAc (2 mL) was added HCl / EtOAc (4 M, 10.00 mL, 190 equiv). The mixture was stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 10%~40%, 8 min) to give L-24b (0.1 g, 117.51 ​​umol, 55.88% yield) as a white solid. 1 H NMR (400 MHZ, DMSO-d 6 )δ7.32-7.27(m,2H),7.22(d,J=8.0Hz,2H),6.68(s,2H),4.73-4.63(m,4H),4.43-4.34(m,4H),4.13-4.04(m,2H),3.83(s,3H),3.26 -3.18(m, 4H), 2.77-2.63(m, 4H), 2.60-2.51(m, 2H), 2.24(d, J=2.4Hz, 6H), 2.07-1.96(m, 4H), 1.94-1.76(m, 2H), 1.46-1.35(m, 6H). HPLC: 96.010% (220nm), 96.753% (254nm). LC / MS [M+H] 851.4 (calculated); LC / MS [M+H] 851.4 (observed).

[0286] Preparation of BBI-L-24 To a solution of L-24b (80.0 mg, 74.1 umol, 1 eq, 2 TFA) in MeOH (5 mL) was added 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (142 mg, 222 umol, 3 eq) at 15° C. After addition, the mixture was stirred at this temperature for 30 min and then added NaBH 3CN (14.0 mg, 222 umol, 3 equiv.) was added. The resulting mixture was stirred at 15° C. for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80*30 mm*3 um, mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8 min) to give BBI-L-24 (40.1 mg, 23.6 umol, 31.8% yield, 2 TFA) as a white solid. 1 H NMR(400MHz,MeOD)δ7.36-7.29(m,2H),7.23(d,J=5.2Hz,2H),6.88(s,2H), 6.68(s,2H),4.67(q,J=6.0Hz,4H),4.45-4.31(m,4H),4.16(s,2H),4.10(br t,J=6.0Hz,2H),3.89-3.77(m,5H),3.72-3.66(m,4H),3.66-3.55(m,32H),3.52(t,J=5.6Hz,2H),3.47-3.3 3(m,8H),3.03-2.90(m,4H),2.84-2.72(m,2H),2.23(d,J=2.0Hz,6H),2.08-1.87(m,6H),1.49-1.34(m,6H). HPLC: 97.38% (220nm). LC / MS[M+H]1471.7 (calculated value), LC / MS[M+H]1471.7 (observed value).

[0287] Example L-26: Synthesis of 7-[3-[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]ethyl]piperazin-1-yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-1-[(E)-4-[2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]benzimidazole-5-carboxamide, BBI-L-26 [ka] [ka] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-(2-nitroanilino)but-2-enyl]carbamate, L-26a To a solution of 1-fluoro-2-nitro-benzene (1 g, 7.09 mmol, 746 uL, 1 equiv) in DMF (10 mL) was added DIEA (2.75 g, 21.26 mmol, 3.7 mL, 3 equiv) and tert-butyl N-[(E)-4-aminobut-2-enyl]-N-tert-butoxycarbonyl-carbamate, 3j (2.23 g, 7.80 mmol, 1.1 equiv), then stirred at 50° C. for 16 h. The reaction mixture was cooled to 20° C. with H 2 The reaction was quenched by adding O (100 mL) and then extracted with EtOAc (50 mL x 3). The combined organic layers were washed with H 2 20 mL) and brine (20 mL). 2 SO 4 The mixture was dried over ice, filtered, and concentrated under reduced pressure to give a residue which was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent of 0-5% ethyl acetate / petroleum ether gradient, 100 mL / min) to give L-26a (2 g, 4.91 mmol, 69.26% yield) as a brown solid. 1 H NMR(400MHz,MeOD)δ8.12(dd,J=1.6,8.8Hz,1H),7.46(td,J=1.6,7.2,8.8Hz,1H),6.96(d,J=8.8Hz,1H),6. 67(d,J=1.6,7.2,8.8Hz,1H),5.80-5.72(m,2H),4.17(d,J=4.0Hz,2H),4.03(d,J=4.0Hz,2H),1.45(s,18H). LC / MS[M+H]408.2 (calculated value), LC / MS[M+H]408.2 (observed value).

[0288] Preparation of (E)-N'-(2-nitrophenyl)but-2-ene-1,4-diamine, L-26b To a solution of L-26b (500 mg, 1.23 mmol, 1 equiv) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 15.34 mL, 50 equiv). The mixture was stirred at 20° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give L-26b (500 mg, crude) as a white solid. 1 H NMR(400MHz,MeOD)δ8.14(dd,J=1.6,8.4Hz,1H),7.49(td,J=1.6,7.2,8.4Hz,1H),6.97(d,J=8.0Hz,1H),6.70(td,J=1.6,7.2,8.4Hz,1H),6.14-6.02(m ,1H),5.86-5.70(m,1H),4.11(dd,J=1.2,4.4Hz,2H),3.57(d,J=6.4Hz,2H). LC / MS[M+H]208.1 (calculated value), LC / MS[M+H]208.1 (observed value).

[0289] Preparation of tert-butyl 4-[3-[5-carbamoyl-3-nitro-2-[[(E)-4-(2-nitroanilino)but-2-enyl]amino]phenoxy]propyl]piperazine-1-carboxylate, L-26c L-26b (0.5g, 1.78mmol, 1eq, 2HCl), DIEA (1.15g, 8.92mmol, 1.55mL, 5eq) and NaHCO 3 A solution of (375 mg, 4.46 mmol, 174 uL, 2.5 equiv) in n-BuOH (10 mL) was stirred at 15° C. for 20 min. Then tert-butyl 4-[3-(5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl]piperazine-1-carboxylate, 3 g (632 mg, 1.43 mmol, 0.8 equiv) was added. The mixture was stirred at 120° C. for 16 h. The reaction mixture was concentrated under reduced pressure to remove n-BuOH, and the residue was diluted with H at 20° C. 2 The reaction was quenched by adding O (50 mL) and then extracted with EtOAc (10 mL x 2). The combined organic layers were washed with brine (10 mL) and diluted with Na 2 SO 4Drying over ice, filtration and concentration under reduced pressure gave L-26c (550 mg, 896.26 umol, 50.22% yield) as a yellow solid. LC / MS [M+H] 614.3 (calculated); LC / MS [M+H] 614.2 (observed).

[0290] Preparation of tert-butyl 4-[3-[3-amino-2-[[(E)-4-(2-aminoanilino)but-2-enyl]amino]-5-carbamoyl-phenoxy]propyl]piperazine-1-carboxylate, L-26d MeOH (1 mL) and THF (1 mL) and H 2 To a solution of L-26c (550 mg, 896 umol, 1 equiv) in O (0.5 mL), 2 CO 3 (570 mg, 5.38 mmol, 6 equiv) and disodium dithionite (1.56 g, 8.96 mmol, 1.95 mL, 10 equiv) were added and then stirred at 20° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated to remove THF and MeOH. The residue was purified by H 2 Dilution with 2H2O (10 mL) and filtration of the precipitate afforded L-26d (550 mg, crude) as a yellow solid: LC / MS [M+H] 554.3 (calculated), LC / MS [M+H] 554.3 (observed).

[0291] Preparation of tert-butyl 4-[3-[6-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-3-[(E)-4-[2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, L-26e A solution of L-26d (550 mg, 993 umol, 1 equiv.) and 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate, 3o (213 mg, 1.09 mmol, 1.1 equiv.) in DMF (5 mL) was stirred at 0° C. for 10 min, and then added with Et 3N (301 mg, 2.98 mmol, 414 uL, 3 equiv.) and EDCI (571 mg, 2.98 mmol, 3 equiv.) were added. The resulting mixture was stirred at 20° C. for 16 h. The reaction mixture was diluted with saturated sodium bicarbonate (10 mL) and H 2 The reaction was quenched by adding O (10 mL) and the precipitate was filtered to give L-26e (560 mg, 639 umol, 64.36% yield) as a white solid. LC / MS [M+H] 876.5 (calculated); LC / MS [M+H] 876.4 (observed).

[0292] Preparation of 2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-1-[(E)-4-[2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-7-(3-piperazin-1-ylpropoxy)benzimidazole-5-carboxamide, L-26f To a solution of L-26e (560 mg, 639 umol, 1 equiv) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 7.99 mL, 50 equiv), then stirred at 20° C. for 2 h. The reaction mixture was concentrated and the residue was purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 5%-40%, 8 min) to give L-26e (96.7 mg, 124.63 umol, 19.50% yield) as a white solid. 1H NMR(400MHz,MeOD)δ7.57(d,J=1.2Hz,1H),7.48(d,J=7.6Hz,1H),7.35-7.24(m,3H),7.22-7.16(m,1H), 6.63(s,1H),6.57(s,1H),6.00-5.90(m,1H),5.84-5.74(m,1H),5.06(d,J=5.2Hz,2H),4.83-4.79(m,2H) ),4.69-4.54(m,4H),4.04(t,J=6.4Hz,2H),3.33(s,2H),3.27-3.23(m,4H),2.86-2.69(m,4H),2.61(t, J=7.2Hz,2H),2.23(s,3H),2.21(s,3H),1.88-1.77(m,2H),1.38(t,J=7.2Hz,3H),1.33(t,J=7.2Hz,3H). LC / MS[M+H]776.4 (calculated value); LC / MS[M+H]776.4 (observed value).

[0293] Preparation of BBI-L-26 A solution of L-26e (30 mg, 29.9 umol, 1 equiv, 2 TFA) and 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (47.6 mg, 74.7 umol, 2.5 equiv) in MeOH (3 mL) was stirred at 20 °C for 10 min, followed by addition of NaBH 3 CN (5.63 mg, 89.6 umol, 3 equiv.) was added. The mixture was stirred at 20° C. for another 0.5 h. The reaction 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 BBI-L-26 (14.9 mg, 10.67 umol, 35.70% yield) as a white solid. 1H NMR(400MHz,MeOD)δ7.57(d,J=1.2Hz,1H),7.48(d,J=8.0Hz,1H),7.35-7.30(m,2H),7.27(t,J=7.6Hz,1H),7.22-7.16(m,1H),6 .86(s,2H),6.62(s,1H),6.56(s,1H),6.05-5.87(m,1H),5.86-5.70(m,1H),5.06(d,J=4.8Hz,2H),4.65-4.54(m,4H),4.15(s,2H) ),4.05(t,J=6.0Hz,2H),3.86-3.78(m,2H),3.67(d,J=2.8Hz,4H),3.64-3.54(m,34H),3.51(t,J=5.6Hz,4H),3.37-3.33(m,4H), 3.21-2.75(m,6H),2.66(t,J=6.8Hz,2H),2.21(d,J=8.0Hz,6H),1.88-1.78(m,2H),1.37(t,J=7.2Hz,3H),1.32(t,J=7.2Hz,3H). LC / MS[M+H] 1396.7 (calculated value); LC / MS[M+H] 1396.7 (observed value).

[0294] Example L-28: 1-[(E)-4-[5-carbamoyl-7-[3-[4-[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]piperazine-1 -yl]propoxy]-2-[[2-(2-hydroxyethyl)-5-methyl-pyrazole-3-carbonyl]amino]benzimidazol-1-yl]but-2-enyl]-2-[[2-(2-hydroxyethyl)-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazole-5-carboxamide, BBI-L-28 [ka] [ka] Preparation of ethyl 2-(2-acetoxyethyl)-5-methyl-pyrazole-3-carboxylate, L-28b To a solution of ethyl 3-methyl-1H-pyrazole-5-carboxylate, L-28a (3 g, 19.5 mmol, 1 equiv.) and 2-hydroxyethyl acetate (2.03 g, 19.5 mmol, 1.83 mL, 1 equiv.) in THF (30 mL) was added triphenylphosphine, PPh 3 (15.3 g, 58.4 mmol, 3 equiv.) and diisopropyl azo-dicarboxylate, DIAD (11.80 g, 58.4 mmol, 11.4 mL, 3 equiv.) were added. The mixture was stirred at 0° C. for 2 h. The reaction mixture was diluted with H 2 The reaction was quenched by adding O (50 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (100 mL) and Na 2 SO 4 The mixture was dried over ice, filtered, and concentrated under reduced pressure to give a residue that was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent of 0-50% ethyl acetate / petroleum ether gradient, 60 mL / min) to give L-28b (5 g, crude) as a colorless oil. 1 H NMR (400MHz, CDCl3) δ6.63(s,1H),4.75(t,J=5.4Hz,2H),4.41(t,J=5.4Hz,2H),4.32(q,J=7.2Hz,2H),2.27(s,3H),1.99(s,3H),1.36(t,J=7.2Hz,3H).

[0295] Preparation of 2-(2-hydroxyethyl)-5-methyl-pyrazole-3-carboxylic acid, L-28c MeOH (30 mL) and H 2 A solution of L-28b (5 g, 20.8 mmol, 1 equiv.) in HO (5 mL) was added to LiOH. 2 O (2.62 g, 62.4 mmol, 3 equiv.) was added and then stirred for 2 h at 20° C. The pH of the reaction mixture was adjusted to about 1 with HCl (4 M) at 0° C., and the desired solid was precipitated from the mixture and filtered to give L-28c (3 g, 17.6 mmol, 84.71% yield) as a white solid.1 H NMR (400MHz, DMSO-d 6 )δ8.87(s,1H),6.57(s,1H),4.46(t,J=6.4Hz,2H),3.66(t,J=6.4Hz,2H),2.16(s,3H).

[0296] Preparation of 2-(2-acetoxyethyl)-5-methyl-pyrazole-3-carboxylic acid, L-28d To a solution of L-28c (3 g, 17.6 mmol, 1 equiv) in DCM (50 mL) was added acetic anhydride (3.60 g, 35.3 mmol, 3.30 mL, 2 equiv) and pyridine (2.79 g, 35.3 mmol, 2.85 mL, 2 equiv). The mixture was stirred at 20° C. for 1 h. The pH of the reaction mixture was adjusted to approximately 1 with HCl (4 M) at 0° C., and the desired solid was precipitated from the mixture and filtered to give L-28d (3 g, 14.14 mmol, 80.19% yield) as a white solid. 1 H NMR (400MHz, DMSO-d 6 )δ8.88(s,1H),6.60(s,1H),4.64(t,J=5.2Hz,2H),4.30(t,J=5.2Hz,2H),2.17(s,3H),1.92(s,3H).

[0297] Preparation of 2-(5-chlorocarbonyl-3-methyl-pyrazol-1-yl)ethyl acetate, L-28e A solution of L-28d (1 g, 4.71 mmol, 1 equiv.) in DCM (10 mL) was diluted with DMF (344 mg, 4.71 mmol, 363 uL, 1 equiv.) and oxalyl dichloride, (COCl) 2 (1.20 g, 9.42 mmol, 825 uL, 2 equiv) was added and then stirred for 1 h at 20° C. The reaction mixture was concentrated under reduced pressure to give L-28e as a yellow oil.

[0298] Preparation of 2-(5-carbonylisothiocyanatidoyl-3-methyl-pyrazol-1-yl)ethyl acetate, L-28f A solution of L-28e (1 g, 4.34 mmol, 1 equiv) and potassium thiocyanate, KSCN (843 mg, 8.67 mmol, 843 uL, 2 equiv) in MeCN (5 mL) was stirred at 20 °C for 1 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent of 0-30% ethyl acetate / petroleum ether gradient, 45 mL / min) to give L-28f (0.4 g, 1.58 mmol, 36.43% yield) as a yellow oil.

[0299] Preparation of tert-butyl 4-[3-[2-[[2-(2-acetoxyethyl)-5-methyl-pyrazole-3-carbonyl]amino]-3-[(E)-4-[2-[[2-(2-acetoxyethyl)-5-methyl-pyrazole-3-carbonyl]amino]-5-carbamoyl-7-methoxy-benzimidazol-1-yl]but-2-enyl]-6-carbamoyl-benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, L-28g A solution of tert-butyl 4-[3-[3-amino-2-[[(E)-4-(2-amino-4-carbamoyl-6-methoxy-anilino)but-2-enyl]amino]-5-carbamoyl-phenoxy]propyl]piperazine-1-carboxylate, 3n (0.3 g, 479 umol, 1 eq.) and L-28f (242 mg, 957 umol, 2 eq.) in DMF (5 mL) was stirred at 0° C. for 0.5 h. Then, EDCI (459 mg, 2.39 mmol, 5 eq.) and Et 3 N (242 mg, 2.39 mmol, 333 uL, 5 equiv) was added. The resulting mixture was stirred at 25° C. for an additional 12 h. The reaction mixture was added with 10 mL of NaHCO at 0° C. 3 The reaction was quenched by adding aqueous solution and the desired solid product was precipitated from the mixture and filtered to give L-28g (0.4 g, 375 umol, 78.46% yield) as a yellow solid.

[0300] Preparation of 1-[(E)-4-[5-carbamoyl-2-[[2-(2-hydroxyethyl)-5-methyl-pyrazole-3-carbonyl]amino]-7-(3-piperazin-1-ylpropoxy)benzimidazol-1-yl]but-2-enyl]-2-[[2-(2-hydroxyethyl)-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazole-5-carboxamide, L-28h To a solution of L-28g (0.4 mg, 375 umol, 1 eq) in EtOAc (10 mL) was added HCl / EtOAc (4 M, 5.0 mL, 53.3 eq), then stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; B%: 5%-35%, 8 min) to give L-28h (0.05 g, 56.8 umol, 15.11% yield) as a white solid. 1 H NMR (400MHz, DMSO-d 6 )δ13.06-12.67(m,2H),8.95-8.44(m,2H),7.96(d,J=9.6Hz,3H),7.65(s,3H),7.36(s ,2H),7.28(d,J=11.6Hz,2H),6.55(d,J=7.2Hz,2H),5.85-5.80(m,2H),4.91(dd,J=3.6 ,9.6Hz,4H),4.61(d,J=2.8Hz,4H),3.95(d,J=5.2Hz,2H),3.72(t,J=6.4Hz,4H),3.67( s, 3H), 3.15-3.10 (m, 4H), 2.58-2.50 (m, 6H), 2.12 (d, J=5.2Hz, 6H), 1.75-1.70 (m, 2H). HPLC: 98.124% (220nm), 98.808% (254nm). LC / MS[M+H]881.4 (calculated value); LC / MS[M+H]881.4 (observed value).

[0301] Preparation of BBI-L-28 A solution of L-28h (35 mg, 31.6 umol, 1 equiv, 2 TFA) and 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (40.2 mg, 63.1 equiv, 2 equiv) in MeOH (1 mL) was diluted with sodium cyanoborohydride, NaBH 3 CN (5.95mg, 94.7umol, 3eq) was added and then stirred at 20℃ for 12h. The reaction mixture was filtered. The residue was purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 5%~35%, 8min) to give BBI-L-28 as a white solid. 1 H NMR (400MHz, DMSO-d 6 )δ13.01-12.71(m,1H),8.21(t,J=4.6Hz,1H),7.96(d,J=11.8Hz,2H),7.65(s,2H),7.37(s,2H) ,7.29(s,1H),7.25(s,1H),7.08(s,2H),6.55(d,J=7.6Hz,2H),5.98-5.68(m,2H),4.93-4.90(4, 3H),4.63-4.59(m,4H),4.01(s,2H),3.95-3.90(m,2H),3.72(t,J=6.4Hz,4H),3.66(s,3H),3.5 5-3.45(m,38H),3.44-3.40(m,6H),3.26-3.14(m,6H),2.12(d,J=6.8Hz,6H),1.80-1.60(m,2H). HPLC: 96.878% (220nm), 99.512% (254nm). LC / MS[M+H]1501.7 (calculated value), LC / MS[M+H]1501.8 (observed value).

[0302] Example L-31: Synthesis of 1-[(E)-4-[5-carbamoyl-7-[3-[4-[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]piperazin-1-yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-2-[[2-(2,3-dihydroxypropyl)-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazole-5-carboxamide, BBI-L-31 [ka] [ka] [ka] Preparation of ethyl 2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carboxylate, L-31b To a solution of ethyl 3-methyl-1H-pyrazole-5-carboxylate, L-31a (5 g, 32.43 mmol, 1 equiv.) in THF (100 mL), PPh 3 (25.52 g, 97.30 mmol, 3 equiv) and (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (5.14 g, 38.92 mmol, 4.8 mL, 1.2 equiv) were added, followed by DEAD (11.30 g, 64.87 mmol, 11.8 mL, 2 equiv). The mixture was stirred at 15° C. for 16 h. The reaction of the mixture was cooled to 10° C. for 2 h. 2 The mixture was quenched with O (300 mL) and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine (100 mL) and 2 SO 4The mixture was dried over ice, filtered, and concentrated under reduced pressure to give a residue that was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® silica flash column, eluent of 0-8% ethyl acetate / petroleum ether gradient, 100 mL / min) to give L-31b (5 g, 18.64 mmol, 57.46% yield) as a colorless oil. 1 H NMR (400 MHz, CDCl 3 )δ6.60(s,1H),4.75(dd,J=5.6,13.2Hz,1H),4.59-4.51(m,1H),4.51-4.42(m,1H),4.32(q,J=7.2 Hz,2H),4.01(dd,J=6.0,8.4Hz,1H),3.83(dd,J=6.0,8.4Hz,1H),2.26(s,3H),1.42-1.28(m,9H). LC / MS[M+H]269.1 (calculated value), LC / MS[M+H]269.0 (observed value).

[0303] Preparation of 2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carboxylic acid, L-31c THF (20 mL) and H 2 A solution of L-31b (2.5 g, 9.32 mmol, 1 equiv.) in HO (5 mL) was added to LiOH H 2 2H2O (1.96 g, 46.59 mmol, 5 equiv.) was added and then stirred at 15° C. for 1 h. The mixture was concentrated to remove THF, then the pH of the aqueous phase was adjusted to pH=about 5 with TFA and extracted with EtOAc (50 mL×2). The combined organic layers were washed with Na 2 SO 4 The mixture was dried at rt, filtered and concentrated under reduced pressure to give the crude product L-31c (2.05 g, 8.53 mmol, 91.57% yield) as a white solid, which was used in the next step without further purification. 1H NMR (400 MHz, MeOD) δ 6.64 (s, 1H), 4.72 (dd, J = 6.0, 13.6 Hz, 1H), 4.59-4.49 (m, 1H), 4.42 (q, J = 6.0 Hz, 1H), 4.01 (dd, J = 6.0, 8.4 Hz, 1H), 3.80 (dd, J = 6.0, 8.4 Hz, 1H), 2.24 (s, 3H), 1.33 (s, 3H), 1.28 (s, 3H). LC / MS [M+H] 241.1 (calculated), LC / MS [M+H] 241.1 (observed).

[0304] Preparation of 2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carbonyl chloride, L-31d To a solution of L-31c (1 g, 4.16 mmol, 1 equiv) in DCM (15 mL) was added 1-chloro-N,N,2-trimethyl-prop-1-en-1-amine (1.11 g, 8.32 mmol, 1.1 mL, 2 equiv) at 0° C., then stirred for 5 h at 15° C. The mixture was concentrated to give L-31d (0.6 g, 2.32 mmol, 55.72% yield) as a pale yellow oil, which was used in the next step without further purification.

[0305] Preparation of 2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carbonyl isothiocyanate, L-31e To a solution of L-31d (0.6 g, 2.32 mmol, 1 equiv.) in MeCN (2 mL) was added potassium thiocyanate, KSCN (676.2 mg, 6.96 mmol, 3 equiv.), then stirred at 15 °C for 1 h. The mixture was filtered and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent of 0-40% ethyl acetate / petroleum ether gradient, 75 mL / min) to give L-31e (0.5 g, 1.78 mmol, 76.63% yield) as a pale yellow oil. 1 H NMR (400 MHz, CDCl 3)δ6.76(s,1H),4.74(dd,J=6.0,13.2Hz,1H),4.59-4.43(m,2H),4.06(dd,J=6.0 ,8.8Hz,1H),3.83(dd,J=5.6,8.8Hz,1H),2.30(s,3H),1.39(s,3H),1.33(s,3H).

[0306] Preparation of tert-butyl N-[(E)-4-(4-carbamoyl-2-methoxy-6-nitro-anilino)but-2-enyl]carbamate, L-31g To a solution of 4-chloro-3-methoxy-5-nitro-benzamide, L-31f (4 g, 17.35 mmol, 1 equiv.) and tert-butyl N-[(E)-4-aminobut-2-enyl]carbamate (4.52 g, 24.28 mmol, 1.4 equiv.) in n-BuOH (70 mL) was added diisopropylethylamine, DIEA (11.21 g, 86.73 mmol, 15.11 mL, 5 equiv.), followed by addition of N 2 The reaction mixture was stirred at 120° C. for 18 h under 100% H O. The reaction mixture was concentrated to remove n-BuOH, and the residue was diluted with 100 mL of H O at 15° C. 2 The crude product was diluted with O, stirred for 30 min, and filtered to give the crude product, which was triturated with EtOAc (50 mL) at 15 °C for 30 min to give L-31g (6 g, 15.77 mmol, 90.93% yield) as a red solid. 1 H NMR (400MHz, DMSO-d 6 ,δ8.18(d,J=2.0Hz,1H),8.01(s,1H),7.74(t,J=6.0Hz,1H),7.55(d,J=1.6Hz,1H),7.31(s,1H) ,6.92(s,1H),5.53(s,2H),4.09(d,J=4.0Hz,2H),3.87(s,3H),3.48(s,2H),1.44-1.30(m,9H). LC / MS [M+H] 381.2 (calculated value), LC / MS [M-55] 325.0 (observed value).

[0307] Preparation of tert-butyl N-[(E)-4-(2-amino-4-carbamoyl-6-methoxy-anilino)but-2-enyl]carbamate, L-31h Dissolve L-31g (6 g, 15.77 mmol, 1 equiv.) in MeOH (30 mL), THF (30 mL) and H 2 In a solution of NaO (15 mL) 2 CO 3 (6.69 g, 63.09 mmol, 4 equiv.) and disodium dithionite (19.22 g, 110.4 mmol, 7 equiv.) were added. The mixture was stirred at 15° C. for 1 h. The mixture was concentrated to remove THF and MeOH, then H 2 O (50 mL) was added, it was stirred for 10 min and filtered to give the crude product L-31h (4 g, 11.42 mmol, 72.37% yield) as an off-white solid, which was used in the next step without further purification. 1 H NMR (400MHz, MeOD) δ6.93(d,J=2.0Hz,1H),6.90(d,J=2.0Hz,1H),5.74-5.57(m,2H),3.85(s,3H),3.60-3.57(m,4H),1.43(s,9H). LC / MS[M+H]351.2 (calculated value), LC / MS[M+H]351.1 (observed value).

[0308] Preparation of 3-amino-4-[[(E)-4-aminobut-2-enyl]amino]-5-methoxy-benzamide, L-31i To a solution of L-31h (1.2 g, 3.42 mmol, 1 equiv) in EtOAc (15 mL) was added HCl / EtOAc (4 M, 42.8 mL, 50 equiv), then stirred at 15 °C for 0.5 h, and the mixture was concentrated to give L-31i (1.1 g, 3.40 mmol, 99.38% yield, 2HCl) as a white solid, which was used in the next step without further purification. LC / MS [M+H] 251.2 (calculated), LC / MS [M+H] 251.1 (observed).

[0309] Preparation of tert-butyl 4-[3-[2-[[(E)-4-(2-amino-4-carbamoyl-6-methoxy-anilino)but-2-enyl]amino]-5-carbamoyl-3-nitro-phenoxy]propyl]piperazine-1-carboxylate, L-31j A solution of L-31i (1.1 g, 3.40 mmol, 1 equiv, 2HCl) and DIEA (3.52 g, 27.2 mmol, 4.7 mL, 8 equiv) in n-BuOH (30 mL) was stirred at 15° C. for 0.5 h, then tert-butyl 4-[3-(5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl]piperazine-1-carboxylate (829 mg, 1.87 mmol, 0.55 equiv) was added and the mixture was stirred at 120° C. for 12 h. The mixture was concentrated in vacuo to remove n-BuOH, then the residue was diluted with 20 mL of water and extracted with DCM / i-PrOH=3 / 1 (30 mL×3). The combined organic layer was washed with 20 mL of brine and diluted with Na 2 SO 4 The crude product was dried over hexane, filtered, and concentrated under reduced pressure to give a residue, which was triturated with MTBE / EtOAc=5 / 1 (30 ml) at 15° C. for 10 min to give L-31j (1 g, 1.52 mmol, 44.74% yield) as a red solid, which was used in the next step without further purification. 1 H NMR(400MHz,MeOD)δ8.32(d,J=2.0Hz,1H),7.50(d,J=2.0Hz,1H),6.92(d,J =2.0Hz,1H),6.85(d,J=2.0Hz,1H),5.77-5.61(m,2H),4.23(d,J=3.6Hz,2H) ,4.04(t,J=6.0Hz,2H),3.77(s,3H),3.71-3.57(m,2H),3.48-3.44(m,4H),2 .54(t,J=7.2Hz,2H),2.46(t,J=5.2Hz,4H),2.06-1.98(m,2H),1.47(s,9H). LC / MS [M+H] 657.3 (calculated); LC / MS [M+H] 657.3 (observed).

[0310] Preparation of tert-butyl 4-[3-[5-carbamoyl-2-[[(E)-4-[5-carbamoyl-2-[[2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]amino]-3-nitro-phenoxy]propyl]piperazine-1-carboxylate, L-31k To a solution of L-31e (111 mg, 396 umol, 1.3 equiv.) in DMF (4 mL), L-31j (200 mg, 305 umol, 1 equiv.) was added and stirred at 0° C. for 30 min, followed by addition of Et 3 N (92.5 mg, 914 umol, 127 uL, 3 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDCI, EDAC, EDC, CAS Registry Number 25952-53-8 (175 mg, 914 umol, 3 equiv.) were added. The mixture was stirred at 15° C. for 16 h. NaHCO 3 The reaction was quenched with aqueous solution (5 mL) and water (5 mL) and the desired solid was precipitated from the mixture and filtered to give L-31k (0.2 g, 221 umol, 72.65% yield) as a pale yellow solid: LC / MS [M+H] 904.4 (calculated), LC / MS [M+H] 904.3 (observed).

[0311] Preparation of tert-butyl 4-[3-[3-amino-5-carbamoyl-2-[[(E)-4-[5-carbamoyl-2-[[2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]amino]phenoxy]propyl]piperazine-1-carboxylate, L-31l L-31k (0.2 g, 221 umol, 1 equiv.) in THF (1 mL), MeOH (1 mL), and H 2 A solution of NaO (1 mL) 2 CO 3 (93.8 mg, 885 umol, 4 equiv.) and disodium dithionite (270 mg, 1.55 mmol, 7 equiv.) were added and then stirred at 15° C. for 0.5 h. The mixture was concentrated to remove THF and MeOH. The desired solid was precipitated from the aqueous phase, filtered, and the cake was diluted with H 2 O (5 mL) to give L-31l (170 mg, 194.51 umol, 87.92% yield) as a yellow solid. LC / MS [M+H] 874.4 (calculated); LC / MS [M+H] 874.4 (observed).

[0312] Preparation of tert-butyl 4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[[2-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, L-31m A solution of L-31l (170 mg, 195 umol, 1 equiv.) and 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate, 3o (49.4 mg, 253 umol, 1.3 equiv.) in DMF (2 mL) was stirred at 0° C. for 0.5 h, then added Et 3 N (59.1 mg, 584 umol, 81.2 uL, 3 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDCI, EDAC, EDC, CAS Registry Number 25952-53-8 (112 mg, 584 umol, 3 equiv.) were added. The resulting mixture was stirred at 15° C. for 16 hours. NaHCO 3 The reaction was quenched with aqueous solution (5 mL) and water (5 mL). The desired solid was precipitated from the mixture and filtered to give L-31m (0.15 g, 144.91 umol, 74.50% yield) as a yellow solid. LC / MS [M+H] 1035.5 (calculated); LC / MS [M+H] 1035.5 (observed).

[0313] Preparation of 1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-(3-piperazin-1-ylpropoxy)benzimidazol-1-yl]but-2-enyl]-2-[[2-(2,3-dihydroxypropyl)-5-methyl-pyrazole-3-carbonyl]amino]-7-methoxy-benzimidazole-5-carboxamide, L-31n To a solution of L-31m (0.15 g, 145 umol, 1 equiv) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 7.25 mL, 200 equiv), then stirred at 15° C. for 0.5 h. The mixture was concentrated and the residue was purified by preparative HPLC (column: Phenomenex Luna 80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; B%: 5%-30%, 8 min) to give L-31n (75 mg, 66.78 umol, 46.09% yield, 2 TFA) as a white solid. 1 H NMR(400MHz,MeOD)δ7.60(d,J=1.2Hz,1H),7.56(d,J=1.2Hz,1H),7.26(d,J=1.2Hz,1H),7.22(d,J=1.2Hz ,1H),6.65(s,1H),6.63(s,1H),5.92-5.82(m,2H),5.05-5.01(m,4H),4.79-4.72(m,1H),4.69-4.59(m,3H) ),4.09-4.01(m,1H),3.90(t,J=5.6Hz,2H),3.69(s,3H),3.54(t,J=4.8Hz,2H),3.20(t,J=5.2Hz,4H),2.7 0-2.64(m,4H),2.51(t,J=7.2Hz,2H),2.22(s,3H),2.20(s,3H),1.78-1.69(m,2H),1.39(t,J=7.2Hz,3H). LC / MS[M+H]895.4 (calculated value); LC / MS[M+H]895.4 (observed value).

[0314] Preparation of BBI-L-31 A solution of 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (42.5 mg, 66.8 umol, 2.5 equiv.) in MeOH (0.3 mL) was added with L-31n (30 mg, 26.7 umol, 1 equiv., 2 TFA) and NaBH 3CN (5.04 mg, 80.1 umol, 3 equiv.) was added and then stirred for 12 h at 15° C. 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 BBI-L-31 (15.6 mg, 10.29 umol, 38.53% yield) as a pale yellow oil. 1 H NMR(400MHz,MeOD)δ7.63(s,1H),7.59(s,1H),7.31(s,1H),7.27(s,1H),6.89(s,2H),6.66(s,1H),6.61(s,1H),5. 96-5.83(m,2H),5.10-5.02(m,4H),4.79-4.61(m,4H),4.21-4.16(m,2H),4.11-4.02(m,1H),3.96(t,J=6.0Hz,2H), 3.86-3.81(m,2H),3.75(s,3H),3.72-3.69(m,4H),3.67-3.59(m,38H),3.56-3.52(m,4H),3.39-3.36(m,3H),2.99 -2.72(m,4H),2.62(t,J=6.8Hz,2H),2.24(s,3H),2.22(s,3H),1.79(td,J=6.8,13.6Hz,2H),1.41(t,J=7.2Hz,3H). LC / MS[M+H]1515.7 (calculated value), LC / MS[M+H]1515.7 (observed value)

[0315] Example L-34: Synthesis of 1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-7-[3-[4-[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]ethyl]piperazin-1-yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazole-5-carboxamide, BBI-L-34 [ka] [ka] Preparation of tert-butyl 4-[3-[2-[[(E)-4-aminobut-2-enyl]amino]-5-carbamoyl-3-nitro-phenoxy]propyl]piperazine-1-carboxylate, L-34a To a solution of (E)-but-2-ene-1,4-diamine (8.98 g, 56.45 mmol, 5 equiv, 2HCl) in n-BuOH (150 mL) was added DIEA (29.18 g, 225.79 mmol, 39.3 mL, 20 equiv) and tert-butyl 4-[3-(5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl]piperazine-1-carboxylate, 3g (5 g, 11.29 mmol, 1 equiv). The mixture was diluted with N 2 The mixture was stirred at 120°C for 12 hours under reduced pressure. The mixture was filtered to remove insoluble materials. The filtrate was concentrated to give a residue. The residue was purified by preparative HPLC (column: Xtimate C18 10u 250mm*80mm; mobile phase: [water (NH4HCO3)-ACN]; B%: 10%~50%, 27 min) to give L-34a (4g, 8.12mmol, 35.97% yield) as a red solid. LC / MS [M+H] 493.3 (calculated); LC / MS [M+H] 493.2 (observed).

[0316] Preparation of tert-butyl 4-[3-[5-carbamoyl-2-[[(E)-4-(4-methoxycarbonyl-2-nitro-anilino)but-2-enyl]amino]-3-nitro-phenoxy]propyl]piperazine-1-carboxylate, L-34b To a solution of L-34a (400 mg, 812 umol, 1 equiv) in n-BuOH (1.20 g, 16.2 mmol, 1.49 mL, 20 equiv) was added DIEA (524 mg, 4.06 mmol, 707 uL, 5 equiv) and methyl 4-fluoro-3-nitro-benzoate (242 mg, 1.22 mmol, 1.5 equiv). The mixture was cooled to 37° C. for 1 hour and then cooled to 37° C. 2 The mixture was stirred at 120° C. for 12 hours under 50° C. The reaction mixture was concentrated to remove n-BuOH. The residue was diluted with H 2The mixture was diluted with 200 mL of EtOAc (10 mL×3) and then extracted with EtOAc (10 mL×3). 2 SO 4 The mixture was dried at rt, filtered and concentrated under reduced pressure to give L-34b (600 mg, crude) as a yellow solid. LC / MS [M+H] 672.3 (calculated); LC / MS [M+H] 672.3 (observed).

[0317] Preparation of tert-butyl 4-[3-[3-amino-2-[[(E)-4-(2-amino-4-methoxycarbonyl-anilino)but-2-enyl]amino]-5-carbamoyl-phenoxy]propyl]piperazine-1-carboxylate, L-34c L-34b (600 mg, 893 umol, 1 equiv.) in MeOH (3 mL), THF (3 mL) and H 2 A solution of NaO (1.5 mL) 2 CO 3 (473 mg, 4.47 mmol, 5 equiv) and disodium dithionite (1.09 g, 6.25 mmol, 7 equiv) were added and then stirred at 15° C. for 1 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with EtOAc (10 mL), filtered to remove insoluble material, and the filtrate was concentrated under reduced pressure to give L-34c (570 mg, crude) as a white solid. LC / MS [M+H] 612.3 (calculated); LC / MS [M+H] 612.3 (observed).

[0318] Preparation of 1-[(E)-4-[7-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propoxy]-5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazole-5-carboxylate, methyl ester, L-34d To a solution of L-34c (570 mg, 931 mmol, 1 equiv) in DMF (5 mL) was added 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate (400 mg, 2.05 mmol, 2.2 equiv). After the addition, the mixture was stirred at 0° C. for 30 min, then diluted with Et3 N (565 mg, 5.59 mmol, 778 uL, 6 eq.) and EDCI (1.07 g, 5.59 mmol, 6 eq.) were added. The resulting mixture was stirred at 15° C. for an additional 12 h. Saturated NaHCO 3 (20 mL), the aqueous phase was extracted with EtOAc (50 mL×3), and the combined organic phase was washed with brine (30 mL) and Na 2 SO 4 Drying over rt, filtration and concentration gave L-34d (800 mg, crude) as a yellow solid. LC / MS [M+H] 934.5 (calculated); LC / MS [M+H] 934.5 (observed).

[0319] Preparation of 1-[(E)-4-[7-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propoxy]-5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazole-5-carboxylic acid, L-34e H 2 A solution of L-34d (700 mg, 749 mmol, 1 equiv.) in 2 mL of HO and 10 mL of THF was treated with lithium hydroxide hydrate, LiOH. 2 2H2O (125 mg, 3.00 mmol, 4 equiv.) was added and then stirred at 50° C. for 4 h. The reaction mixture was quenched with HCl (2M) until the pH was about 5, and the mixture was concentrated under reduced pressure to give a residue, which was then purified by H 2 Trituration with 2H2O (10 mL) and filtration gave L-34e (800 mg, crude) as a white solid. LC / MS [M+H] 920.5 (calculated); LC / MS [M+H] 920.5 (observed).

[0320] Preparation of tert-butyl 4-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]piperazine-1-carboxylate, L-34f To a solution of L-34e (250 mg, 272 umol, 1 equiv.) in DMF (0.5 mL) was added NH4Cl (145 mg, 2.72 mmol, 10 equiv.), DIEA (105 mg, 815 umol, 142 uL, 3 equiv.) and 1-bis(dimethylamino)methylene]-1H-1,2,3-triazo[4,5-b]pyridinium 3-oxide hexafluorophosphate, azabenzotriazole tetramethyluronium, HATU, CAS Registry Number 148893-10-1 (155 mg, 408 umol, 1.5 equiv.). The mixture was stirred at 15° C. for 1 h. The mixture was poured into water (15 mL), the aqueous phase was extracted with EtOAc (20 mL×3), and the combined organic phase was washed with brine (15 mL) and sodium chloride. 2 SO 4 Drying over ice, filtration and concentration gave L-34f (250 mg, crude) as a white solid. LC / MS [M+H] 919.4 (calculated); LC / MS [M+H] 919.4 (observed).

[0321] Preparation of 1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-(3-piperazin-1-ylpropoxy)benzimidazole-5-carboxamide, L-34g To a solution of L-34f (250 mg, 272 umol, 1 eq) in EtOAc (1 mL) was added HCl / EtOAc (4M, 4.17 mL, 61.3 eq). The mixture was stirred at 25° C. for 0.5 h. The mixture was concentrated in vacuo to give a residue. The residue was purified by preparative HPLC (column: Phenomenex C18 80*30 mm*3 um; mobile phase: [water (TFA)-ACN]; B%: 1%-25%, 8 min) to give L-34g (21 mg, 25.64 umol, 9.43% yield) as a white solid. 1 H NMR(400MHz,MeOD)δ7.94(d,J=1.2Hz,1H),7.72(dd,J=1.6,8.4Hz,1H),7.53(d,J=1.2Hz,1H),7.35(d ,J=8.4Hz,1H),7.26(s,1H),6.58(s,1H),6.53(s,1H),6.00-5.86(m,1H),5.76-5.73(m,1H),5.02(br d,J=4.0Hz,2H),4.92--4.84(m,2H),4.65-4.48(m,4H),4.01(br t,J=6.0Hz,2H),3.36(br t,J=4.8Hz,4H),3.02-2.98(m,4H),2.80(br t,J=7.2Hz,2H),2.18(d,J=9.5Hz,6H),1.94-1.79(m,2H),1.43-1.25(m,6H). HPLC: 96.66% (220nm). LC / MS[M+H]819.4 (calculated value); LC / MS[M+H]819.4 (observed value).

[0322] Preparation of BBI-L-34 To a solution of L-34g (50 mg, 61.06 umol, 1 equiv.) in MeOH (1 mL), 2-(2,5-dioxopyrrol-1-yl)-N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-oxoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (38.87 mg, 61.06 umol, 1 equiv.) was added at 0° C., and the mixture was stirred at 0° C. for 15 minutes. NaBH3CN (11.51 mg, 183.17 umol, 3 equiv.) was then added, and the resulting mixture was stirred at 15° C. for 16 hours. The mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: 10%-40%, 8 min) to give BBI-L-34 (60mg, 35.98umol, yield 58.93%, 2TFA) as a white solid. 1 H NMR(400MHz,MeOD)δ7.97(d,J=1.6Hz,1H),7.75(dd,J=1.6,8.4Hz,1H),7.56(d,J=1.2Hz,1H),7.39(d,J=8.4Hz,1H),7.31(s,1H),6.87(s,2H), 6.62(s,1H),6.56(s,1H),6.02-5.89(m,1H),5.86-5.73(m,1H),5.09-5 .03(m,2H),4.90-4.80(m,2H),4.67-4.53(m,4H),4.16(s,2H),4.05(br t,J=6.0Hz,2H),3.89-3.80(m,2H),3.74-3.46(m,42H),3.37-3.33(m,4H),2.98-2.75(m,4H),2.66(br t,J=7.2Hz,2H),2.20(d,J=9.6Hz,6H),1.87-1.76(m,2H),1.38(t,J=7.2Hz,3H),1.32(t,J=7.2Hz,3H). HPLC: 97.16% (220nm). LC / MS[M+H]1439.7 (calculated value), LC / MS[M+H]1439.9 (observed value).

[0323] Example 201: Preparation of immune complexes (IC) In an exemplary procedure, for preparation of lysine-based conjugation, the antibody is buffer exchanged into conjugation buffer containing 100 mM borate, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3 using Zeba™ spin desalting columns (Thermo Fisher Scientific). The concentration of the buffer-exchanged antibody is adjusted to approximately 5-25 mg / ml using conjugation buffer and sterile filtered. The bis-benzimidazole-linker (BBI-L) intermediate compound of formula (II) is dissolved in either dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5-20 mM. For conjugation, the antibody is mixed with 4-20 molar equivalents of BBI-L. In some cases, additional DMA or DMSO up to 20% (v / v) was added to improve the solubility of BBI-L in the conjugation buffer. The reaction is allowed to proceed for approximately 30 minutes to 4 hours at 20°C or 30°C or 37°C. The resulting conjugate is purified from unreacted BBI-L using two successive 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).

[0324] In an exemplary procedure, for preparation of cysteine-based conjugation, the antibody is 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 antibody was adjusted to approximately 5-20 mg / ml using conjugation buffer and sterile filtered. BBI-L is 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 BBI-L. In some cases, additional DMA or DMSO up to 20% (v / v) was added to improve the solubility of BBI-L in the binding buffer. The reaction is allowed to proceed at 20 °C for approximately 30 min to 4 h. The resulting complex is purified from unreacted BBI-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).

[0325] After conjugation, the complex may be further purified using size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential filtration, and combinations thereof to potentially remove unreacted BBI-L and / or high molecular weight aggregates.

[0326] In another exemplary procedure, the antibody is buffer exchanged into binding 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). The buffer is then used to adjust the eluate to a concentration of about 1-10 mg / ml each, which is then sterile filtered. The antibody is pre-heated to 20-30° C. and rapidly mixed with 2-20 (e.g., 7-10) molar equivalents of a bis-benzimidazole-linker (BBI-L) intermediate compound of formula (II). The reaction is allowed to proceed for about 16 hours at 30° C., and the immune complex (IC) is separated from the reactants by passing it through two successive G-25 desalting columns equilibrated with phosphate buffered saline (PBS) at pH 7.2 to provide the immune complex (IC) of Table 2. Adjuvant to antibody ratios (DAR) were determined by liquid chromatography mass spectrometry using an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) C4 reversed-phase column interfaced to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0327] For conjugation, the antibody may 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 may be used. BBI-L is dissolved in a solvent system that includes at least one polar aprotic solvent described elsewhere herein. In some such embodiments, BBI-L is dissolved in Tris buffer pH 8 (e.g., 50 mM Tris) to a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM, and within these ranges, for example, about 5 mM to about 50 mM or about 10 mM to about 30 mM. In some embodiments, BBI-L is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide), or acetonitrile, or another suitable dipolar aprotic solvent.

[0328] Alternatively, in the conjugation reaction, an equivalent excess of the BBI-L solution may be diluted and combined with the antibody solution. The BBI-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 equivalents of the thienoazepine-linker intermediate to the antibody may be about 1.5:1, about 3:1, about 5:1, about 10:1, about 15:1, or about 20:1, and within these ranges, for example, about 1.5:1 to about 20:1, about 1.5:1 to about 15:1, about 1.5:1 to about 10:1, about 3:1 to about 15:1, about 3:1 to about 10:1, about 5:1 to about 15:1, or about 5:1 to about 10:1. Completion of the reaction may be suitably monitored by methods known in the art, such as LC-MS. The conjugation reaction is usually completed within about 1 hour to about 16 hours. After the reaction is completed, a reagent may be added to the reaction mixture to quench the reaction. When the antibody thiol groups react with a thiol-reactive group such as the maleimide of BBI-L, the unreacted antibody thiol groups may be reacted with a capping reagent. An example of a suitable capping reagent is ethylmaleimide.

[0329] 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, chromatofocusing, 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 ion exchange column, followed by washing, for example, with at least 10 column volumes of 20 mM sodium succinate, pH 5. The complex may be suitably eluted with a buffer such as PBS.

[0330] Example 202: Functional evaluation of immune complexes The immunoconjugates of the present invention can be evaluated in a co-culture assay using primary human peripheral blood mononuclear cells (PBMCs) co-cultured with target antigen-expressing tumor cells. Briefly, PBMCs are freshly isolated from healthy human donor blood (Stanford Blood Center) by density centrifugation. PBMCs are then co-cultured with antigen-expressing tumor cells at an effector to target ratio of 10:1 in complete medium (RPMI supplemented with 10% FBS) and incubated overnight with a range of concentrations of the test substances indicated. Activation is measured by secretion of pro-inflammatory cytokines such as IFNλ1 and TNFα by BioLegend LEGENDPLEX™ cytokine bead assay.

[0331] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and as if set forth in its entirety herein.

Claims

1. An immune complex comprising an antibody covalently attached by a linker to one or more STING agonist moieties, of formula I: Ab[L-D] ｐ I or a pharmaceutically acceptable salt thereof, said immune complex, wherein, Ab is said antibody; p is an integer from 1 to 8; D is said STING agonist moiety having the following formula; 【Chemical Formula 1】 X ａ and X ｂ are independently selected from 5-membered heteroaryl optionally substituted by R ５ ; R １ and R ４ are independently H, F, Cl, Br, I, -CN, -OH, -O-(C １ -C ６ alkyl) and R ５ selected from the group consisting of; R ２ａ and R ２ｂ are H, -C(=O)N(R ６ ) ２ and R ５ independently selected from; X ａ X ｂ R １ R ４ R ２ａ R ２ｂ one of is substituted by R ５ ; R ３ is F, Cl, -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 ) ２C optionally substituted by one or more groups selected from １ -C ６ -alkyl-diyl, -(C １ -C ３ -alkyl-diyl)-O-(C １ -C ３ -alkyl-diyl)-, C ２ -C ６ -alkenyl-diyl and C ２ -C ６ -alkynyl-diyl; R ５ is -(C １ -C １２ -alkyl-diyl)-*; -(C １ -C １２ -alkyl-diyl)-N(R ６ )-*; -(C １ -C １２ -alkyl-diyl)-O-*; -(C １ -C １２ -alkyl-diyl)-(C ２ -C ２０ -heterocyclyl-diyl)-*; -O-(C １ -C １２ -alkyl-diyl)-*; -O-(C １ -C １２ -alkyl-diyl)-N(R ６ )-*; -O-(C １ -C １２ -alkyl-diyl)-O-*; -O-(C １ -C １２ -alkyl-diyl)-(C ２ -C ２０ -heterocyclyl-diyl)-*; -O-(C １ -C １２ -alkyl-diyl)-(C ２ -C ２０ -heterocyclyl-diyl)-N(R ６ )-*; -OC(=O)N(R ６ )-*; -OC(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-N(R ６ )-*; -N(R ６ )-*; -N(R ６ )-(C １ -C １２ alkyldiyl)-*; -N(R ６ )-(C １ -C １２ alkyldiyl)-N(R ６ )-*; -N(R ６ )-(C １ -C １２ alkyldiyl)-O-*; -N(R ６ )-(C １ -C １２ alkyldiyl)(C ２ -C ２０ heterocyclyldiyl)-*; -C(=O)N(R ６ )-*; -C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-*; -C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)N(R ６ )-*; -C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-O-*; -(C ２ -C ２０ heterocyclyldiyl)-*; -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-*; and -S(=O) ２ -(C ２ -C ２０ heterocyclyldiyl)-(C １ -C １２ alkyldiyl)-N(R ６ ), selected from the group consisting of; The asterisk * indicates the binding site of L; R ６ is independently H or C １ -C ６ alkyl; L is -C(=O)-PEG-; -C(=O)-PEG-C(=O)N(R ６ )(C １ -C １２ alkyldiyl)-C(=O)-Gluc-; -C(=O)-PEG-O-; -C(=O)-PEG-O-C(=O)-; -C(=O)-PEG-C(=O)-; -C(=O)-PEG-C(=O)-PEP-; -C(=O)-PEG-N(R ６ ); -C(=O)-PEG-N(R ６ )-C(=O)-; -C(=O)-PEG-N(R ６ )-PEG-C(=O)-PEP-; -C(=O)-PEG-N ＋ (R ６ ) ２ -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-C(=O)N(R ６ )(C １ -C １２ (Alkyldiyl)-C(=O)-PEP-; -C(=O)-PEG-SS-(C １ -C １２ Alkyldiyl)-OC(=O)-; -C(=O)-PEG-SS-(C １ -C １２ Alkyldiyl)-C(=O)-; -C(=O)-(C １ -C １２ Alkyldiyl)-C(=O)-PEP-; -C(=O)-(C １ -C １２ Alkyldiyl)-C(=O)-PEP-N(R ６ )- (C １ -C １２ Alkyldiyl)-; -C(=O)-(C １ -C １２ Alkyldiyl)-C(=O)-PEP-N(R ６ )- (C １ -C １２ Alkyldiyl)-N(R ５ )-C(=O); -C(=O)-(C １ -C １２ Alkyldiyl)-C(=O)-PEP-N(R ６ )- (C １ -C １２ Alkyldiyl)-N(R ６ )C(=O)-(C ２ -C ５ Monoheterocyclyldiyl)-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )- (C １ -C １２ Alkyldiyl)-C(=O)-Gluc-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-O-C(=O)-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-N(R ５ )-C(=O)-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-PEP-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ alkyl diyl)-OC(=O)-; -Succinimidyl-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )(C １ -C １２ alkyl diyl)-; -Succinimidyl-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )(C １ -C １２ alkyl diyl)-C(=O)-PEP-; -Succinimidyl-(CH ２ ) ｍ-C(=O)-PEP-N(R ６ )(C １ -C １２ -alkyldiyl)N(R ６ )(C=O)-; and -succinimidyl-(CH ２ )( ｍ -C(=O)-PEP-N(R ６ )(C １ -C １２ -alkyldiyl)N(R ６ )(C=O)-(C ２ -C ５ -monoheterocyclediyl)- selected from the group consisting of; PEG has the formula: -(CH ２ CH ２ O) ｎ -(CH ２ )( ｍ - where m is an integer from 1 to 5 and n is an integer from 2 to 50; Gluc is [Chemical Formula 2] has; PEP has the formula: [Chemical Formula 3] has, AA is independently selected from natural or non-natural 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 substituted by one or more groups selected from F, Cl, NO ２ , -OH, -OCH ３ and glucuronic acid having the following structure, C ６ -C ２０ -aryldiyl and C １ -C ２０ -heteroaryldiyl selected from, [Chemical Formula 4] R ７ is -CH(R ８ )O-, -CH ２ -, -CH ２ N(R ８ )-, and -CH(R ８selected from the group consisting of O—C(═O)—, R ８ is H, C １ —C ６ alkyl, C(═O)—C １ —C ６ alkyl, and —C(═O)N(R ９ ) ２ selected from, R ９ is independently H, C １ —C １２ alkyl, and —(CH ２ CH ２ O) ｎ —(CH ２ ) ｍ —OH selected from the group consisting of, m is an integer from 1 to 5, n is an integer from 2 to 50, or two R ９ groups together form a 5- or 6-membered heterocyclic ring; y is an integer from 2 to 12; z is 0 or 1; alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl and heteroaryldiyl are independently and optionally 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 ) ２CH 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 ), -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ , -CH ２ NHSO ２ CH ３ , -CH ２ NHCH ３ , -CH ２ N(CH ３ ) ２ , -CO ２ H, -COCH ３ , -CO ２ CH ３ , -CO ２ C(CH ３ ) ３ , -COCH(OH)CH ３ , -CONH ２ , -CONHCH ３ , -CON(CH ３ ) ２ , -C(CH ３ ) ２ CONH ２ , -NH ２ , -NHCH ３ , -N(CH ３ ) ２ , -NHCOCH ３ , -N(CH ３ )COCH ３ , -NHSO ２ CH ３ , -N(CH ３ )C(CH ３ ) ２ CONH ２ , -N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ , -NHC(=NH)H, -NHC(=NH)CH ３ , -NHC(=NH)NH ２ , -NHC(=O)NH ２ , -NO ２ , =O, -OH, -OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, -OCH ２ CH ２ N(CH ３ O) ２ , -O(CH ２ CH ２ O) ｎ -(CH ２ ) ｍ CO ２ H, -O(CH ２ CH ２ O) ｎ , -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, the immune complex being substituted with one or more groups independently selected from

2. The immune complex according to claim 1, wherein the antibody is an antibody construct having an antigen-binding domain that binds to a target selected from PD-L1, HER2, CEA, and Trop2.

3. The immune complex according to claim 2, wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, ravulizumab, and sacituzumab.

4. X ａ and X ｂ are independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl, the immune complex according to any one of claims 1 to 3.

5. X ａ and X ｂ are each, -CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , and -CH ２ CH(CH ３ ) ２ is pyrazolyl substituted by one or more groups selected from, the immune complex according to claim 4.

6. X ａ and X ｂ one of is substituted with R ５ , the immune complex according to any one of claims 1 to 3.

7. R １ is, -OCH ３ , -OCH ２ CH ３ , -OCH ２ CH ２ OCH ３ , -OCH ２ CH ２ OH, and -OCH ２ CH ２ N(CH ３ ) ２ selected from the group consisting of, the immune complex according to any one of claims 1 to 3.

8. R １ is, -OCH ３The immune complex according to claim 7, which is or F.

9. R ２ａ and R ２ｂ each being -C(=O)NH ２ The immune complex according to any one of claims 1 to 3.

10. R ２ａ and R ２ｂ One of which is R ５ The immune complex according to any one of claims 1 to 3, which is substituted.

11. R ３ being -CH ２ CH ２ -, -CH=CH-, and -C≡C-; The immune complex according to any one of claims 1 to 3, which is selected from.

12. R ３ being substituted with one or more groups selected from F, -OH and -OCH ３ C ２ -C ４ Alkenyldiyl; The immune complex according to any one of claims 1 to 3.

13. R ４ being -O-(C １ -C １２ -Alkyldiyl)-(C ２ -C ２０ Heterocyclyldiyl)-*; The immune complex according to any one of claims 1 to 3.

14. C １ -C １２ Alkyldiyl is propyldiyl, and C ２ -C ２０ Heterocyclyldiyl is piperidyiyl; The immune complex according to claim 13.

15. R １ and R ４ One of which is R ５ The immune complex according to any one of claims 1 to 3, which is substituted.

16. The immune complex according to any one of claims 1 to 3, wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

17. The immune complex according to any one of claims 1 to 3, wherein L binds to the cysteine thiol of the antibody.

18. The immune complex according to any one of claims 1 to 3, wherein for the PEG, m is 1 or 2 and n is an integer from 2 to 10.

19. The immune complex according to claim 18, wherein n is 10.

20. The immune complex according to any one of claims 1 to 3, wherein L contains PEP, PEP is a dipeptide, and has the following formula: 【Chemical Formula 5】

21. The immune complex according to any one of claims 1 to 3, wherein L contains PEP, PEP is a tripeptide, and has the following formula: 【Chemical Formula 6】

22. The immune complex according to any one of claims 1 to 3, wherein L contains PEP, PEP is a tetrapeptide, and has the following formula: 【Chemical Formula 7】

23. L is selected from the following structures: 【Chemical Formula 8】 In the formula, the wavy line indicates the bonding point to R ５ The immune complex according to any one of claims 1 to 3.

24. A STING agonist-linker intermediate compound having Formula II, 【Chemical Formula 9】 In the formula, X ａ and X ｂ are independently R ５selected from 5-membered heteroaryl optionally substituted by; R １ and R ４ are independently selected from the group consisting of F, Cl, Br, I, -CN, -OH, -O-(C １ -C ６ -alkyl) and R ５ ; R ２ａ and R ２ｂ are independently -C(=O)N(R ６ ) ２ , and R ５ ; X ａ , X ｂ , R １ , R ４ , R ２ａ , R ２ｂ one of is substituted by R ５ ; R ３ is optionally substituted by one or more groups selected from F, Cl, -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 C １ -C ６ -alkyldiyl, -(C １ -C ３ -alkyldiyl)-O-(C １ -C ３ -alkyldiyl)-, C ２ -C ６ -alkenyldiyl and C ２ -C ６ -alkynyldiyl; R ５ is -(C １ -C １２ -alkyldiyl)-L; -(C １ -C １２-(Alkyldiyl)-N(R ６ ); -(C １ -C １２ -Alkyldiyl)-O-L; -(C １ -C １２ -Alkyldiyl)-(C ２ -C ２０ -Heterocyclyldiyl)-L; -O-(C １ -C １２ -Alkyldiyl)-L; -O-(C １ -C １２ -Alkyldiyl)-N(R ６ ); -O-(C １ -C １２ -Alkyldiyl)-O-L; -O-(C １ -C １２ -Alkyldiyl)-(C ２ -C ２０ -Heterocyclyldiyl)-L; -O-(C １ -C １２ -Alkyldiyl)-(C ２ -C ２０ -Heterocyclyldiyl)-N(R ６ ); -OC(=O)N(R ６ ); -OC(=O)N(R ６ )-(C １ -C １２ -Alkyldiyl)-N(R ６ ); -N(R ６ ); -N(R ６ )-(C １ -C １２ -Alkyldiyl)-L; -N(R ６ )-(C １ -C １２ -Alkyldiyl)-N(R ６ ); -N(R ６ )-(C １ -C １２-(alkyl diyl)-O-L; -N(R ６ )-(C １ -C １２ alkyl diyl)-(C ２ -C ２０ heterocyclic diyl)-L; -C(=O)N(R ６ )-L; -C(=O)N(R ６ )-(C １ -C １２ alkyl diyl)-L; -C(=O)N(R ６ )-(C １ -C １２ alkyl diyl)-N(R ６ )-L; -C(=O)N(R ６ )-(C １ -C １２ alkyl diyl)-O-L; -(C ２ -C ２０ heterocyclic diyl)-L; -S(=O) ２ -(C ２ -C ２０ heterocyclic diyl)-L; and -S(=O) ２ -(C ２ -C ２０ heterocyclic diyl)-(C １ -C １２ alkyl diyl)-N(R ６ )-L selected from the group consisting of; R ６ is independently H or C １ -C ６ alkyl; L is, Q-C(=O)-PEG-; Q-C(=O)-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyl diyl)-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 alkyl diyl)-; Q-C(=O)-PEG-C(=O)-PEP-N(R ６ )(C １ -C １２ alkyl diyl)N(R ６ )C(=O)-(C ２ -C ５ monoheterocyclic diyl)-; Q-C(=O)-PEG-C(=O)N(R ６ )(C １ -C １２ alkyl diyl)-C(=O)-PEP-; Q-C(=O)-PEG-SS-(C １ -C １２ alkyl diyl)-OC(=O)-; Q-C(=O)-PEG-SS-(C １ -C １２ alkyl diyl)-C(=O)-; Q-C(=O)-(C １ -C １２ alkyl diyl)-C(=O)-PEP-; Q-C(=O)-(C １ -C １２ alkyl diyl)-C(=O)-PEP-N(R ６ )(C １ -C １２-(Alkyldiyl)-; Q-C(=O)-(C １ -C １２ -Alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ -Alkyldiyl)-N(R ５ )-C(=O); Q-C(=O)-(C １ -C １２ -Alkyldiyl)-C(=O)-PEP-N(R ６ )-(C １ -C １２ -Alkyldiyl)-N(R ６ )C(=O)-(C ２ -C ５ -Monoheterocyclyldiyl)-; 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)-Glu-; 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(R ５ )-C(=O)-; Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)-PEP-; Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-SS-(C １ -C １２ alkyldiyl)-OC(=O)-; Q-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)-; Q-(CH ２ ) ｍ -C(=O)N(R ６ )-PEG-C(=O)N(R ６ )-(C １ -C １２ alkyldiyl)-C(=O)-PEP-; Q-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ )C(═O)—; and Q-(CH ２ ) ｍ -C(=O)-PEP-N(R ６ )-(C １ -C １２ Alkyldiyl)N(R ６ ) C(=O)-(C ２ -C ５ is a linker selected from the group consisting of: PEG has the formula: -(CH ２ CH ２ O) ｎ - (CH ２ ) ｍ - m is an integer from 1 to 5 and n is an integer from 2 to 50; Gluc is [C10] has; PEP has the formula: [Chemical Formula 11] has, AA is independently selected from natural or non-natural amino acid side chains, or one or more of the nitrogen atoms adjacent to AA form a 5-membered ring proline amino acid, and the wavy line indicates the bonding point; Cyc is F, Cl, NO ２ , -OH, -OCH ３ and is optionally substituted by one or more groups selected from glucuronic acid having the following structure C ６ -C ２０ aryl diyl and C １ -C ２０ heteroaryl diyl, [Chemical Formula 12] R ７ is -CH(R ８ )O-, -CH ２ -, -CH ２ N(R ８ )-, and -CH(R ８ )O-C(=O)-, selected from the group consisting of; R ８ is H, C １ -C ６ alkyl, C(=O)-C １ -C ６ alkyl, and -C(=O)N(R ９ ) ２ selected from; R ９ is independently H, C １ -C １２ alkyl, and -(CH ２ CH ２ O) ｎ -(CH ２ ) ｍ -OH, selected from the group consisting of; m is an integer from 1 to 5, n is an integer from 2 to 50, or two R ９ groups together form a 5- or 6-membered heterocyclic ring; y is an integer from 2 to 12; z is 0 or 1; Q is F, Cl, NO ２and SO ３ － selected from the group consisting of N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, maleimide, and phenoxy, and substituted with one or more groups independently selected from Alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclic, carbocyclicdiyl, heterocyclic, heterocyclicdiyl, heteroaryl, and heteroaryldiyl are each independently and optionally F, Cl, Br, I, -CN, -CH ３ , -CH ２ CH ３ , -CH=CH ２ , -C≡CH, -C≡CCH ３ , -CH ２ CH ２ CH ３ , -CH(CH ３ ) ２ , -CH ２ CH(CH ３ ) ２ , -CH ２ OH, -CH ２ OCH ３ , -CH ２ CH ２ OH, -C(CH ３ ) ２ OH, -CH(OH)CH(CH ３ ) ２ , -C(CH ３ ) ２ CH ２ OH, -CH ２ CH ２ SO ２ CH ３ , -CH ２ OP(O)(OH) ２ , -CH ２ F, -CHF ２ , -CF ３ , -CH ２ CF ３ , -CH ２ CHF ２ , -CH(CH ３ )CN, -C(CH ３ ) ２ CN, -CH ２ CN, -CH ２ NH ２ 、 - CH ２ NHSO ２ CH ３ 、 - CH ２ NHCH ３ 、 - CH ２ N(CH ３ ) ２ 、 - CO ２ H、 - COCH ３ 、 - CO ２ CH ３ 、 - CO ２ C(CH ３ ) ３ 、 - COCH(OH)CH ３ 、 - CONH ２ 、 - CONHCH ３ 、 - CON(CH ３ ) ２ 、 - C(CH ３ ) ２ CONH ２ 、 - NH ２ 、 - NHCH ３ 、 - N(CH ３ ) ２ 、 - NHCOCH ３ 、 - N(CH ３ )COCH ３ 、 - NHS(O) ２ CH ３ 、 - N(CH ３ )C(CH ３ ) ２ CONH ２ 、 - N(CH ３ )CH ２ CH ２ S(O) ２ CH ３ 、 - NHC(=NH)H、 - NHC(=NH)CH ３ 、 - NHC(=NH)NH ２ 、 - NHC(=O)NH ２ 、 - NO ２ 、 = O、 - OH、 - OCH ３ 、 - OCH ２ CH ３ 、 - OCH ２ CH ２ OCH ３ 、 - OCH ２ CH ２ 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 The STING agonist-linker intermediate compound is substituted with one or more groups independently selected from H.

25. Q, [C13] 25. The STING agonist-linker intermediate compound of claim 24, selected from:

26. Q is F, Cl, NO ２ , and S.O. ３ － 25. The STING agonist-linker intermediate compound of claim 24, which is phenoxy substituted with one or more groups independently selected from:

27. 25. The STING agonist-linker intermediate compound of claim 24, wherein Q is 2,3,5,6-tetrafluorophenoxy or 2,3,5,6-tetrafluoro-4-sulfonato-phenoxy.

28. 25. The STING agonist-linker intermediate compound of claim 24, wherein Q is maleimide.

29. L has the structure: [C14] Selected from, with a wavy line indicating the junction to R ５ The STING agonist-linker intermediate compound according to claim 24, showing the junction point to R.

30. An STING agonist-linker intermediate compound selected from.

31. An immune complex prepared by the binding of an antibody to the STING agonist-linker intermediate compound according to any one of claims 24 to 30.

32. A pharmaceutical composition comprising a therapeutically effective amount of the immune complex according to any one of claims 1 to 3 and one or more pharmaceutically acceptable diluents, vehicles, carriers or excipients.

33. A medicament for treating cancer, comprising the immune complex according to any one of claims 1 to 3, wherein the cancer is sensitive to the inflammatory response induced by STING agonism.

34. A medicament for treating cancer, comprising the immune complex according to any one of claims 1 to 3, wherein the cancer is selected from bladder cancer, salivary gland cancer, endometrial 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.

35. A method for preparing the immune complex of formula I according to claim 1, wherein the STING agonist-linker intermediate compound according to claim 24 is bound to an antibody.

36. The STING agonist-linker intermediate compound.

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